EP1421170A2 - Compositions d'administration therapeutique et methodes d'utilisation associees - Google Patents

Compositions d'administration therapeutique et methodes d'utilisation associees

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Publication number
EP1421170A2
EP1421170A2 EP02765919A EP02765919A EP1421170A2 EP 1421170 A2 EP1421170 A2 EP 1421170A2 EP 02765919 A EP02765919 A EP 02765919A EP 02765919 A EP02765919 A EP 02765919A EP 1421170 A2 EP1421170 A2 EP 1421170A2
Authority
EP
European Patent Office
Prior art keywords
molecular weight
compound
approximately
weight
polyoxypropylene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02765919A
Other languages
German (de)
English (en)
Inventor
Martin R. Emanuele
Konstantin G. Kousoulas
Hameedsulthan S. Allaudeen
Mark Newman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
LadRx Corp
Original Assignee
CytRx Corp
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Filing date
Publication date
Priority claimed from US09/919,504 external-priority patent/US20020123476A1/en
Priority claimed from US09/929,819 external-priority patent/US6933286B2/en
Application filed by CytRx Corp filed Critical CytRx Corp
Publication of EP1421170A2 publication Critical patent/EP1421170A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the present invention relates to compositions and methods for treating infectious diseases and genetic disorders through gene therapy and intracellular delivery of nucleic acids.
  • oligonucleotides that are complementary to certain gene messages or viral sequences known as "antisense” compounds, have been shown to have an inhibitory effect against viruses.
  • antisense compounds By creating an antisense compound that hybridizes with the targeted RNA message of cells or viruses the translation of the message into protein can be interrupted or prevented. In this fashion gene activity can be modulated.
  • antisense oligonucleotides have inhibited infections by herpes-viruses, influenza viruses and the human immunodeficiency virus that causes AIDS. It may also be possible to target antisense oligonucleotides against mutated oncogenes. Antisense technology also holds the potential for regulating growth and development. However, in order for the gene therapy to work, antisense therapeutic compounds must be delivered across cellular plasma membranes to the cytosol.
  • Gene activity is also modified using sense DNA in a technique known as gene therapy.
  • Defective genes are replaced or supplemented by the administration of "good" or normal genes that are not subject to the defect.
  • the administered normal genes which insert into a chromosome, or may be present in extracellular DNA, produce normal RNA, which in turn leads to normal gene product.
  • gene defects and deficiencies in the production of gene product may be corrected.
  • Still further gene therapy has the potential to augment the normal genetic complement of a cell. For example, it has been proposed that one way to combat HIV is to introduce into an infected person's T cells a gene that makes the cells resistant to HIV infection.
  • gene therapy is sometimes called "intracellular immunization.”
  • Genetic material such as polynucleotides may be administered to a mammal to elicit an immune response against the gene product of the administered nucleic acid sequence.
  • gene vaccines elicit an immune response in the following manner. First, the nucleic acid sequence is administered to a human or animal. Next, the administered sequence is expressed to form gene product within the human or animal.
  • the gene product inside the human or animal is recognized as foreign material and the immune system of the human or animal mounts an immunological response against the gene product.
  • this approach currently is not feasible due to a lack of effective gene delivery systems that facilitate the delivery of genetic material across both cellular and nuclear membranes.
  • gene therapy may be used as a method of delivering drugs in vivo.
  • genes that code for therapeutic compounds can be delivered to endothelial cells, the gene products would have facilitated access to the blood stream.
  • genes are delivered to cells e vivo and then reintroduced to the animal.
  • Retroviral vectors can be used to deliver genes ex vivo to isolated cells, which are then infused back into the patient.
  • retroviral vectors have some drawbacks, such as being able to deliver genes only to dividing cells, random integration of the gene to be delivered, potentially causing unwanted genetic alterations, and possibly reverting back to an infectious wild-type retroviral form.
  • Another drawback of antisense gene therapy is that it is effective at the messenger RNA level, which means that antisense oligonucleotides must be introduced in a quantity to interact with all or a substantial number of the mRNA in the cytosol, and that such treatment is only effective during active synthesis of mRNA.
  • triplex DNA technology utilizes oligonucleotides and compounds that specifically bind to particular regions of duplex DNA, thereby inactivating the targeted gene.
  • a drawback of triplex DNA technology is that the oligonucleotide or compound must pass through not only the cellular membrane, but also the microbial membrane in the case of treating microbial infections, or the nuclear membrane in the case of altering eukaryotic gene function or expression of foreign DNA integrated into chromosomal DNA.
  • Ribozymes are catalytic RNA molecules that consist of a hybridizing region and an enzymatic region. Ribozymes may in the future be engineered so as to specifically bind to a targeted region of nucleic acid sequence and cut or otherwise enzymatically modify the sequence so as to alter its expression or translation into gene product.
  • Ribozymes There is a great need, therefore, for improved delivery systems for genetic material such as genes, polynucleotides, and antisense oligonucleotides that can be used in gene therapy. More specifically, there is a need for non-toxic compositions having surfactant properties that can facilitate the transport of genetic compounds and other drugs and therapeutic compounds across cellular membranes.
  • HIV Acquired Immune Deficiency Syndrome
  • HTLV- III Human T Lymphotropic Virus III
  • HIV has ribonucleic acid, or RNA, as its genetic material.
  • RNA messenger RNA
  • reverse transcriptase exploits the viral RNA as a template to assemble a corresponding molecule of DNA.
  • the DNA travels through the cell nucleus and inserts itself among the host chromosomes, where it provides the basis for viral replication.
  • the host cell is often a T4 lymphocyte, a white blood cell that has a central and regulatory role in the immune system.
  • T4 lymphocyte a white blood cell that has a central and regulatory role in the immune system.
  • the virus may remain latent until the lymphocyte is immunologically stimulated by a secondary infection. Then the virus reproducing itself rapidly killing or rendering ineffective the host cell.
  • the resulting depletion of the T4 cells, and loss of activity leaves the patient vulnerable to "opportunistic" infections by an agent that would not normally harm a healthy person.
  • the virus damages the host by many other mechanisms as well.
  • AZT is known to have serious side effects and its efficacy in mitigating the AIDS disease has been questioned.
  • the efficacy of AZT and other antiviral and antimicrobial drugs could be increased if improved means and methods for delivering therapeutic agents to the site of infection were available.
  • the present invention includes a method of delivering therapeutic drugs to a human or animal for treating disease states such as, but not limited to, bacterial infection and infections caused by HIV and other DNA and RNA viruses.
  • the present invention relates particularly to compositions and methods for treating infectious diseases and genetic disorders through gene therapy and intracellular delivery of antisense oligonucleotides or other nucleic acid sequences.
  • a is an integer such that the hydrophobe represented by (C3H6O) has a molecular weight of approximately 750 and approximately 15,000, preferably between approximately 2250 and approximately 15,000, more preferably between approximately 3250 and approximately 15,000, and b is an integer such that the hydrophile portion represented by (C2H4O) constitutes approximately 1% to approximately 50% by weight of the compound, preferably approximately 5% to approximately 20%.
  • Another aspect of the present invention comprises the surface active nonionic block copolymer of the present invention also comprises a reverse triblock copolymer of polyoxypropylene/polyoxyethylene having the formula:
  • Another embodiment of the surface active nonionic block copolymer of the present invention comprises the reverse triblock copolymer of polyoxypropylene/polyoxyethylene having the formula:
  • Yet another embodiment of the present invention comprises the reverse triblock copolymer of polyoxypropylene/polyoxyethylene having the formula:
  • a preferred embodiment of the present invention comprises the reverse triblock copolymer polyoxypropylene/polyoxyethylene having the formula:
  • POP POE POP wherein "b” represents a number such that the molecular weight of the hydrophobe (C 3 H ⁇ O) b is between approximately 5,000 and 6,000, and "a” represents a number such that the percentage of hydrophile (C 2 H 4 ⁇ ) a is between approximately 14% and 16%.
  • the present invention comprises a therapeutic delivery composition effective for treating a disease state comprising an administerable admixture of an effective amount of a therapeutic compound capable of altering nucleic acid sequence function and an effective amount of a biologically-active copolymer comprising a copolymer of polyoxyethylene (POE) which is hydrophilic and polyoxypropylene (POP) which is hydrophobic.
  • POE polyoxyethylene
  • POP polyoxypropylene
  • the block copolymer is built on a tetrafunctional ethylenediamine initiator.
  • the block copolymers that comprise the biologically-active copolymers of the present invention have the following general formulas:
  • the mean aggregate molecular weight of the hydrophobe portion of the octablock copolymer consisting of polyoxypropylene (C3H6 ⁇ )b (POP) is between approximately 5000 and 7000 daltons; a is a number such that the hydrophile portion represented by polyoxyethylene (C2H4 ⁇ ) a (POE) constitutes between approximately 10% and 40% of the total molecular weight of the compound; and b is a number such that the polyoxypropylene (C3H6 ⁇ )b (POP) portion of the total molecular weight of the octablock copolymer constitutes between approximately 60% and 90% of the compound.
  • the biologically- active copolymer comprises a polymer of hydrophilic polyoxyethylene (POE) built on an ethylene diamine initiator. Polymers of hydrophobic polyoxypropylene (POP) are then added to the blocks of hydrophilic polyoxyethylene (POE). This results in an octablock copolymer with the following general formula:
  • a is a number such that the hydrophile portion represented by polyoxyethylene (C2H 4 0)a (POE) constitutes between approximately 10% to 40% of the total molecular weight of the compound;
  • the mean aggregate molecular weight of the hydrophobe portion of the octablock copolymer consisting of polyoxypropylene (C3H6 ⁇ )b (POP) is between approximately 5000 and 7000 daltons;
  • b is a number such that the polyoxypropylene (C3H6 ⁇ )b (POP) portion of the total molecular weight of the octablock copolymer constitutes between approximately 60% and 90% of the compound.
  • a particularly useful composition is an admixture of a compound capable of altering gene expression and/or protein translation, such as nucleic acids, vector constructs, an antisense oligonucleotide, a triplex DNA compound, a ribozyme or other compound capable of altering nucleic acid sequence function, and either of the above-described block copolymer, the above-described octablock copolymer, or a combination thereof.
  • the composition of the present invention can be administered by a number of routes including, but not limited topical, transdermal, oral, trans-mucosal, subcutaneous injection, intravenous injection, intraperitoneal injection and intramuscular injection.
  • an object of the invention is to provide a therapeutic drug delivery vehicle.
  • Another object of the present invention is to provide compositions that facilitate delivery of one or more therapeutic nucleic acid sequence function altering agents into the interior of a cell, such as a phagocytic cell, when admixed with a therapeutic agent.
  • Another object of the present invention is to provide compositions that act synergistically with a delivered agent once inside a cell.
  • Still another object of the invention is to provide nonionic block copolymers and octablock copolymers having surfactant properties that facilitate the transmission and introduction across cellular plasma membranes of nucleic acid sequences and compounds capable of altering nucleic acid sequence function.
  • a further object of the present invention is to provide compositions and a method for treating genetic and physiologic disorders using nucleic acid sequences and antisense oligonucleotides in combination with nonionic block copolymers, octablock copolymers, or a combination thereof.
  • Yet another object of the present invention is to provide a method of treating viral infections in humans or animals.
  • Another object of the present invention is a compound and method that is effective in inhibiting the replication of viruses in both animals and humans.
  • Another object of the present invention is to provide a compound and method that is effective in inhibiting the replication of HIV and other RNA and DNA viruses.
  • Yet another object of the present invention is to provide a method of treating microbial infections in humans or animals.
  • Fig. 1 is a grid illustrating block copolymers by molecular weight of hydrophobe and percent hydrophile.
  • Fig. 2 is a grid illustrating preferred therapeutic delivery block copolymers by molecular weight of hydrophobe and percent hydrophile.
  • Fig. 3 is a grid illustrating more preferred therapeutic delivery block copolymers by molecular weight of hydrophobe and percent hydrophile.
  • the present invention includes gene therapy compositions that are admixtures of a nonionic block copolymer, an octablock copolymer, or a combination thereof, and nucleic acid sequences or compounds capable of altering nucleic acid sequence function, and methods of delivering these compositions to a human or animal in need thereof for the intracellular alteration of gene expression and/or protein translation.
  • polyoxyethylene- polyoxypropylene block copolymers having a low percentage of polyoxyethylene, block copolymers, and combinations thereof facilitate the transport of DNA and other compounds into cells and thus are useful for the intracellular delivery of therapeutic agents in vivo for the treatment of disease. It is believed that both the block copolymers and the octablock copolymers are particularly useful in helping to reseal membranes and thus increase the percent survival of cells wherein nucleic acid sequences or other compounds have been intracellularly introduced.
  • compositions comprising the nonionic block copolymers and/or octablock copolymers of the present invention and nucleic acid sequences are less susceptible to the degrading effects of DNAase than nucleic acid sequences alone.
  • the present invention also comprises therapeutic compositions and methods which kill or inhibit the growth of microorganisms and alter the expression or function nucleic acid sequences.
  • An example of the bacteria that the present invention is effective against is mycobacteria species, such as Mycobacterium tuberculosis, Mycobacterium avium, an ⁇ Mycobacterium leprae.
  • microorganisms that the invention is effective against include, but are not limited to, Chlamydia trachomatis, Chlamydia pneumoniae, Listeria monocytogenes, Candida albicans, Cryptococcus neoformans, Toxoplasma gondii, Pneumocystis carinii, Herpes simplex virus type 1, Cytomegalovirus, influenza virus type A and B, and respiratory syncytial virus.
  • the present invention includes therapeutic compositions and methods for treating DNA viruses and RNA viruses, and infections and infectious diseases caused by such viruses in a human or animal, including infections caused by HIV or herpes or antigenically-related strains thereof.
  • Antigenically-related strains are strains that crossreact with antibodies specific for HIV.
  • One skilled in the art can readily determine viral strains that are antigenically-related to HIV by conducting standard immunoassay tests using anti-HIV antibodies and the viral strain to be analyzed, and looking for positive cross-reactivity.
  • the therapeutic compositions comprising the surface active copolymers and/or octablock copolymers disclosed herein are effective in inhibiting or suppressing the replication of such viruses in cells.
  • the present invention includes a therapeutic composition useful for delivering antimicrobial drugs and treating disease states comprising an admixture of a surface active nonionic block copolymer, or an octablock copolymer, or a combination thereof and a compound capable of altering nucleic acid sequence function; and may further comprise an antibiotic or therapeutic drug.
  • a therapeutic composition useful for delivering antimicrobial drugs and treating disease states comprising an admixture of a surface active nonionic block copolymer, or an octablock copolymer, or a combination thereof and a compound capable of altering nucleic acid sequence function; and may further comprise an antibiotic or therapeutic drug.
  • compounds capable of altering nucleic acid sequence function include nucleic acids, vector constructs such as expression vectors or vectors containing functional nucleic acid sequences, genes, oligonucleotides, antisense oligonucleotides, triplex DNA compounds, and ribozymes.
  • Drugs that can be used with the nonionic copolymers of the present invention include, but are not limited to, rifampin, isoniazid, ethambutol, gentamicin, tetracycline, erythromycin, pyrazinamide, streptomycin, clofazimine, rifabutin, fluoroquinolones such as ofloxacin and sparfloxacin, azithromycin, clarithromycin, dapsone, doxycyline, ciprofloxacin, ampicillin, amphotericin B, fluconazole, ketoconazole, fluconazole, pyrimethamine, sulfadiazine, clindamycin, azithromycin, paromycin, diclazaril, clarithromycin, atovaquone, pentamidine, acyclovir, trifluorouridine, AZT, DDI, DDC, and other antiviral nucleoside analogs, foscornat, ganci
  • Cryptococcus neoformans Amphotericin B, ketoconazole, fluconazole
  • HIV AZT HIV AZT, DDI, DDC, foscarnat, viral protease inhibitors, peptides, antisense oligonucleotides, triplex and other nucleic acid sequences
  • surfactants and low molecular weight alcohols are added to the therapeutic admixture of antimicrobial drug and nonionic block copolymer, block copolymer, or combination thereof.
  • surfactants useful in the present invention include Tween 80 and emulsions with fatty acids such as phospholipids, cholate and amino acids.
  • the preferred surfactant is Tween 80.
  • Surfactants are added to the admixture at a concentration ranging from approximately 0.1% to approximately 5% v/v. The preferred surfactant concentration is approximately 2%.
  • the term "low molecular weight alcohols” means alcohols having two to eight carbons.
  • An example of a low molecular weight alcohols useful in the present invention is ethanol, which is the preferred low molecular weight alcohol.
  • Low molecular weight alcohols are added to the admixture at a concentration ranging from approximately 0.5% to approximately 5% v/v.
  • the preferred low molecular weight alcohol concentration is between approximately 1% and approximately 3% v/v.
  • the present invention also includes compositions and methods for immunizing animals or humans, otherwise termed DNA vaccination.
  • Immunization is accomplished by administering a composition comprising the gene that codes for the gene product to be immunized against contained in an expression, in combination with a block copolymer that promotes and facilitates uptake of genetic material across cell membranes.
  • the introduced gene is expressed, resulting in the production of an antigenic gene product.
  • compositions comprising nonionic block copolymers, octablock copolymers, or a combination thereof and genes that code for compounds effective for killing, reducing or retarding cancer, such as lymphokines, may be administered to humans or animals for the treatment of cancer.
  • the present invention comprises a surface active copolymer that is preferably an ethylene oxide-propylene oxide condensation product with the following general formula:
  • a is an integer such that the hydrophobe represented by (C3H6O) has a molecular weight of between approximately 750 and approximately 20,000
  • b is an integer such that the hydrophile portion represented by (C2H4O) constitutes approximately 1% to approximately 50% by weight of the compound.
  • the present invention also comprises a therapeutic delivery composition useful for altering gene expression and/or protein translation comprising an administerable admixture of an effective amount of an anti- sense oligonucleotide or other nucleic acid sequence, and an effective amount of a nonionic block copolymer having the following general formula:
  • a is an integer such that the hydrophobe represented by (C3H6O) has a molecular weight of approximately 750 and approximately 20,000, preferably between approximately 2250 and approximately 15,000, more preferably between approximately 3250 and approximately 15,000, and most preferably between approximately 10,000 and approximately 15,000
  • b is an integer such that the hydrophile portion represented by (C2H4O) constitutes approximately 1% to approximately 50% by weight of the compound, preferably approximately 5% to approximately 20%.
  • the term admixture as used herein means any combination of therapeutic drug and nonionic block copolymer, including solutions, suspensions, or encapsulations of drug in copolymer micelles.
  • An effective amount is an amount sufficient to alter the activity and/or the amount of gene product produced by the gene or genes sought to be modulated in a human or animal.
  • the present invention also comprises a therapeutic delivery composition useful for immunizing an animal or human against a particular gene product comprising an administerable admixture of an effective amount of an expression vector, the gene that codes for the gene product to be immunized against contained in the expression vector, and an effective amount of a nonionic block copolymer having the following general formula:
  • a is an integer such that the hydrophobe represented by (C3H6O) has a molecular weight of approximately 750 and approximately 20,000, preferably between approximately 2250 and approximately 15,000, more preferably between approximately 3250 and approximately 15,000, and most preferably between approximately 10,000 and approximately 15,000
  • b is an integer such that the hydrophile portion represented by (C2H 4 O) constitutes approximately 1% to approximately 50% by weight of the compound, preferably approximately 5% to approximately 20%.
  • An effective amount is an amount sufficient to elicit an immunological response against the gene product of the nucleic acid sequence administered to the human or animal.
  • the surface active nonionic block copolymer of the present invention comprises a reverse triblock copolymer of polyoxypropylene/polyoxyethylene having the formula:
  • POP POE POP wherein "b” represents a number such that the molecular weight of the hydrophobe (C 3 H 6 ⁇ ) b is between approximately 750 and approximately 20,000, preferably between approximately 2250 and approximately 15,000, more preferably between approximately 3250 and approximately 15,000, preferably 2,000 and 10,000, and perferably between approximately 10,000 and approximately 15,000, and "a” represents a number such that the percentage of hydrophile (C 2 H 4 ⁇ ) a is between approximately 2% and 30%.
  • Another embodiment of the surface active nonionic block copolymer present invention comprises the reverse triblock copolymer of polyoxypropylene/polyoxyethylene having the formula:
  • Yet another embodiment of the present invention comprises the reverse triblock copolymer of polyoxypropylene/polyoxyethylene having the formula:
  • a preferred embodiment of the present invention comprises the reverse triblock copolymer polyoxypropylene/polyoxyethylene having the formula:
  • compositions of the present invention include, but are not limited to aqueous solutions, suspensions or emulsions, such as oil-in-water emulsions.
  • the polymer blocks are formed by condensation of ethylene oxide and propylene oxide, at elevated temperature and pressure, in the presence of a catalyst. There is some statistical variation in the number of monomer units which combine to form a polymer chain in each copolymer.
  • Ethylene oxide-propylene oxide condensation products which may be employed in the present invention are included in Table II. It is to be understood that these compounds are merely representative of the compounds that can be used to practice the present invention and do not include all possible compounds that could be used to practice the present invention.
  • the high molecular weight copolymers listed in Table II that do not have a BASF tradename are novel compositions that have never been synthesized before.
  • a grid illustrating the range of copolymer encompassed by the present invention based upon the molecular weight of the hydrophobe portion and the percent hydrophile, and showing selected nonionic block copolymers appears as Figure 1.
  • the polymer blocks are formed by condensation, at elevated temperature and pressure, of ethylene oxide and propylene oxide in the presence of a catalyst. There is some statistical variation in the number of monomer units which combine to form a polymer chain in each copolymer.
  • the molecular weights given are approximations of the average size of copolymer molecules in each preparation. A further description of the preparation of these block copolymers is found in U.S.
  • the non-ionic copolymers have negative thermal coefficients of solubility. In the cold, the kinetic energy of water molecules is reduced and they form weak hydrogen bonds with the oxygen of the POP blocks. This hydration of the hydrophobe promotes solubility at low temperatures. As the temperature rises, the "cloud point" is reached; the increased kinetic energy of the water breaks the hydrogen bonds, the polymer becomes insoluble and micelles form.
  • the biologically-active octablock copolymers of the present invention comprise a surface active compound with four hydrophobic segments and a small proportion of hydrophile. Typical examples have eight segments or octablock structure with a core of either a hydrophobic or hydrophilic central structure and a hydrophilic or hydrophobic outer structure.
  • the entire molecule is poorly soluble in water and is either a nonionic or weakly cationic surface active agent.
  • the steric configuration and physiochemical properties of the molecule, rather than the chemical nature of the constituent parts, are thought to be responsible for the biologic effects of the copolymer.
  • the octablock copolymers of the present invention comprise blocks of polyoxypropylene and polyoxyethylene built on an alkylenediamine initiator.
  • the blocks of polyoxypropylene (POP) and polyoxyethylene (POE) have the following structures:
  • the polymer blocks are formed by condensation of ethylene oxide and propylene oxide onto a tetra unctional ethylene diamine initiator at elevated temperature and pressure in the presence of a basic catalyst. There is some statistical variation in the number of monomer units which combine to form a polymer chain in each copolymer. The molecular weights given are approximations of the average weight of copolymer molecule in each preparation. A further description of the preparation of these block copolymers is found in U.S. Patent No. 2,674,619 and U.S. Patent No. 2,979,528. (Also see "A Review of Block Polymer Surfactants", Schmolka, I.R., J. Am. Oil Chemists' Soc, 54: 110-116 (1977) m ⁇ Block and Graft Copolymerization, Volume 2 edited by R.J. Ceresa, John Wiley &
  • the block copolymer comprises a polymer of hydrophobic polyoxypropylene (POP) built on an ethylenediamine initiator. Polymers of hydrophilic polyoxyethylene (POE) are then built on the block of hydrophobic polyoxypropylene (POP). This results in an octablock copolymer with the following general formula: (C 2 H 4 0) a (C 3 H 6 0) b / (C 3 H 6 0) b (C 2 H 4 0) a
  • the mean aggregate molecular weight of the hydrophobe portion of the octablock copolymer consisting of polyoxypropylene (C3H6 ⁇ )b (POP) is between approximately 5000 and 7000 daltons; a is a number such that the hydrophile portion represented by polyoxyethylene (C2H4 ⁇ )a (POE) constitutes between approximately 10% to 40% of the total molecular weight of the compound; and b is a number such that the polyoxypropylene (C3H6 ⁇ )b (POP) portion of the total molecular weight of the octablock copolymer constitutes between approximately 60% and 90% of the compound.
  • the octablock copolymer comprises a polymer of hydrophilic polyoxyethylene (POE) built on an ethylene diamine initiator. Polymers of hydrophobic polyoxypropylene (POP) are then built on the block of hydrophilic polyethylene (POE). This results in a reverse octablock copolymer with the following general formula:
  • the molecular weight of the hydrophobe portion of the octablock copolymer consisting of polyoxypropylene (C3H6 ⁇ )b (POP) is between approximately 5000 and 7000 daltons; a is a number such that the hydrophile portion represented by polyoxyethylene (C2H4 ⁇ ) a (POE) constitutes between approximately 10% and 40% of the total molecular weight of the compound; and b is a number such that the polyoxypropylene (C3H6 ⁇ ) (POP) portion of the octablock copolymer constitutes between approximately 60% and 90% of the compound.
  • This type of polymer is called reverse copolymer because its structure is the reverse of octablock copolymers that have polyoxypropylene (POP) in the center flanked by blocks of polyoxyethylene (POE).
  • POP polyoxypropylene
  • POE polyoxyethylene
  • the (C 2 H 4 0) portion of the copolymer can constitute as low as 5% of the octablock copolymer.
  • the octablock copolymers comprising the biologically active copolymers of the present invention include, but are not limited to, the block copolymers Tetronic® and reverse Tetronic® manufactured by the BASF
  • a is a number such that the hydrophile portion represented by polyoxyethylene (C2H4 ⁇ )a (POE) constitutes between approximately 5% and 20% of the total molecular weight of the compound;
  • b is a number such that the polyoxypropylene (C3H6 ⁇ )b (POP) portion of the octablock copolymer constitutes between approximately 80% and 95% of the compound.
  • a preferred biologically active copolymer is the octablock copolymer Tl 10R1 (BASF Corporation, Parsippany, NJ) which corresponds to the following formula:
  • the mean molecular weight of the hydrophobe portion of the octablock copolymer represented by polyoxypropylene (C3H6 ⁇ )b (POP) is approximately 5,220 daltons; a is a number such that the hydrophile portion represented by polyoxyethylene (C2H4 ⁇ ) a (POE) constitutes approximately 10% of the compound by weight; and b is a number such that the polyoxypropylene (C3H6 ⁇ ) D (POP) portion of the octablock copolymer constitutes approximately 90% of the compound by weight.
  • a preferred biologically active copolymers is the octablock copolymer T130R2 (BASF Corporation, Parsippany, NJ) which corresponds to the following formula:
  • the mean molecular weight of the hydrophobe portion of the octablock copolymer represented by polyoxypropylene (C3H6 ⁇ )b (POP) is approximately 5750 daltons; a is a number such that the hydrophile portion represented by polyoxyethylene (C2H4 ⁇ )a (POE) constitutes approximately 20% of the compound by weight; and b is a number such that the polyoxypropylene (C3H6 ⁇ )b (POP) portion of the octablock copolymer constitutes approximately 80% of the compound by weight.
  • Another preferred embodiment of the biologically active copolymers of the present invention is the compound designated T1501 (BASF Corporation, Parsippany, NJ) which corresponds to the following formula:
  • POE POP POP POE wherein: the mean molecular weight of the hydrophobe portion of the octablock copolymer represented by polyoxypropylene (C3H6 ⁇ ) (POP) is approximately 6750 daltons; a is a number such that the hydrophile portion represented by polyoxyethylene (C2H4 ⁇ )a (POE) constitutes approximately 10% of the compound by weight; and b is a number such that the polyoxypropylene (C3H6 ⁇ )b (POP) portion of the octablock copolymer constitutes approximately 90% of the compound by weight.
  • C3H6 ⁇ polyoxypropylene
  • the most preferred embodiment of the biologically active copolymers of the present invention is the octablock copolymer T150R1
  • the mean molecular weight of the hydrophobe portion of the octablock copolymer represented by polyoxypropylene (C3H6 ⁇ )b (POP) is approximately 6750 daltons; a is a number such that the hydrophile portion represented by polyoxyethylene (C2H4 ⁇ )a (POE) constitutes approximately 10% of the compound by weight; and b is a number such that the polyoxypropylene (C3H6 ⁇ )b (POP) portion of the octablock copolymer constitutes approximately 90% of the compound by weight.
  • the present invention also comprises a therapeutic delivery composition useful for immunizing an animal or human against a particular gene product comprising an administerable admixture of an effective amount of an expression vector, the gene that codes for the gene product to be immunized against contained in the expression vector, and an effective amount of an octablock copolymer of the present invention.
  • the present invention also comprises a therapeutic delivery composition useful for immunizing an animal or human against a particular gene product comprising an administerable admixture of an effective amount of an expression vector, the gene that codes for the gene product to be immunized against contained in the expression vector, and an effective amount of an octablock copolymer of the present invention.
  • the octablock copolymers of the present invention, including biological activity thereof, are further discussed in U.S. Patent No. 5,494,660, which is incorporated herein in its entirety by reference.
  • the copolymers including both the nonionic block copolymers and the octablock copolymers of the present invention, which are therapeutic themselves, can form physical structures that can be combined or loaded with an additional, distinct therapeutic agent. Consequently, the nonionic block copolymers and the octablock copolymers of the present invention can be used as therapeutic drug delivery vehicles.
  • Admixtures of therapeutic drugs with non-ionic block and octablock copolymers have the advantage of synergistic activity of at least two therapeutic agents.
  • copolymers having specific characteristics can be selected for use with particular therapeutic drugs.
  • CRL- 8131 which is hydrophobic, is an excellent carrier for hydrophobic antibiotics such as rifampin.
  • other agents which are not distinctly hydrophobic can be used according to the present invention.
  • a therapeutic delivery vehicle is prepared using any of the surface active nonionic block copolymers and/or octablock copolymers of the present invention in combination with any of a variety of antimicrobial agents.
  • CRL-8131 is used at a concentration of approximately 3% to approximately 5% to construct a therapeutic delivery vehicle.
  • Therapeutic delivery vehicles made using copolymers that are more hydrophilic than CRL-8131 normally require a higher concentration (approximately 5% to approximately 10%) of the copolymer.
  • copolymer-based micelles as a therapeutic drug delivery vehicle is particularly desirable because the micelles are accumulated readily and are present for an extended period of time, in macrophages, the site of HIV and other viral infections and a major target for viral therapy.
  • therapeutic copolymer-based therapeutic compositions include CRL-8131 combined with 2% Tween 80 and 1% ethanol, and CRL- 8142 combined with 1 % Tween 80 and 5% ethanol.
  • Nucleic acid sequences or other compounds capable of altering nucleic acid sequence function are administered to a human or animal to alter gene expression and/or modify the amount or activity of gene product.
  • antisense oligonucleotides admixed with either or both of the above-described copolymers yield compositions useful for delivery of the antisense oligonucleotides for the purpose of altering or regulating gene expression and/or protein translation.
  • nucleic acid sequences such as genes can be administered which incorporate into the chromosome replacing or augmenting the defective gene.
  • the intracellularly administered gene may reside in the cell and be expressed in an extrachromosomal element.
  • the present invention also provides novel compositions and methods for immunizing an animal or human.
  • the compositions comprise an expression vector, a gene that codes for the gene product to be immunized against contained in the expression vector, and a block copolymer, which includes the nonionic block copolymers, the octablock copolymers, or a combination thereof, effective for transferring genetic material such as expression vectors across the membrane of cells.
  • the method of immunizing an animal or human comprises administering of the expression vector-containing copolymer composition to the animal or human.
  • a preferred mode of administration is by intraperitoneal injection.
  • This embodiment of the invention provides means for the delivery of genetic sequences capable of expressing an antigenic gene product directly into human or animal cells, either in vivo or ex vivo with subsequent reintroduction into the human or animal. Once introduced into the cells the production of antigenic gene product induces and maintains an immune response by the human or animal against the introduced gene product.
  • compositions and methods of the invention useful for gene therapy and compositions and methods of the invention useful for gene-mediated immunization. It should be appreciated that other embodiments and uses will be apparent to those skilled in the art andthat the invention is not limited to these specific illustrative examples.
  • Example I A therapeutic delivery vehicle is prepared by combining any of the surface active nonionic block copolymers, such as CRL-8131 with any of a variety of compounds capable of altering nucleic acid sequence function.
  • CRL-8131 a concentration of three to five percent weight per volume is desirable to construct the therapeutic vehicle.
  • hydrophilic copolymer a five to ten percent weight per volume.
  • CRL-8131 300 milligrams of CRL-8131 was added to 10 ml of 0.9% NaCl and the mixture is solubilized by storage at temperatures of 2-4°C until a clear solution is formed.
  • An appropriate amount of a compound capable of altering nucleic acid gene function is added to the mixture and micelles associating the copolymer and the compound are formed by raising the temperature above 5°C and allowing the suspension of micelles to equilibrate. The equilibrated suspension is suitable for administration.
  • an antisense oligonucleotide sequence such as one of those disclosed by Matsukara, M. et al.Proc. Natl. Acad. Sci. USA 84:7706-7710 (1987), which is expressly incorporated herein in its entirety by reference, is combined with the copolymer to form a micelle composition.
  • phosphorothioate or methylphosphonate derivatives of a sequence complimentary to regions of the art/trs genes of HIV having the sequence 5'-TCGTCGCTGTCTCG-3' are prepared according to the method of Matsukura et al. 300 milligrams of CRL-8131 is added to 10 ml of 0.9% NaCl and the mixture is solubilized by storage at temperatures of 2-4°C until a clear solution is formed. The desired antisense oligonucleotide subsequently is mixed with the copolymer solution to provide a concentration effective in inhibiting viral activity when administered to a patient infected with the HIV virus.
  • the effective amount of antisense compound will be such that the final concentration in the blood is in the range of 1 ⁇ M to 100 ⁇ M, although other effective amounts of antisense compounds outside this range may be found for specific antisense compounds.
  • One skilled in the art can readily test the relative effectiveness of any particular antisense oligonucleotide according to the vivo test of Matsukura, et al.
  • oligonucleotide concentrations of approximately 6 mM to 600 mM are required in the composition when 1 ml injections are to be administered.
  • Lower oligonucleotide compositions can be used with larger administration volumes.
  • Example II The antiinfective antisense oligonucleotide composition of
  • Example I is administered to HIV patients by any route effective to reduce viral activity.
  • the preferred route of administration is by intravenous injection.
  • the antisense composition may be administered multiple times a day to ensure that an effective amount of the antisense oligonucleotide is maintained.
  • Example III A gene therapy composition for treating an animal or human suffering from the effects of a defective or missing gene is made by combining a copolymer, such as CRL-8131 with a normal copy of the defective gene.
  • a copolymer such as CRL-8131
  • a gene therapy composition is made that contains a normal copy of the adenosine deaminase gene.
  • the gene therapy composition is made by mixing a copolymer prepared as described above in Example I with the desired gene, removing blood from the human or animal, transfecting blood cells with the ADA gene-containing composition, and reintroducing the transfected blood cells into the human or animal.
  • the introduced gene is expressed in vivo, alleviating the effects of the original gene deficiency.
  • Example III the gene therapy composition of Example III is combined with isolated T-lymphocytes to form T-lymphocytes containing the ADA gene.
  • the ADA gene-containing T-lymphocytes are subsequently administered, for example by injection, into the patient suffering from adenosine deaminase deficiency.
  • the administered cells express the ADA and produce adenosine deaminase, thus augmenting the supply of the enzyme in the patient and correcting the deficiency.
  • Example V DNA vaccination is carried out essentially as described for gene therapy in Examples III or IV, except that the gene that is introduced into the host expresses an antigenic gene product that is recognized as foreign by the host animal, thus eliciting an immune response.
  • Example VI A composition comprising copolymer CRL-8131 and an expression vector containing the gD gene of Herpes simplex virus type-1 was used in a transfection experiment.
  • DNA transfection normally is performed using standard calcium chloride and DEAE dextran precipitation techniques.
  • DEAE dextran is used to rough up the cell membrane and calcium is used to precipitate DNA onto the cell surface, facilitating DNA uptake into the cells. This procedure is generally toxic to the cells, however, and causes substantial cellular mortality.
  • Vero cells were incubated in DEAE dextran for 30 seconds. A mixture of copolymer and an expression vector containing glycoprotein gD DNA of Herpes simplex virus type-1 was added to Vero cells immediately after the removal of DEAE dextran. It was found that up to 40% of the cells were effectively transfected with the gD gene.
  • Example VII Other studies have also demonstrated that block copolymers are effective in transferring genetic material across cellular membranes in vivo. DNA vaccine-induced immunization was successful when an expression vector containing the gD gene of Herpes simplex virus type-1 combined with copolymer was injected intraperitoneally into rabbits every two weeks. Sera was collected and tested for the presence of anti-gD antibody. Low levels of anti-gD antibody were detected after 4 weeks of inoculation in this fashion. These results demonstrate that genetic material administered intraperitoneally with a copolymer delivery vehicle is taken up by cellstn vivo and expressed to give the gene product in quantities sufficient to elicit an immune response.
  • DNAse protection experiments Five different compounds (CRL 1122, 3362, 3632, 9352, and 8131) were used in experiments to test the degree of protection. DNA was mixed with compounds at 4°C, and after 15 min. at 37°C DNAse I (1 ⁇ l of 10 mg/ml solution) was added. After 30 min. of incubation at 37°C, DNAse I was removed by treatment with proteinase K (3 ⁇ l of 10 mg/ml solution). Controls were: DNAse I in the absence of nonionic block copolymer and DNA alone without any DNAse I treatment. DNA was protected from DNAse I degradation in all samples in which nonionic block copolymers were present. The best protection of DNA was achieved with CRL-3362 and 8131.
  • DNA copolymer compositions did not migrate in horizontal agarose electrophoresis and remained within the wells (stained with ethidium bromide). Effective protection against DNAse I action was achieved in solutions of 1 volume DNA solution (1 ⁇ g/ml) to 5 volumes of nonionic block copolymer (30 ⁇ g/ml). The estimated amount of protection varied from experiment to experiment and was estimated to be within 15-40% of total DNA. Additional experiments showed that DNA-copolymer compounds failed to transform E. coli competent cells via the calcium method. Phenol also failed to dissolve nonionic block copolymer away from DNA. DNA bound to NBC can be precipitated by adding 5 volumes of isopropyl alcohol.
  • Example IX Transfection experiments Typical transfection experiments for transient expression of herpes viral glycoprotein genes and other genes of interest involved the following procedure.
  • Cells such as COS (African monkey kidney cells; CV1) are seeded on 6-well plates. Transfection is performed when cells are 50-80% confluent (still in log growth phase). Cells are first washed with PBS buffer, they are incubated with 0.5 ml of DEAE-Dextran solution (500 mg/ml) for 1-2 minutes, this solution is aspirated and DNA precipitate is added to cells. DNA to be transfected is mixed for 30 min. at room temperature with CaCh at controlled pH conditions to form a fine precipitate.
  • DEAE-Dextran solution 500 mg/ml
  • This solution is mixed with 1 ml of growth medium (DMEM) and put onto cells for 4 hours at 37°C. At this time, the cells are shocked with 15% glycerol and subsequently washed with PBS. This osmotic shock facilitates the taking up of CaCl 2 -DNA precipitate into cells. Cells are then washed again with PBS, and incubated with growth media at 37°C for 48 hours.
  • DMEM growth medium
  • Gene expression is detected in most cases using specific monoclonal antibodies directed against the expressed proteins using indirect immunofluorescence.
  • the expressed proteins can be also labeled with radioactive tracers and immunoprecipitated or detected in westerns.
  • DNA alone, dextran alone, copolymer alone, and DNA plus dextran had negligible transfection of less than 0.2%.
  • the positive control of DNA plus dextran plus glycerol has transfection of 2% while various copolymers plus DNA were successful in transfecting DNA into cells up to 2.5 times better than the control, as shown in Table III:

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Abstract

L'invention concerne des compositions et des méthodes de traitement des maladies infectieuses et génétiques par thérapie génique ainsi que par administration intracellulaire d'oligonucléotides antisens ou d'autres séquences d'acides nucléiques. L'invention concerne une composition d'administration thérapeutique pouvant traiter un état pathologique. Cette composition contient un mélange d'apport d'une dose efficace d'un composé thérapeutique pouvant modifier la fonction de la séquence d'acides nucléiques, ainsi qu'une dose efficace d'un copolymère bloc représenté par la formule générale (I) ou (II), dans laquelle le poids moléculaire moyen global de la portion de copolymère octabloc représentée par polyoxypropyléne est compris entre environ 5000 et environ 7000 daltons; a représente un nombre tel que la portion représentée par polyoxyéthylène constitue entre environ 10 % et environ 40 % du composé en poids; et b représente un nombre tel que la portion de polyoxypropylène du poids moléculaire total du copolymère octabloc constitue entre environ 60 % et environ 90 % du composé en poids. Par ailleurs, l'invention concerne des compositions et des procédés faisant intervenir des copolymères bloc inverses actifs. Ces copolymères inverses comportent un noyau central de polyoxypropylène (POP) pourvu de part et d'autre de polyoxyéthylène (POE). Ces copolymères bloc inverses sont représentés par la formule (III) dans laquelle « b » représente un nombre tel que le poids moléculaire de l'hydrophobe (C3H6O)b est compris entre environ 2000 et 20 000, e « a » représentant un nombre tel que le pourcentage de l'hydrophile (C2H4O)a est compris entre environ 1 et 90 %.
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US09/919,504 US20020123476A1 (en) 1991-03-19 2001-07-31 Therapeutic delivery compositions and methods of use thereof
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US09/929,819 US6933286B2 (en) 1991-03-19 2001-08-14 Therapeutic delivery compositions and methods of use thereof
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US5234683A (en) * 1985-06-18 1993-08-10 Emory University Method of stimulating the immune system
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US6093391A (en) * 1992-10-08 2000-07-25 Supratek Pharma, Inc. Peptide copolymer compositions
US5656611A (en) * 1994-11-18 1997-08-12 Supratek Pharma Inc. Polynucleotide compositions
US6359054B1 (en) * 1994-11-18 2002-03-19 Supratek Pharma Inc. Polynucleotide compositions for intramuscular administration
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