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WO2001037665A1 - Compositions et methodes d'administration de medicaments au moyen de molecules de liaison amphiphiles - Google Patents

Compositions et methodes d'administration de medicaments au moyen de molecules de liaison amphiphiles Download PDF

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Publication number
WO2001037665A1
WO2001037665A1 PCT/US2000/032612 US0032612W WO0137665A1 WO 2001037665 A1 WO2001037665 A1 WO 2001037665A1 US 0032612 W US0032612 W US 0032612W WO 0137665 A1 WO0137665 A1 WO 0137665A1
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dna
amphiphile
gene
cell
cells
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PCT/US2000/032612
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English (en)
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Jon A. Wolff
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Mirus Corporation
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Priority to EP00982310A priority Critical patent/EP1233671A4/fr
Publication of WO2001037665A1 publication Critical patent/WO2001037665A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • 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
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • the present invention relates to the delivery of desired compounds (e.g., drugs and nucleic acids) into cells using noncovalent delivery systems.
  • desired compounds e.g., drugs and nucleic acids
  • the present invention provides compositions and methods for the delivery and release of a compound of interest to a cell.
  • a variety of methods and routes of administration have been developed to deliver pharmaceuticals that include small molecular drugs and biologically active compounds such as peptides, hormones, proteins, and enzymes to their site of action.
  • Parenteral routes of administration include intravascular (intravenous, intraarterial), intramuscular, intraparenchymal, intradermal, subdermal, subcutaneous, intratumor, intraperitoneal, and intralymphatic injections that use a syringe and a needle or catheter.
  • the blood circulatory system provides systemic spread of the pharmaceutical.
  • Polyethylene glycol and other hydrophilic polymers have provided protection of the pharmaceutical in the blood stream by preventing its interaction with blood components and to increase the circulatory time of the pharmaceutical by preventing opsonization , phagocytosis and uptake by the reticuloendothelial system.
  • the enzyme adenosine deaminase has been covalently modified with polyethylene glycol to increase the circulatory time and persistence of this enzyme in the treatment of patients with adenosine deaminase deficiency.
  • Liposomes were also used as drug delivery vehicles for low molecular weight drugs and macromolecules such as amphote ⁇ cin B for systemic fungal infections and candidiasis Inclusion of anti-cancer drugs such as ad ⁇ amycin have been developed to increase their delivery to tumors and reduce it to other tissue sites (e g heart) thereby decreasing their toxicity pH-sensitive polymers have been used m conjunction with posomes for the triggered release of an encapsulatede drug
  • hydrophobically-modified N- lsopropylacrylamide-methacryhc acid copolymer can render regular egg phosphatidyl chloline hposomes pH-sensitive by pH-dependent interaction of grafted aliphatic chains with hpid bilayer (O Meyer et al FEBS Lett , 421 , 61, 1998)
  • Gene or polynucleotide transfer is the cardinal process of gene therapy
  • the gene needs to be transferred across the cell membrane and enter the nucleus where the gene can be expressed
  • Gene transfer methods currently being explored included viral vectors and physical-chemical methods
  • Viruses have evolved over millions of year to transfer their genes into mammalian cells Viruses can be modified to carry a desired gene and become a "vector" for gene therapy Using standard recombinant techniques, the harmful or superfluous viral genes can be removed and replaced with the desired normal gene This was first accomplished with mouse retroviruses The development of retroviral vectors were the catalyst that promoted current gene therapy efforts However, they cannot infect all cell types very efficiently, especially in vivo Other viral vectors based on Herpes virus are being developed to enable more efficient gene transfer into brain cells Adenoviral and adenoassociated vectors are being developed to mfect lung and other cells
  • the desired gene is placed within bacte ⁇ al plasmid DNA along with a mammalian promoter, enhancer, and other sequences that enable the gene to be expressed in mammalian cells
  • Several milligrams of the plasmid DNA contaimng all these sequences can be prepared and pu ⁇ fied from the bacterial cultures
  • the plasmid DNA containing the desired gene can be incorporated into hpid vesicles (hposomes including catiomc pids such as Lipofectin) that then transfer the plasmid DNA into the target cell
  • Plasmid DNA can also be complexed with proteins that target the plasmid DNA to specific tissues )ust as certain proteins are taken up (endocytosed) by specific cells
  • plasmid DNA can be complexed with polymers such as polylysme and polyethylemmine Another plasmid-based technique involves "shooting" the plasmid DNA on
  • Gene therapy approaches can be classified into direct and indirect methods Some of these gene transfer methods are most effective when directly injected into a tissue space Direct methods using many of the above gene transfer techniques are being used to target tumors, muscle, liver, lung, and brain Other methods are most effective when applied to cells or tissues that have been removed from the body and the genetically-modified cells are then transplanted back into the body Indirect approaches in conjunction with retroviral vectors are being developed to transfer genes into bone marrow cells, lymphocytes, hepatocytes, myoblasts and skin cells
  • Gene therapy promises to be a revolutionary advance in the treatment of disease It is a fundamentally new approach for treating disease that is different from the conventional surgical and pharmaceutical therapies
  • gene therapy is a relatively simple approach If someone has a defective gene, then gene therapy would fix the defective gene
  • the disease state would be modified by manipulating genes instead of their products, l e proteins, enzymes, enzyme substrates and enzyme products
  • the initial motivation for gene therapy was the treatment of genetic disorders, it is becoming increasingly apparent that gene therapy will be useful for the treatment of a broad range of acquired diseases such as cancer, infectious disorders (AIDS), heart disease, arthritis, and neurodegenerative disorders (Parkinson's and Alzheimer's)
  • Gene therapy promises to take full-advantage of the major advances brought about by molecular biology While, biochemistry is mainly concerned with how the cell obtains the energy and matter that is required for normal function, molecular biology is mainly concerned with how the cell gets the information to perform its functions Molecular biology wants to discover the flow of information in the cell Using the metaphor of computers, the cell is the hardware while the genes are the software In this sense, the purpose of gene therapy is to provide the cell with a new program (genetic information) so as to reprogram a dysfunctional cell to perform a normal function The addition of a new cellular function is provided by the insertion of a foreign gene that expresses a foreign protein or a native protein at amounts that are not present in the patient
  • RNA messenger RNA
  • Anti-sense methods use a RNA sequence or an ohgonucleotide that is made complementary to the target mRNA sequence and therefore binds specifically to the target messenger RNA
  • Ribozymes destroy a specific mRNA by a different mechanism
  • Ribozymes are RNA's that contain sequence complementary to the target messenger RNA plus a RNA sequence that acts as an enzyme to cleave the messenger RNA, thus destroying it and preventing it from being translated
  • cytokine genes that stimulate the immune system to destroy the cancer cells
  • the cytokme genes would be transferred into the lymphocytes by removing the lymphocytes from the body and infecting them with a retroviral vector carrying the cytokme gene
  • the tumor cells would be similarly genetically modified by this indirect approach to express cytokines within the tumor
  • Direct approaches involving the expression of cytokines in tumor cells in situ are also being considered
  • Other genes besides cytokines may be able to induce an immune response against the cancer
  • HLA-B7 gene which encodes a potent immunogen
  • “Suicide” genes are genes that kill cells that express the gene
  • the diphtheria toxin gene directly kills cells
  • the Herpes thymidme kinase (TK) gene kills cells in conjunction with acyclovir (a drug used to treat Herpes viral infections)
  • Other gene therapy approaches take advantage of our knowledge of oncogenes and suppressor tumor genes- also known as anti- oncogenes
  • the loss of a functioning anti- oncogene plays a decisive role in childhood tumors such as retinoblastoma, osteosarcoma and Wilms tumor and may play an important role in more common tumors such as lung, colon and breast cancer Introduction of the normal anti- oncogene back mto these tumor cells may convert them back to normal cells
  • the activation of oncogenes also plays an important role in the development of cancers Since these oncogenes operate in a "dominant” fashion, treatment will require mactivation of the abnormal oncogene This can be done using either
  • Gene therapy can be used as a type of vaccination to prevent infectious diseases and cancer
  • a foreign gene is transferred into a cell and the protein is made, the foreign protein is presented to the immune system differently from simply injecting the foreign protein into the body This different presentation is more likely to cause a cell-mediated immune response which is important for fighting latent viral infections such as human immunodeficiency virus (HIV causes AIDS), Herpes and cytomegalovirus Expression of the viral gene withm a cell simulates a viral infection and induces a more effective immune response by fooling the body that the cell is actually infected by the virus, without the danger of an actual viral infection
  • HIV human immunodeficiency virus
  • Herpes and cytomegalovirus Expression of the viral gene withm a cell simulates a viral infection and induces a more effective immune response by fooling the body that the cell is actually infected by the virus, without the danger of an actual viral infection
  • One direct approach uses the direct intramuscular injection of naked plasmid DNA to express a viral gene in muscle
  • nucleic acid or other compound that modifies the genetic instruction can co ⁇ ect a mutated gene or mutate a functioning gene
  • Polymers are used for drug delivery for a vanety of therapeutic purposes
  • Polymers have also been used in research for the delivery of nucleic acids (polynucleotides and o gonucleotides) to cells with an eventual goal of providing therapeutic processes
  • Such processes have been termed gene therapy or anti-sense therapy
  • One of the several methods of nucleic acid delivery to the cells is the use of DNA-polycation complexes
  • catiomc proteins like histones and protamines or synthetic polymers like polylysme, polyarginine, polyor thine, DEAE dextran, polybrene, and polyethylemmine may be effective mtracellular delivery agents while small polycations like sperm e are ineffective
  • the following are some important principles involving the mechanism by which polycations facilitate uptake of DNA
  • Polycations provide attachment of DNA to the cell surface
  • the polymer forms a cross-bridge between the polyaniomc nucleic acids and the polyaniomc surfaces of the cells
  • the main mechanism of DNA translocation to the mtracellular space might be non-specific adsorptive endocytosis which may be more effective then liquid endocytosis or receptor-mediated endocytosis
  • polycations are a convenient linker for attaching specific hgands to DNA and as result, DNA- polycation complexes can be targeted to specific cell types
  • Polycations can also facilitate DNA condensation
  • the volume which one DNA molecule occupies in a complex with polycations is drastically lower than the volume of a free DNA molecule
  • the size of a DNA/polymer complex is probably critical for gene delivery in vivo
  • DNA needs to cross the endothehal barrier and reach the parenchymal cells of interest
  • the largest endothelia fenestrae (holes in the endothehal barrier) occur in the liver and have an average diameter of 100 n
  • the trans- epithehal pores in other organs are much smaller, for example, muscle endothehum can be desc ⁇ bed as a structure which has a large number of small pores with a radius of 4 nm, and a very low number of large pores with a radius of 20-30 nm
  • the size of the DNA complexes is also important for the cellular uptake process After binding to the cells the DNA- polycation complex should be taken up by endocytosis Since the endocytic vesicles have a homo
  • DNA condensation is favored when 90% or more of the charges along the sugar- phosphate backbone are neutralized
  • Toroids have been considered an attractive form for gene delivery because they have the smallest size While the size of DNA toroids produced within single preparations has been shown to vary considerably, toroid size is unaffected by the length of DNA being condensed DNA molecules from 400 bp to genomic length produce toroids similar in size Therefore one toroid can include from one to several DNA molecules The kinetics of DNA collapse by polycations that resulted in toroids is very slow For example DNA condensation by Co(NH3)6Cl3 needs 2 hours at room temperature
  • DNA condensation is not clear
  • the electrostatic force between unperturbed helices a ⁇ ses p ⁇ ma ⁇ ly from a counte ⁇ on fluctuation mechanism requinng multivalent cations and plays a major role in DNA condensation
  • the hydration forces predominate over electrostatic forces when the DNA helices approach closer then a few water diameters
  • DNA condensation is a more complicated process than the case of low molecular weight polycations
  • Different polycationic proteins can generate toroid and rod formation with different size DNA at a ratio of positive to negative charge of two to five T4 DNA complexes with polyargimne or histone can form two types of structures, an elongated structure with a long axis length of about 350 nm (like free DNA) and dense spherical particles Both forms exist simultaneously in the same solution
  • the reason for the co-existence of the two forms can be explained as an uneven distribution of the polycation chains among the DNA molecules
  • the uneven distnbution generates two thermodynamically
  • DNA-cation particles have been created to circumvent the nonspecific interactions of the DNA-cation particle and the toxicity of cationic particles
  • modifications include attachment of ste ⁇ c stabilizers, e g polyethylene glycol, which inhibit nonspecific interactions between the cation and biological polyamons
  • Another example is recharging the DNA particle by the additions of polyamons, which interact with the cationic particle, thereby lowering its surface charge, l e recharging of the DNA particle U S 09/328,975
  • Another example is cross-linking the polymers and thereby caging the complex desc ⁇ bed in U S 08/778,657, U S 09/000,692, U S 09/070299, and U S 09/464,871
  • Nucleic acid particles can be formed by the formation of chemical bonds and template polyme ⁇ zation desc ⁇ bed in U S 08/778,657, U S 09/000,692, U S 09/070299, and U S 09/464,871
  • a potential problem with these modifications is that they may be irreversible rende ⁇ ng the particle unable to interact with the cell to be transfected, and/or incapable of escaping from the lysosome once taken into a cell, and/or incapable of entering the nucleus once inside the cell
  • a method for formation of DNA particles that is reversible under conditions found in the cell may allow for effective delivery of DNA
  • the conditions that cause the reversal of particle formation may be, but not limited to, the pH, ionic strength, oxidative or reductive conditions or agents, or enzymatic activity
  • the stability of DNA nanoassembhes based on DNA condensation is generally low in vivo because they easily engage in polyion exchange reactions with strong polyamons
  • the process of exchange consists of two stages 1 ) rapid formation of a triple DNA-polycation- polyamon complex, 2) slow substitution of one same -charge polyion with another At equilibrium conditions, the whole process eventually results in formation of a new binary complex and an excess of a third polyion
  • the presence of low molecular weight salt can greatly accelerate such exchange reactions, which often result in complete disassembly of condensed DNA particles Hence, it is desirable to obtain more colloidally stable structures where DNA would stay in its condensed form in complex with co ⁇ esponding polycation independently of environment conditions
  • DNA condensation upon neutralization of only 90% of the polymer's phosphates results m the presence of unpaired positive charges in the DNA particles If the polycation contains such reactive groups, such as primary amines, these unpaired positive charges may be modified This modification allows practically limitless possibilities of modulating colloidal properties of DNA particles via chemical modifications of the complex
  • DNA/PLL DNA-poly-L-lysme
  • DTBP cleavable cross-linking reagent dimethy 1-3,3 '- dithiobispropiommidate
  • DNA/polycation particles VS Trubetskoy, A Loomis, JE Hagstrom, VG Budker, JA Wolff Nucleic Acids Res 27 3090-3095, 1999
  • DNA/pC DNA/PLL (1 3 charge ratio) formed in low salt 25 mM HEPES buffer and recharged with increasing amounts of various polyamons
  • the DNA particles were characterized after addition of a third polyion component to a DNA/polycation complex using a new DNA condensation assay (VS Trubetskoy, PM Slattum, JE Hagstrom, JA Wolff, VG Budker Anal Biochem 267 309-313, 1999) and static light scatte ⁇ ng It has been found that certain polyamons such as poly(methacryhc acid) and poly(aspart ⁇ c acid) decondensed DNA in DNA/PLL complex
  • DNA/PLL particles du ⁇ ng titration with SPLL revealed the change of particle surface charge at approximately the charge equivalency point
  • addition of low charge density polyanion to the cationic DNA PLL particles results in particle surface charge reversal while maintaining condensed DNA core intact
  • DNA/polycation complexes can be both recharged and crosslinked or caged U S 08/778,657, U S 09/000,692, U S 97/24089, U S 09/070299, and U S 09/464,871
  • cationic lipids are amphipathic compounds that contain a hydrophobic domain, a spacer, and positively-charged amine
  • the hydrophobic domains are typically hydrocarbon chains such as fatty acids derived from oleic or mynstic acid
  • the hydrocarbon chains are often joined either by ether or ester bonds to a spacer such as glycerol Quaternary amines often compose the cationic groups
  • the cationic lipids are mixed with a fusogemc lipid such as DOPE (dioleoyl phosphatidyl ethanolamme) to form hposomes
  • DOPE dioleoyl phosphatidyl ethanolamme
  • the mixtures are mixed in chloroform that is then d ⁇ ed Water is added to the dried lipid film and umlamellar hposomes form du ⁇ ng somcation Multilamellar cationic hposomes and cationic hposome
  • cationic lipids contain a spermine group for binding to DNA DOSPA
  • the cationic lipid within the Lipofect AMINE formulation contains a spermine linked via a amide bond and ethyl group to a t ⁇ methyl, quaternary amine
  • DOGS dioctadecylglycinespermine
  • Cationic hposomes may deliver DNA either directly across the plasma membrane or via the endosome compartment Regardless of its exact entry point, much of the DNA withm cationic hposomes does accumulate in the endosome compartment Several approaches have been investigated to prevent loss of the foreign DNA in the endosomal compartment by protecting it from hydrolytic digestion within the endosomes or enabling its escape from endosomes mto the cytoplasm They include the use of acidotropic (lysomotrophic), weak amines such as chloroquine that presumably prevent DNA degradation by inhibiting endosomal acidification [Legendre, J & Szoka, F Delivery of plasmid DNA into mammalian cell lines using pH-sensitive hposomes Comparison with cationic hposomes Pharmaceut Res 9, 1235-1242 (1992)] Viral fusion peptides or whole virus have been included to disrupt endosomes or promote fusion of hposomes with endosomes, and facilitate release of DNA into the cytoplasm [K
  • anionic, pH-sensitive hposomes that have been designed to destabilize or fuse with the endosome membrane at acidic pH [Duzgunes, N , Straubmger, R M , Baldwin, P A & Papahadjopoulos, D PH- sensitive hposomes (eds Wilschub, J & Hoekstra, D ) p 713-730 (Marcel Deker INC, 1991 )] All of the anionic, pH-sensitive hposomes have utilized phosphatidylethanolamme (PE) bilayers that are stabilized at non-acidic pH by the addition of lipids that contain a carboxyhc acid group Liposomes containing only PE are prone to the inverted hexagonal phase (H j) In pH-sensitive, anionic liposomes,
  • PE phosphatidylethanolamme
  • pH sensitivity can be broadly defined as any change in polymer's physico-chemical properties over certain range of pH More na ⁇ ow definition demands significant changes in the polymer's ability to retain (release) a bioactive substance (drug) in a physiologically tolerated pH range (usually pH 5 5 - 8) pH-sensitivity presumes the presence of lomzable groups in the polymer (polyion) All polyions can be divided into three catego ⁇ es based on their ability to donate or accept protons in aqueous solutions polyacids, polybases and polyampholytes Use of pH-sensitive polyacids in drug delivery applications usually relies on their ability to become soluble with the pH increase (acid/salt conversion), to form complex with other polymers over change of pH or undergo significant change in hydrophobicity/hydrophihcity
  • Copolymers of polymethacryhc acid are known as polymers which are insoluble at lower pH but readily solubihzed at higher pH, so they are used as ente ⁇ c coatings designed to dissolve at higher intestinal pH (Z Hu et al J Drug Target , 7, 223, 1999)
  • a typical example of pH-dependent complexation is copolymers of polyacrylate(graft)ethyleneglycol which can be formulated into various pH-sensitive hydrogels which exhibit pH-dependent swelling and drug release (F Madsen et al , Biomate ⁇ als, 20, 1701 , 1999)
  • Hydrophobically-modified N-isopropylacrylamide- methacryhc acid copolymer can render regular egg PC liposomes pH-sensitive by pH- dependent interaction of grafted aliphatic chains with lipid bilayer (O Meyer et al , FEBS Lett , 421, 61, 1998) Polymers with pH -mediated hydrophobicity (like polyethylacry
  • a cellular transport step that has attracted attention for gene transfer is that of DNA release from mtracellular compartments such as endosomes (early and late), lysosomes, phagosomes, vesicle, endoplasmic reticulum, golgi apparatus, trans golgi network (TGN), and sarcoplasmic reticulum Release includes movement out of an mtracellular compartment into cytoplasm or mto an organelle such as the nucleus
  • mtracellular compartments such as endosomes (early and late), lysosomes, phagosomes, vesicle, endoplasmic reticulum, golgi apparatus, trans golgi network (TGN), and sarcoplasmic reticulum Release
  • mtracellular compartments such as endosomes (early and late), lysosomes, phagosomes, vesicle, endoplasmic reticulum, golgi apparatus, trans golg
  • Viruses such as adenovirus have been used to induce gene release from endosomes or other mtracellular compartments (D Cunel, Agarwal, S , Wagner, E , and Cotten, M PNAS 88 8850, 1991) Rhmovirus has also been used for this purpose (W Zauner et al J Virology 69 1085-92, 1995) Viral components such as influenza virus hemagglutimn subunit HA-2 analogs has also been used to induce endosomal release (E Wagner et al PNAS 89 7934, 1992) Amphipathic peptides resembling the N-terminal HA-2 sequence has been studied (K Mechtler and E Wagner, New J Chem 21 105- 1 1 1 , 1997) Parts of the pseudonmonas exotoxin and dipthe ⁇ a toxm have also been used for drug delivery (I Pastan and
  • Nucleic acid and gene delivery may involve the biological pH gradient that is active withm organisms as a factor in delivering a polynucleotide to a cell
  • Different pathways that may be affected by the pH gradient include cellular transport mechanisms, endosomal disruption/breakdown, and particle disassembly (release of the DNA)
  • Other gradients that can be useful m gene therapy research involve ionic gradients that are related to cells For example, both Na and K have large concentration gradients that exist across the cell membrane Systems containing metal-binding groups can utilize such gradients to influence delivery of a polynucleotide to a cell Changes in the osmotic pressure in the endosome also have been used to disrupt membranes and allow for transport across membrane layer
  • Buffering of the endosome pH may cause these changes in osmotic pressure
  • the "proton sponge" effect of PEI O Boussif et al Proc Natl Acad Sci USA 92, 7297, 1995
  • certain polyamons Murtfiy, N , Robichaud, J R , Ti ⁇ ell, D A , Stayton, P S , Hoffman, A S Journal of Controlled Release 1999, 61, 137
  • both extra and intracellularly may be used to deliver the gene of interest either by aiding in the delivery to the cell or escape from mtracellular compartments
  • Proteases found in serum, lysosome and cytoplasm, may be used to disrupt the particle and allow its interaction with the cell surface or cause it fracture the mtracellular compartment, e g endosome or lysosome, allowing the gene to be released intracellularly
  • the invention relates to noncovalent amphiphile binding systems for use in biologic systems More particularly, amphiphile-bindmg agents and polymers of amphrphile-binding agents are utilized in the delivery of molecules, polymers, nucleic acids and genes to cells
  • Described in a preferred embodiment is a process for obtaining an expression product by de ve ⁇ ng a polynucleotide to a cell, comprising the step of associating an amphiphile binding agent, an amphiphile, and a polynucleotide to form a complex Then, dehvenng the complex to the cell and expressing the polynucleotide m the cell
  • a complex for delivering and expressing DNA in a mammal, comprising an amphiphile binding agent, an amphiphile, and DNA in complex
  • Another preferred embodiment is a process for obtaining an expression product in vivo, comprising forming a complex with a cyclodextnn, an amphiphile and a polynucleotide Then, delivering the complex to a cell in a mammal which expresses the polynucleotide
  • Cyclodext ⁇ ns are naturally occumng cyclic ohgomers of glucose in 1 -4 ⁇ linkages (structure 1)
  • the cyclic structure is to ⁇ oidal in shape with the center of the to ⁇ oid relatively nonpolar compared to water For this reason, cyclodextnns will bind to nonpolar sections of amphipathic compounds, also known as amphiphihc compounds or amphiphiles, in water
  • Amphiphiles are compounds that contain both hydrophilic and hydrophobic functional groups Examples include lipids, acyl-glycerol, sterols, polyethyleneglycol, and ammo acids Hydrophilic groups indicate in qualitative terms that the chemical moiety is water-preferring Typically, such chemical groups are water soluble, and are hydrogen bond donors or acceptors with water Examples of hydrophilic groups include compounds with the following chemical moieties, carbohydrates, polyoxyethylene, peptides, ohgonucleot
  • Nucleic acid includes DNA (plasmid DNA, antisense, ohgonucleotides) and RNA ( ⁇ bozymes, ohgonucleotides, artificial messenger RNA)
  • these nonviral complexes may be grouped into two classes cationic lipid complexes ( poplexes) and catiomc polymer (polyplexes) complexes
  • poplexes cationic lipid complexes
  • polyplexes catiomc polymer
  • the polyaniomc DNA is complexed with a cation
  • the cations are associated noncovalently by hydrophobic hpid- hpid interactions to form a polycation
  • polymer complexes the positive charges are attached covalently to form a polycation
  • Nucleic acids are delivered to cells for the purpose of gene therapy and antisense therapy
  • cyclodextnns form complexes with amphipathic molecules that may be positively or negatively charged Therefore, a polymer composed of cyclodextnns will become a polyion, a noncovalent amphiphihc electrolyte, when associated with a charged amphiphile For example, association between a polymer composed of cyclodextnns and a catiomc amphiphile will result in a polycation that may interact with DNA
  • a cyclodext ⁇ n-containmg polymers are constructed by reacting cyclodextnn with epichlorohydrin under alkaline conditions to produce cyclodext ⁇ n- epichlorohydnn copolymer This cyclodextnn- epichlorohydrin copolymer, compacts pDN A upon addition of cations such as 1 -adamantanamine or 1 -dodecylamine The complex of cyclodextn
  • Amphiphile binding agents may also be used to create anionic noncovalent amphiphihc polyelectrolytes Association between a polymer composed of cyclodextnns and an anionic amphiphile will result in a polyanion that will interact with a positively-charged DNA-polycation complex, I e "recharge" the DNA complex
  • the complex between cyclodextnn- epichlorohyd ⁇ n copolymer and 4-t-butylbenzo ⁇ c acid, to form an anionic noncovalent amphiphihc polyelectrolyte was added to particles of DNA and poly-L-lysine The resulting particles were found to transfect cells in vitro
  • anionic amphiphiles that are polymers that are bound to monome ⁇ c or polymeric amphiphile binding agents may be used to "recharge" DNA particles
  • succinyloleoylpoly- L-lysine is an anionic polymeric amphiphile which complexes with
  • cyclodextnn the basis for the DNA-polyion interaction
  • cyclodext ⁇ n-based polyions may have properties (e g surface charge and stability) different from standard polyions
  • the polyions de ⁇ ved from cyclodextrin-containing polymers and charged amphiphiles are reversible
  • the existence of the polyion is dependent upon the concentration of the cyclodextrin-containing polymer and the charged amphiphile, such that the disruption of the polyion maybe t ⁇ gger by simple dilution of either cyclodextnn or charged amphiphile
  • Monome ⁇ c cyclodextnns may also be incorporated into nucleic acid complexes by association with amphiphile molecules in a DNA complex
  • the cyclodextnns are not the basis for the DNA-electrolyte interactions, but may be used to change the properties of the DNA-electrolyte complex, e g stability or surface charge
  • cell targeting hgands aid m transport to a cell but may not be necessary, and may inhibit, transport mto a cell In all of these cases, the reversible attachment of the interaction modifier, through a labile bond, would be beneficial
  • the present invention provides for the transfer of polynucleotides, and other biologically active compounds into cells in culture (also known as ". « ⁇ iti o") Compounds or kits for the transfection of cells in culture is commonly sold as “transfection reagents” or “transfection kits”
  • the present invention also provides for the transfer of polynucleotides, and biologically active compounds into cells within tissues in situ and m ⁇ ⁇ vo, and delivered intravasculary (U S patent application serial number 08/571,536), lntrarte ⁇ ally, intravenous, orally, intraduodenaly.
  • Compounds for the transfection of cells in vivo in a whole organism can be sold as "in vivo transfection reagents" or ". « vivo transfection kits” or as a pharmaceutical for gene therapy
  • Amphiphile binding agents are compounds with molecular weight 1,300 or less that bind through a noncovalent interaction amphiphihc compounds in water The basis for this interaction is contact between hydrophobic portions of the amphiphile with hydrophobic portions of the amphiphile binding agent In particular ⁇ , ⁇ and ⁇ -cyclodext ⁇ ns. and their derivatives, are amphiphile binding agents
  • Polyermic amphiphile binding agent is a polymer composed of monomers that are amphiphile binding agents
  • Noncovalent amphiphihc polyelectrolytes are systems composed of amphiphile binding agents and charged amphiphiles, which are bound by the amphiphile binding agents The interaction between charged amphiphile and polymer results in a complex that has a different charge than the amphiphile binding agent alone
  • the amphiphile binding agent ma ⁇ be uncharged, charge positive or neutral, but upon interaction with a charged amphiphile the charge of the complex is different than the amphiphile binding agent alone
  • a biologically active compound is a compound having the potential to react with biological components More particularly, biologically active compounds utilized in this specification are designed to change the natural processes associated with a living cell
  • a cellular natural process is a process that is associated with a cell before delivery of a biologically active compound
  • the cellular production of, or inhibition of a material, such as a protein, caused by a human assisting a molecule to an in vivo cell is an example of a delivered biologically active compound
  • Pharmaceuticals, proteins, peptides, polypeptides, enzyme inhibitors, hormones, cytokines, antigens, viruses, ohgonucleotides, enzymes and nucleic acids are examples of biologically active compounds
  • Peptide and polypeptide refer to a series of ammo acid residues, more than two, connected to one another by amide bonds between the beta or alpha-ammo group and carboxyl group of contiguous ammo acid residues
  • the amino acids may be naturally occumng or synthetic
  • Polypeptide includes proteins and peptides, modified proteins and peptides, and non-natural proteins and peptides
  • Enzymes are proteins evolved by the cells of living organisms for the specific function of catalyzing chemical reactions A chemical reaction is defined as the formation or cleavage of covalent or ionic bonds
  • Bioactn e compounds may be used interchangeably with biologically active compound for purposes of this application
  • the delivery of a biologically active compound is commonly known as “drug delivery” “Delivered” means that the biologically active compound becomes associated with the cell or organism
  • Delivery means that the biologically active compound becomes associated with the cell or organism
  • the compound can be in the circulatory system, intravessel, extracellular, on the membrane of the cell or inside the cytoplasm, nucleus, or other organelle of the cell
  • Parenteral routes of administration include intravascular (intravenous, mtraartal), intramuscular, mtraparenchymal, intradermal, subdermal, subcutaneous, mtratumor lntrape ⁇ toneal, intrathecal, subdural, epidural, and mtralymphatic inactions that use a synnge and a needle or catheter
  • An intravascular route of administration enables a polymer or polynucleotide to be delivered to cells more evenly distributed and more efficiently expressed than direct injections
  • Intravascular herein means withm a tubular structure called a vessel that is connected to a tissue or organ within the body Within the cavity of the tubular structure, a bodily fluid flows to or from the body part
  • bodily fluid include blood, cerebrospinal fluid (CSF), lymphatic fluid, or bile
  • vessels include arteries, arte ⁇ oles, capillaries, venules, sinusoids, veins, lymphatics, and bile ducts
  • Delivery system is the means by which a biologically active compound becomes delivered That is all compounds, including the biologically active compound itself, that are required for delivery and all procedures required for delivery including the form (such volume and phase (solid, liquid, or gas)) and method of administration (such as but not limited to oral or subcutaneous methods of delivery)
  • nucleic acid is a term of art that refers to a polymer containing at least two nucleotides
  • Nucleotides contain a sugar deoxynbose (DNA ) or ⁇ bose (RNA), a base, and a phosphate group Nucleotides are linked together through the phosphate groups
  • Bases include purmes and py ⁇ midmes, which further include natural compounds adenine, thymine, guanine, cytosme, uracil, inosme, and natural analogs, and synthetic denvatives of purmes and pynmidmes, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhahdes Nucleotides are the monome ⁇ c units of nucleic acid polymers A "polynucleotide” is distinguished here from an "o gonucleotide
  • DNA may be in the form of anti-sense, plasmid DNA, parts of a plasmid DNA, product of a polymerase chain reaction (PCR), vectors (PI, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chime ⁇ c sequences, chromosomal DNA, or derivatives of these groups
  • RNA may be in the form of ohgonucleotide RNA, tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, ⁇ bozymes, chime ⁇ c sequences, or derivatives of these groups
  • Anti-sense is a polynucleotide that interferes with the function of DNA and/or RNA This may result in suppression of expression
  • Natural nucleic acids have a phosphate backbone
  • artificial nucleic acids may contain other types of backbones and bases These include PNAs (peptide nucleic acids), phosphothionates, and other variants of the phosphate backbone of native nucleic acids
  • DNA and RNA may be single, double, tnple, or quadruple stranded
  • recombinant DNA molecule refers to a DNA molecule that is comprised of segments of DNA joined together by means of molecular biological techniques
  • “Expression cassette” refers to a natural or recombinantly produced polynucleotide molecule that is capable of expressing prote ⁇ n(s)
  • a DNA expression cassette typically includes a promoter (allowing transcription initiation), and a sequence encoding one or more proteins
  • the expression cassette may include tranc ⁇ ptional enhancers, non-coding sequences, splicing signals, transcription termination signals, and polyadenylation signals
  • An RNA expression cassette typically includes a translation initiation codon (allowing translation initiation), and a sequence encoding one or more proteins
  • the expression cassette may include translation termination signals, a polyadenosine sequence, internal ⁇ bosome entry sites (IRES), and non-coding sequences
  • a nucleic acid can be used to modify the genomic or extrachromosomal DNA sequences This can be achieved by delivering a nucleic acid
  • the term "gene” refers to a nucleic acid (e g , DNA) sequence that comprises coding sequences necessary for the production of a polypeptide or precursor (e g -myosm heavy chain)
  • the polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e g , enzymatic activity, hgand binding, signal transduction, etc ) of the full-length or fragment are retained
  • the term also encompasses the coding region of a structural gene and the including sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb or more on either end such that the gene corresponds to the length of the full- length mRNA
  • the sequences that are located 5' of the coding region and which are present on the mRNA are refe ⁇ ed to as 5' non-translated sequences
  • nucleic acid molecule encoding As used herein, the terms “nucleic acid molecule encoding,” “DNA sequence encoding,” and “DNA encoding” refer to the order or sequence of deoxy ⁇ bonucleotides along a strand of deoxy ⁇ bonucleic acid The order of these deoxy ⁇ bonucleotides determines the order of amino acids along the polypeptide (piotem) chain The DNA sequence thus codes for the amino acid sequence
  • an oligonucleotide having a nucleotide sequence encoding a gene and “polynucleotide having a nucleotide sequence encoding a gene,” means a nucleic acid sequence comprising the coding region of a gene or in other words the nucleic acid sequence which encodes a gene product
  • the coding region may be present m either a cDNA, genomic DNA or RNA form
  • the oligonucleotide or polynucleotide may be single-stranded (i e , the sense strand) or double-stranded
  • Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and or correct processing of the p ⁇ mary RNA transcript
  • the coding region utilized in the expression vectors of the present invention may contain endogenous enhance
  • the oligonucleotide or polynucleotide may be single-stranded), but may contain both the sense and anti-sense strands (. e , the oligonucleotide or polynucleotide may be double-stranded)
  • RNA expression refers to the process of converting genetic information encoded in a gene into RNA (e g , mRNA, rRNA, tRNA, or snRNA) through
  • transcnption factors that are involved in up-regulation or down-regulation are often called “activators” and “repressors,” respectively Delivery of Nucleic Acids
  • the process of delivering a polynucleotide to a cell has been commonly termed “transfection” or the process of "transfecting” and also it has been termed “transformation”
  • the polynucleotide could be used to produce a change in a cell that can be therapeutic
  • the delivery of polynucleotides or genetic material for therapeutic and research purposes is commonly called “gene therapy”
  • the delivery of nucleic acid can lead to modification of the DNA sequence of the target cell
  • transfection refers to the introduction of foreign DNA into eukaryotic cells Transfection may be accomplished by a vanety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, micromjection, hposome fusion, hpofection, protoplast fusion, retroviral infection, and biohstics
  • stable transfection or 'stably transfected refers to the introduction and integration of foreign DNA into the genome of the transfected cell
  • stable transfectant refers to a cell which has stably integrated foreign DNA into the genomic DNA
  • transient transfection or “transiently transfected” refers to the introduction of foreign DNA into a cell where the foreign DNA fails to integrate into the genome of the transfected cell
  • the foreign DNA persists in the nucleus of the transfected cell for several days Du ⁇ ng this time the foreign DNA is subject to the regulatory controls that govern the expression of endogenous genes in the chromosomes
  • transient transfectant refers to cells which have taken up foreign DNA but have failed to integrate this DNA
  • naked polynucleotides indicates that the polynucleotides are not associated with a transfection reagent or other delivery vehicle that is required for the polynucleotide to be delivered to a cell
  • transfection reagent or “delivery vehicle” is a compound or compounds that b ⁇ nd(s) to or complex(es) with ohgonucleotides, polynucleotides, or other desired compounds and mediates their entry into cells
  • transfection reagents include, but are not limited to, cationic liposomes and lipids, polyamines, calcium phosphate precipitates, histone proteins, polyethylemmine, and polylysme complexes (polyethylemmine and polylysme are both toxic)
  • the transfection reagent has a net positive charge that binds to the polynucleotide's negative charge
  • cationic liposomes or polylysme complexes have net positive charges that enable them to bind to DNA or RNA
  • Enzyme Enzyme is a protein that acts as a catalyst That is a protein that increases the rate of a chemical reaction without itself undergoing any permanent chemical change
  • the chemical reactions that are catalyzed by an enzyme are termed enzymatic reactions and chemical reactions that are not are termed nonenzymatic reactions
  • Two molecules are combined, to form a complex through a process called complexation or complex formation, if the are in contact with one another through noncovalent interactions such as electrostatic interactions, hydrogen bonding interactions, and hydrophobic interactions
  • a molecule is modified, to fo rm a modification through a process called modification, by a second molecule if the two become bonded through a covalent bond That is, the two molecules form a covalent bond between an atom form one molecule and an atom from the second molecule resulting in the formation of a new single molecule
  • a chemical covalent bond is an interaction, bond, between two atoms in which there is a sha ⁇ ng of electron density
  • Osmosis is the passage of a solvent through a semipermeable membrane, a membrane through which solvent can pass but not all solutes, separating two solutions of different concentrations There is a tendency for the separated solutions to become the same concentration as the solvent passes from low concentration to high concentration Osmosis will stop when the two solutions become equal in concentration or when pressure is applied to the solution containing higher concentration
  • the higher concentrated solution is in a closed system, that is when system is of constant volume, there is a build up of pressure as the solvent passes from low to high concentration This build up of pressure is called osmotic pressure Salt
  • a salt is any compound containing ionic bonds, that is bonds in which one or more electrons are transferred completely from one atom to another
  • An interpolyelectrolyte complexe is a noncovalent interaction between polyelectrolytes of opposite charge
  • the charge, polarity, or sign of a compound refers to whether or not a compound has lost one or more electrons (positive charge, polarity, or sign ) or gained one or more electrons (negative charge, polarity, or sign)
  • Cell targeting signal (or abbreviated as the Signal) is defined in this specification as a molecule that modifies a biologically active compounds such as drug or nucleic acid and can direct it to a cell location (such as tissue) or location in a cell (such as the nucleus) either in culture or in a whole orgamsm. By modifying the cellular or tissue location of the foreign gene, the function of the biologically active compound can be enhanced
  • the cell targeting signal can be a protein, peptide, lipid, steroid, sugar, carbohydrate,
  • the cell targeting signal enhances cellular binding to receptors, cytoplasmic transport to the nucleus and nuclear entry or release from endosomes or other mtracellular vesicles
  • Nuclear localizing signals enhance the targeting of the pharmaceutical into proximity of the nucleus and/or its entry into the nucleus
  • Such nuclear transport signals can be a protein or a peptide such as the SV40 large T ag NLS or the nucleoplasmin NLS
  • These nuclear localizing signals interact with a variety of nuclear transport factors such as the NLS receptor (karyophenn alpha) which then interacts with karyophenn beta
  • the nuclear transport proteins themselves could also function as NLS's since they are targeted to the nuclear pore and nucleus
  • karyophenn beta itself could target the DNA to the nuclear pore complex
  • Several peptides have been derived from the SV40 T antigen These include a short NLS (H-CGYGPKKKRKVGG-OH) or long NLS's (H- CKKKSSSDDEATADSQHSTPPKKKRKVEDPKDFPSELLS-OH and H-
  • CKKKWDDEATADSQHSTPPKKKRKVEDPKDFPSELLS-OH Other NLS peptides have been denved from M9 protein (CYNDFGNYNNQSSNFGPMKQGNFGGRSSGPY), El A (H-CKRGPKRPRP-OH), nucleoplasmin (H-CKKAVKRPAATKKAGQAKKKKL-OH),and c-myc (H-CKKKGPAAKRVKLD-OH)
  • Signals that enhance release from mtracellular compartments can cause DNA release from mtracellular compartments such as endosomes (early and late), lysosomes, phagosomes, vesicle, endoplasmic reticulum, golgi apparatus, trans golgi network (TGN), and sarcoplasmic reticulum Release includes movement out of an mtracellular compartment into cytoplasm or into an organelle such as the nucleus Releasing signals include chemicals such as chloroqume, bafilomycm or Brefeldm A 1 and the ER-retaining signal (KDEL sequence), viral components such as influenza virus hemagglutimn subumt HA-2 peptides and other types of amphipathic peptides
  • Cellular receptor signals are any signal that enhances the association of the biologically active compound with a cell This can be accomplished by either increasing the binding of the compound to the cell surface and/or its association with an mtracellular compartment, for example hgands that enhance endocytosis by enhancing binding the cell surface
  • Other proteins such as insulin, EGF, or transfemn can be used for targeting Peptides that include the RGD sequence can be used to target many cells
  • Chemical groups that react with thiol, sulfhydryl, or disulfide groups on cells can also be used to target many types of cells
  • Folate and other vitamins can also be used for targeting Other targeting groups include molecules that interact with membranes such as lipids, fatty acids, cholesterol, dansyl compounds, and amphote ⁇ cin denvatives
  • viral proteins could be used to bind cells
  • An interaction modifier changes the way that a molecule interacts with itself or other molecules, relative to molecule containing no interaction modifier The result of this modification is that self-interactions or interactions with other molecules are either increased or decreased
  • cell targeting signals are interaction modifiers that change the interaction between a molecule and a cell or cellular component
  • Polyethylene glycol is an interaction modifier that decreases interactions between molecules and themselves and with other molecules
  • Reporter or Marker Molecules are compounds that can be easily detected Typically they are fluorescent compounds such as fluorescein, rhodamine, Texas red, cy 5, cy 3 or dansyl compounds They can be molecules that can be detected by infrared, ultraviolet or visible spectroscopy or by antibody interactions or by electron spin resonance Biotm is another reporter molecule that can be detected by labeled avidin Biotin could also be used to attach targeting groups
  • the linkage may be electronically neutral, or may bear a positive or negative charge
  • the chemical moieties can be hydrophilic or hydrophobic Preferred spacer groups include, but are not limited to C1-C12 alkyl, C 1-C 12 alkenyl, C1-C12 alkynyl, C6- C18 aralkyl, C6-C18 aralkenyl, C6-C18 aralkynyl, ester, ether, ketone.
  • alcohol polyol, amide, amine, polyglycol, polyether, polyamine, thiol, thio ether, thioester, phosphorous containing, and heterocyc c
  • Bifunctional molecules commonly referred to as crosshnkers, are used to connect two molecules together, l e form a linkage between two molecules Bifunctional molecules can contain homo or heterobifunctiona ty
  • Crosslinkmg refers to the chemical attachment of two or more molecules with a bifunctional reagent
  • a bifunctional reagent is a molecule with two reactive ends The reactive ends can be identical as in a homobifunctional molecule, or different as in a heterobifucnctional molecule
  • Hydrophilic groups indicate m qualitative terms that the chemical moiety is water-prefemng Typically, such chemical groups are water soluble, and are hydrogen bond donors or acceptors with water Examples of hydrophilic groups include compounds with the following chemical moieties, carbohydrates, polyoxyethylene, peptides, ohgonucleotides and groups containing amines, amides, alkoxy amides, carboxyhc acids, sulfurs, or hydroxyls Hydrophobic groups indicate in qualitative terms that the chemical moiety is water-avoiding Typically, such chemical groups are not water soluble, and tend not to hydrogen bonds Hydrocarbons are hydrophobic groups
  • Detergents or surfactants are water-soluble molecules containing a hydrophobic portion (tail) and a hydrophilic portion (head), which upon addition to water decrease water's surface tension
  • the hydrophobic portion can be alkyl, alkenyl, alkynyl or aromatic
  • the hydrophilic portion can be charged with either net positive (cationic detergents), negative (anionic detergents), uncharged (nonionic detergents), or charge neutral (zwittenomc detergent)
  • anionic detergents are sodium dodecyl sulfate, glycohc acid ethoxylate(4 units) 4-terr-butylphenylether, palmitic acid, and oleic acid
  • catiomc detergents are cetylt ⁇ methylammonium bromide and oleylamme
  • nonionic detergents include, laurylmaltoside, Triton X-100, and Tween
  • zwittenomc detergents include 3-[(3-cholam ⁇ do
  • the surface tension of a liquid is the force acting over the surface of the liquid per unit length of surface that is perpendicular to the force that is acting of the surface
  • Surface charge has the units force per length, e g Newtons/meter
  • Membrane active agents or compounds are compounds (typically a polymer, peptide or protein) that are able alter the membrane structure This change in structure can be shown by the compound inducing one or more of the following effects upon a membrane, an alteration that allows small molecule permeability, pore formation in the membrane, a fusion and/or fission of membranes, an alteration that allows large molecule permeability, or a dissolving of the membrane This alteration can be functionally defined by the compound's activity in at least one the following assays red blood cell lysis (hemolysis), posome leakage, hposome fusion, cell fusion, cell lysis and endosomal release
  • An example of a membrane active agent in our examples is the peptide mehttm, whose membrane activity is demonstrated by its ability to release heme from red blood cells (hemolysis).
  • DM- Mel dimethylmaleamic-modified mellitin
  • membrane active compounds allow for the transport of molecules with molecular weight greater than 50 atomic mass units to cross a membrane This transport may be accomplished by either the total loss of membrane structure, the formation of holes (or pores) m the membrane structure, or the assisted transport of compound through the membrane
  • transport between liposomes, or cell membranes may be accomplished by the fusion of the two membranes and thereby the mixing of the contents of the two membranes
  • Membrane active peptides are peptides that have membrane activity There are many naturally occurring membrane active peptides such as cecropin (insects), magainm, CPF 1 , PGLa, Bombimn BLP-1 (all three from amphibians), mehttm (bees), semmalplasmm (bovine), lndo cidin, bactenecin (both from bovine neutrophils), tachyplesm 1 (crabs), protegnn (porcine leukocytes), and defensins (from human, rabbit, bovine, fungi, and plants) Gramicidin A and gramicidin S (bacillus brevis), the lantibiotics such as nisin (lactococcus lactis), androctomn (scorpion), cardiotoxin I (cobra), cae ⁇ n (frog htoria spectaculara), dermaseptm (frog) Viral peptides have also been shown to have membrane activity, examples include
  • a polymer is a molecule built up by repetitiv e bonding together of smaller units called monomers
  • the term polymer includes both ohgomers which have two to about 80 monomers and polymers having more than 80 monomers
  • the polymer can be linear, branched network, star, comb, or ladder types of polymer
  • the polymer can be a homopolymer m which a single monomer is used or can be copolymer in which two or more monomers are used Types of copolymers include alternating, random, block and graft
  • the main chain of a polymer is composed of the atoms whose bonds are required for propagation of polymer length For example in poly-L-lysme, the carbonyl carbon, a-carbon, and a-amine groups are required for the length of the polymer and are therefore mam chain atoms
  • the side chain of a polymer is composed of the atoms w hose bonds are not required for propagation of polymei length For example in poly-L-lys
  • step polymenzation the polymerization occurs in a stepwise fashion Polymer growth occurs by reaction between monomers, ohgomers and polymers No initiator is needed since there is the same reaction throughout and there is no termination step so that the end groups are still reactive The polymerization rate decreases as the functional groups are consumed
  • step polyme ⁇ zation is done either of two different ays
  • the monomer has both reactive functional groups (A and B) in the same molecule so that
  • A-A + B-B yields -[A-A-B-B]-
  • acylation is defined as the introduction of an acyl group (-COR) onto a molecule
  • Alkylation is defined as the introduction of an alkyl group onto a molecule
  • A is an amme then B can be (but not restncted to) an isothiocyanate, isocyanate, acyl azide, N-hydroxysuccimmide, sulfonyl chlo ⁇ de, aldehyde (including formaldehyde and glutaraldehyde), ketone, epoxide, carbonate, lmidoester.
  • function A is an amine then function B can be acylatmg or alkylatmg agent or ammation
  • function B can be (but not restricted to) an lodoacetyl derivative, maleimide, azi ⁇ dine derivative, acryloyl de ⁇ vative. fluorobenzene derivatives, or disulfide derivativ e (such as a py ⁇ dyl disulfide or 5-th ⁇ o-2- mtrobenzoic ac ⁇ d ⁇ TNB ⁇ derivatives)
  • function B can be (but not restricted to) a diazoacetate or an amine in which a carbodnmide is used
  • Other additives may be utilized such as carbonyld ⁇ midazole, dimethylaminopy ⁇ dme. N-hydroxysuccinimide or alcohol using carbodnmide and dimethylaminopy ⁇ dine
  • function B can be (but not restricted to) an epoxide, oxirane, or an amine m which carbonyld ⁇ midazole or N, N'-disuccmimidyl carbonate, or N-hydroxysuccinimidyl chloroformate or other chloroformates are used
  • function B can be (but not restncted to) an hydrazine, hydrazide derivative, amine (to form a imme or iminium that may or may not be reduced by reducing agents such as NaCNBH3 ) or hydroxyl compound to form a ketal or acetal
  • A-A plus another agent yields -[A-A]-
  • function A is a thiol, sulfhydr l, group then it can be converted to disulfide bonds by oxidizing agents such as iodine (b ) or NaI ⁇ 4 (sodium pe ⁇ odate), or oxygen (O2)
  • Function A can also be an amme that is con erted to a thiol, sulfhydryl, group by reaction with 2- Immothiolate (Traut's reagent) which then undergoes oxidation and disulfide formation
  • Disulfide derivatives (such as a py ⁇ dyl disulfide or 5-th ⁇ o-2-mtrobenzo ⁇ c ac ⁇ d ⁇ TNB
  • Functional group A or B m any of the above examples could also be a photoreactive group such as aryl azides, halogenated aryl azides, diazo, benzophenones, alkynes or dia
  • Monomers containing vinyl, acrylate, methacrylate, acrylamide. methaacrylamide groups can undergo chain reaction which can be radical, anionic , or cationic Chain polymerization can also be accomplished by cycle or ring opening polymerization
  • chain reaction can be radical, anionic , or cationic Chain polymerization can also be accomplished by cycle or ring opening polymerization
  • free radical lmtiatiors could be used that include peroxides, hydroxy peroxides, and azo compounds such as 2,2'-Azob ⁇ s(-am ⁇ d ⁇ nopropane) dihydrochlo ⁇ de ( AAP)
  • a compound is a material made up of two or more elements
  • a wide va ⁇ ety of monomers can be used in the polyme ⁇ zation processes These include positive charged organic monomers such as amines, lmidine, guamdine, imme, hydroxylamme, hydrozyine, heterocycles (like lmidazole, py ⁇ dine, morphohne, py ⁇ midme, or pyrene
  • the amines could be pH-sensitive in that the pKa of the amme is withm the physiologic range of 4 to 8
  • Specific amines include spermine spermidme, N,N'-b ⁇ s(2-ammoethyl)-l ,3-propaned ⁇ am ⁇ ne (AEPD), and 3.3 '-D ⁇ am ⁇ no-N,N- dimethyldipropylammonium bromide
  • Monomers can also be hydrophobic, hydrophilic or amphipathic Monomers can also be intercalating agents such as ac ⁇ dine, thiazole organge. or ethidium bromide
  • the polymers have other groups that increase their utility These groups can be incorporated mto monomers prior to polymer formation or attached to the polymer after its formation These groups include Targeting Groups- such groups are used for targeting the polymer-nucleic acid complexes to specific cells or tissues
  • Targeting Groups- such groups are used for targeting the polymer-nucleic acid complexes to specific cells or tissues
  • targeting agents include agents that target to the asialoglycoprotem receptor by using asiologlycoprotems or galactose residues
  • Other proteins such as insulin, EGF, or transfer ⁇ n can be used for targeting Protein refers to a molecule made up of 2 or more amino acid residues connected one to another as in a polypeptide
  • the amino acids may be naturally occurring or synthetic Peptides that include the RGD sequence can be used to target many cells
  • Chemical groups that react with thiol, sulfhydryl, or disulfide groups on cells can also be used to target many types of cells Folate and other vitamins can also be
  • targeting groups can be used to increase the delivery of the drug or nucleic acid to certain parts of the cell
  • agents can be used to disrupt endosomes and a nuclear localizing signal (NLS) can be used to target the nucleus
  • Ligands have been used to target drugs and genes to cells and to specific cellular receptors
  • the hgand may seek a target withm the cell membrane, on the cell membrane or near a cell
  • Binding of ligands to receptors typically initiates endocytosis
  • Ligands could also be used for DNA delivery that bind to receptors that are not endocytosed
  • peptides containing RGD peptide sequence that bind mteg ⁇ n receptor could be used
  • viral proteins could be used to bind the complex to cells
  • Lipids and steroids could be used to directly insert a complex into cellular membranes
  • the polymers can also contain cleavable groups within themselves When attached to the targeting group, cleavage leads to reduce interaction between the complex and the receptor for the targeting group
  • Cleavable groups include but are not restricted to disulfide bonds, diols, diazo bonds, ester bonds, sulfone bonds, acetals, ketals, enol ethers, enol esters enamines and lmmes
  • a polyelectrolyte, or polyion is a polymer possessing charge, that is the polymer contains a group (or groups) that has either gained or lost one or more electrons
  • a polycation is a polyelectrolyte possessing net positiv e charge, for example poly-L-lysme hydrobromide
  • the polycation can contain monomer units that are charge positiv e, charge neutral, or charge negative, however, the net charge of the polymer must be positive
  • a polycation also can mean a non-polymeric molecule that contains two or more positive charges
  • a polyanion is a polyelectrolyte containing a net negative charge The polyanion can contain monomer units that are charge negative, charge neutral, or charge positive, however.
  • a polyanion can also mean a non-polymenc molecule that contains two or more negative charges
  • polyelectrolyte includes polycation, polyanion, zwittenomc polymers, and neutral polymers
  • zwittenomc refers to the product (salt) of the reaction between an acidic group and a basic group that are part of the same molecule
  • a chelator is a polydentate hgand, a molecule that can occupy more than one site in the coordination sphere of an ion, particularly a metal ion, primary amine, or single proton
  • examples of chelators include crown ethers, cryptates, and non-cyclic polydentate molecules
  • the X and CR1-2 moieties can be substituted, or at a different oxidation states X can be oxygen, nitrogen, or sulfur, carbon, phosphorous or any combination thereof.
  • R can be H, C, O, S, N, P
  • the beginning X atom of the strand is an X atom in the (-X-(CR1- 2)n)m unit, and the terminal CH2 of the new strand is bonded to a second X atom m the (-X- (CRl-2)n)m unit
  • the X and CR1-2 moieties can be substituted, or at a different oxidation states X can be oxygen, nitrogen, or sulfur, carbon, phosphorous or any combination thereof.
  • a polychelator is a polymer associated with a plurality of chelators by an ionic or covalent bond and can include a spacer
  • the polymer can be catiomc, anionic, zwittenomc, neutral, or contain any combination of catiomc, anionic, zwittenomc, or neutral groups with a net charge being cationic, anionic or neutral, and may contain ste ⁇ c stabilizers, peptides, proteins, signals, or amphipathic compound for the formation of micellar. reverse micellar. or umlamellar structures
  • the amphipathic compound can have a hydrophilic segment that is catiomc, anionic. or zwittenomc, and can contain polyme ⁇ zable groups, and a hydrophobic segment that can contain a polyme ⁇ zable group
  • a steric stabilizer is a hydrophilic group that prevents aggregation of a polymer or particle by ste ⁇ cally hmde ⁇ ng particle to particle electrostatic interactions
  • Examples include- alkyl groups, PEG chains, polysaccha ⁇ des, hydrogen molecules, alkyl amines. Electrostatic interactions are the non-covalent association of two or more substances due to attractiv e forces between positive and negative charges
  • Buffers Buffers are made from a weak acid or weak base and their salts Buffer solutions resist changes in pH when additional acid or base is added to the solution
  • a compound is reactive if it is capable of forming either an ionic or a covalent bond with another compound
  • the portions of reactive compounds that are capable of forming covalent bonds are refe ⁇ ed to as reactive functional groups
  • Lipids are compounds that are insoluble m water but soluble in organic solvent which have the general structure composed of two distinct hydrophobic sections, that is two separate sections of uninterrupted carbon-carbon bonds The two hydrophobic sections are connected through a linkage that contains at least one heteroatom, that is an atom that is not carbon (e g nitrogen, oxygen, silicon, and sulfur)
  • Examples include esters and amides of fatty acids and include the glyce ⁇ des ( 1 ,2-d ⁇ oleoylglycerol (DOG)), glycohpids, phosphohpids (dioleoylphosphatidylethanolamme (DOPE ) )
  • Alkyl means any sp -hybridized carbon-containing group, alkenyl means containing
  • aklkynyl means containing tw o or more sp hybndized carbon atoms
  • aralkyl means containing one or more aromatic nng(s) in addition
  • aralkenyl means containing one or more aromatic nng(s) in addition to containing two or more sp ⁇ hybridized carbon atoms
  • aralkynyl means containing one or more aromatic ⁇ ng(s) in addition to containing two or more sp hybndized carbon atoms
  • steroid includes natural and unnatural steroids and steroid derivatives
  • a steroid derivative means a sterol, a sterol in which the hydroxyl moiety has been modified (for example, acylated), or a steroid hormone, or an analog thereof
  • the modification can include spacer groups, linkers, or reactive groups
  • Carbohydrates include natural and unnatural sugars (for example glucose), and sugar derivatives (a sugar derivative means a system m which one or more of the hydroxyl groups on the sugar moiety has been modified (for example, but not limited to, acylated), or a system m which one or more of the hydroxyl groups is not present)
  • sugar derivatives means a system m which one or more of the hydroxyl groups on the sugar moiety has been modified (for example, but not limited to, acylated), or a system m which one or more of the hydroxyl groups is not present
  • a compound is a material made up of two or more elements
  • Electron withdrawing group is any chemical group oi atom composed of electronegative atom(s), that is atoms that tend to attract electrons
  • Electron donating group is any chemical group or atom composed of electropositive atom(s), that is atoms that tend to attract electrons
  • Resonance Stabilization is the ability to distribute charge on multiple atoms through pi bonds
  • the inductive effectiv e, in a molecule, is a shift of electron density due to the polarization of a bond by a nearby electronegative or electropositive atom Activated Carboxylate
  • An activated carboxylate is a carboxyhc acid derivative that reacts with nucleophiles to form a new covalent bond
  • Nucleophiles include nitrogen, oxygen and sulfur-containing compounds to produce ureas, amides, carbonates, carbamates, esters, and thioesters
  • the carboxyhc acid may be activated by various agents including carbodnmides, carbonates, phosphomums, uromums to produce activated carboxylates acyl ureas, acylphosphonates, acid anhyd ⁇ des, and carbonates
  • Activation of carboxyhc acid may be used in conjunction with hydroxy and amine-contaimng compounds to produce activated carboxylates N- hydroxysuccimmide esters, hydroxybenzot ⁇ azole esters, N-hydroxy-5-norbornene-endo-2,3- dicarboximide esters, p-mtrophen ⁇ 1 esters, pentafluorophenyl esters, 4-
  • a nucleophile is a species possessing one or more electron-rich sites, such as an unshared pair of electrons, the negative end of a polar bond, or pi electrons
  • Cleavage, or bond breakage is the loss of a covalent bond between two atoms
  • Cleavable means that a bond is capable of being cleaved
  • a substituted group or a substitution refers to chemical group which is placed onto a parent system instead of a hydrogen atom
  • the methyl group is a substituted group, substituent, or substitution on the parent system benzene
  • the methyl groups on 2,3-d ⁇ methylmale ⁇ c anhydride are substituted groups, or substitutions on the parent compound (or system) maleic anhydnde
  • a primary amme is a mtrogen-contaming compound which is derived by monosubstitution of ammonia (NH3) by a carbon-contaming group
  • a primary amine is a nitrogen-containing compound which is derived bv disubstitution of ammonia (NH3 ) by a carbon-contaming group Examples
  • Example 4" Characterization of particles formed by poly-L-lvsine. epichlorohydnn- - cvclodext ⁇ n copolymer, and 4-. -butylbenzo ⁇ c acid To a solution of epichlorohydnn- ⁇ -cyclodextrm copolymer ( 100 ⁇ g/mL) and poly-L-lysine
  • Example 5 Characterization of particles formed by plasmid DNA, epichlorohydnn- ⁇ - cvclodext ⁇ n copolymer, and oleoylamine.
  • Example 8 In vitro Transfection with DNA-poly-L-lysine- epichlorohydnn- ⁇ -cyclodextnn copolymer in the presence of p-t-butyl-benzoic acid
  • Example 11 In vivo expression of complexes of plasmid DNA, 1 -adamantamine, and ⁇ - cyclodext ⁇ n- epichlorohydnn copolymer
  • Plasmid DNA was labeled with Mirus' Labellt ⁇ digoxin labeling kit according to protocol A complex of digoxin-labeled pCI Luc (2 ⁇ g) and ⁇ -cyclodext ⁇ n (17 mg) were formulated m 2 5 mL in R gers solution Tail vein injections of the complex were performed as previously described (Zhang, G , Budker, V , Wolff, J A Hum Gene Ther 1999, 10 1735 ) Luciferase expression was determined as previously reported (Wolff, J A , Malone, R W , Williams, P , Chong, W , Acsadi, G , Jam, A and Feigner, P L Direct gene transfer into mouse muscle m vivo Science, 1465-1468, 1990 ) A Lumat LB 9507 (EG&G Berthold, Bad- Wildbad, Germany) luminometer was used

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Abstract

La présente invention concerne l'acheminement de composés voulus (tels que des acides nucléiques) dans les cellules au moyen de systèmes d'administration non covalents, dont des acides nucléiques complexants, des agents de liaison amphipathiques et des amphiphiles.
PCT/US2000/032612 1999-11-29 2000-11-29 Compositions et methodes d'administration de medicaments au moyen de molecules de liaison amphiphiles WO2001037665A1 (fr)

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6509323B1 (en) 1998-07-01 2003-01-21 California Institute Of Technology Linear cyclodextrin copolymers
FR2842106A1 (fr) * 2002-07-11 2004-01-16 Centre Nat Rech Scient Dispersions aqueuses de particules nanometriques ou micrometriques pour l'encapsulation de composes chimiques
EP1507874A2 (fr) * 2002-05-28 2005-02-23 Mirus Corporation Compositions et procedes d'inhibition de l'expression genetique au moyen de polynucleotides
US6884789B2 (en) 1998-07-01 2005-04-26 California Institute Of Technology Linear cyclodextrin copolymers
US7018609B2 (en) 2000-12-19 2006-03-28 California Institute Of Technology Compositions containing inclusion complexes
US7375096B1 (en) 1998-12-04 2008-05-20 California Institute Of Technology Method of preparing a supramolecular complex containing a therapeutic agent and a multi-dimensional polymer network
EP2438908A1 (fr) 2010-10-11 2012-04-11 Vectum Pharma, S.L. Compositions d'ancrage pour applications topiques
EP2325193A3 (fr) * 2001-11-02 2012-05-02 Insert Therapeutics, Inc. Procédés et compositions permettant l'utilisation thérapeutique de l'interférence ARN
US8252276B2 (en) 2002-09-06 2012-08-28 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8357377B2 (en) 2002-10-09 2013-01-22 Suzie Hwang Pun Cyclodextrin-based materials, compositions and uses related thereto
US8497365B2 (en) 2007-01-24 2013-07-30 Mark E. Davis Cyclodextrin-based polymers for therapeutics delivery
US11464871B2 (en) 2012-10-02 2022-10-11 Novartis Ag Methods and systems for polymer precipitation and generation of particles

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5283185A (en) * 1991-08-28 1994-02-01 University Of Tennessee Research Corporation Method for delivering nucleic acids into cells
US5650096A (en) * 1994-12-09 1997-07-22 Genzyme Corporation Cationic amphiphiles for intracellular delivery of therapeutic molecules
US5942634A (en) * 1997-05-09 1999-08-24 Genzyme Corporation Cationic amphiphiles for cell transfections
US5965434A (en) * 1994-12-29 1999-10-12 Wolff; Jon A. Amphipathic PH sensitive compounds and delivery systems for delivering biologically active compounds

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002519393A (ja) * 1998-07-02 2002-07-02 ジエンザイム コーポレイション 分極化細胞中における導入遺伝子の発現
AUPQ259399A0 (en) * 1999-09-01 1999-09-23 Lustre Investments Pte Ltd Therapeutic agents

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5283185A (en) * 1991-08-28 1994-02-01 University Of Tennessee Research Corporation Method for delivering nucleic acids into cells
US5650096A (en) * 1994-12-09 1997-07-22 Genzyme Corporation Cationic amphiphiles for intracellular delivery of therapeutic molecules
US5965434A (en) * 1994-12-29 1999-10-12 Wolff; Jon A. Amphipathic PH sensitive compounds and delivery systems for delivering biologically active compounds
US5942634A (en) * 1997-05-09 1999-08-24 Genzyme Corporation Cationic amphiphiles for cell transfections

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
PUYAL ET AL.: "A new cationic liposome encapsulating genetic material A potential delivery system for polynucleotides", EUR. J. BIOCHEM., vol. 228, 1995, pages 697 - 703, XP002938699 *
SAKATA ET AL.: "Effects of saturated and unsaturated fatty acids on apolipoprotein B secretion using cyclodextrin delivery system", FASEB JOURNAL, vol. 11, no. 3, 1997, pages A169, ABSTRACT NO. 984, XP002938700 *
See also references of EP1233671A4 *

Cited By (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6884789B2 (en) 1998-07-01 2005-04-26 California Institute Of Technology Linear cyclodextrin copolymers
US6509323B1 (en) 1998-07-01 2003-01-21 California Institute Of Technology Linear cyclodextrin copolymers
US7091192B1 (en) 1998-07-01 2006-08-15 California Institute Of Technology Linear cyclodextrin copolymers
US7375096B1 (en) 1998-12-04 2008-05-20 California Institute Of Technology Method of preparing a supramolecular complex containing a therapeutic agent and a multi-dimensional polymer network
US8277846B2 (en) 2000-12-19 2012-10-02 California Institute Of Technology Complexing agents for compositions containing inclusion complexes
US7166302B2 (en) 2000-12-19 2007-01-23 California Institute Of Technology Complexing agents for compositions containing inclusion complexes
US7807198B2 (en) 2000-12-19 2010-10-05 California Institute Of Technology Compositions containing inclusion complexes
US7968123B2 (en) 2000-12-19 2011-06-28 California Institute Of Technology Complexing agents for compositions containing inclusion complexes
US8092833B2 (en) 2000-12-19 2012-01-10 California Institute Of Technology Compositions containing inclusion complexes
US7018609B2 (en) 2000-12-19 2006-03-28 California Institute Of Technology Compositions containing inclusion complexes
EP2325193A3 (fr) * 2001-11-02 2012-05-02 Insert Therapeutics, Inc. Procédés et compositions permettant l'utilisation thérapeutique de l'interférence ARN
EP1507874A4 (fr) * 2002-05-28 2006-06-28 Mirus Bio Corp Compositions et procedes d'inhibition de l'expression genetique au moyen de polynucleotides
EP1507874A2 (fr) * 2002-05-28 2005-02-23 Mirus Corporation Compositions et procedes d'inhibition de l'expression genetique au moyen de polynucleotides
WO2004006897A1 (fr) * 2002-07-11 2004-01-22 Centre National De La Recherche Scientifique (C.N.R.S.) Dispersions aqueuses de particules nanometriques ou micrometriques pour l'encapsulation de composes chimiques
US7682635B2 (en) 2002-07-11 2010-03-23 Centre National De La Recherche Scientifique (C.N.R.S.) Aqueous dispersions of nanometric or micrometric particles for encapsulating chemical compounds
FR2842106A1 (fr) * 2002-07-11 2004-01-16 Centre Nat Rech Scient Dispersions aqueuses de particules nanometriques ou micrometriques pour l'encapsulation de composes chimiques
US8314230B2 (en) 2002-09-06 2012-11-20 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8580243B2 (en) 2002-09-06 2013-11-12 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US9550860B2 (en) 2002-09-06 2017-01-24 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8680202B2 (en) 2002-09-06 2014-03-25 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8609081B2 (en) 2002-09-06 2013-12-17 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8389499B2 (en) 2002-09-06 2013-03-05 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8399431B2 (en) 2002-09-06 2013-03-19 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8404662B2 (en) 2002-09-06 2013-03-26 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8475781B2 (en) 2002-09-06 2013-07-02 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8603454B2 (en) 2002-09-06 2013-12-10 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8518388B2 (en) 2002-09-06 2013-08-27 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8580242B2 (en) 2002-09-06 2013-11-12 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8580244B2 (en) 2002-09-06 2013-11-12 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8252276B2 (en) 2002-09-06 2012-08-28 Cerulean Pharma Inc. Cyclodextrin-based polymers for therapeutics delivery
US8357377B2 (en) 2002-10-09 2013-01-22 Suzie Hwang Pun Cyclodextrin-based materials, compositions and uses related thereto
US8497365B2 (en) 2007-01-24 2013-07-30 Mark E. Davis Cyclodextrin-based polymers for therapeutics delivery
US9610360B2 (en) 2007-01-24 2017-04-04 Ceruliean Pharma Inc. Polymer drug conjugates with tether groups for controlled drug delivery
EP2438908A1 (fr) 2010-10-11 2012-04-11 Vectum Pharma, S.L. Compositions d'ancrage pour applications topiques
WO2012049453A2 (fr) 2010-10-11 2012-04-19 Vectum Pharma S.L. Compositions destinées à des applications topiques
US11464871B2 (en) 2012-10-02 2022-10-11 Novartis Ag Methods and systems for polymer precipitation and generation of particles

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