WO2010132047A1 - Guanosine/gmp gels and uses thereof - Google Patents

Guanosine/gmp gels and uses thereof Download PDF

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Publication number
WO2010132047A1
WO2010132047A1 PCT/US2009/004189 US2009004189W WO2010132047A1 WO 2010132047 A1 WO2010132047 A1 WO 2010132047A1 US 2009004189 W US2009004189 W US 2009004189W WO 2010132047 A1 WO2010132047 A1 WO 2010132047A1
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composition
gel
guanosine
gmp
antibody
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PCT/US2009/004189
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French (fr)
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WO2010132047A8 (en
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Linda B. Mcgown
Yuehua Yu
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Rensselaer Polytechnic Institute
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Publication of WO2010132047A8 publication Critical patent/WO2010132047A8/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • 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
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • 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
    • A61K9/0043—Nose
    • 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
    • A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
    • A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the invention relates to guanosine/guanosine monophosphate-based compositions for the delivery of antibodies and other proteins, cells, nucleic acids, particles, and other agents, including controlled-release of the agent.
  • guanosine/guanosine monophosphate (GMP) gels that are useful for dispersing certain molecules (e.g., proteins, nucleic acids) and other agents (e.g., microparticles, nanoparticles) at high concentrations, e.g., for drug delivery. Accordingly, in some embodiments new approaches are provided for overcoming challenges associated with maintaining these agents at high concentrations, namely aggregation, agglomeration, decreased activity, and/or instability. In some embodiments, guanosine/GMP gels are provided that exhibit distinctive temperature-sensitive gelation properties and that are effective for a variety of storage and delivery applications.
  • guanosine/GMP gels are provided that are useful for antibody preparations, particularly high concentration antibody preparations. In other embodiments, guanosine/GMP gels are provided that are useful for nucleic acid preparations. In other embodiments, guanosine/GMP gels are provided that are useful for microparticle or nanoparticle preparations. In other embodiments, guanosine/GMP gels are provided that are useful for cell encapsulation preparations.
  • compositions are provided that are capable of delivering an agent, e.g., an antibody, over an extended period of time at a predetermined rate to a surrounding medium.
  • agent e.g., an antibody
  • guanosine/GMP gels which comprise guanosine and guanosine monophosphate, are provided that are biocompatible and that are capable of releasing agents over an extended period of time at a predetermined rate.
  • methods are provided for controlled release of agents in a subject from biocompatible guanosine/GMP gels.
  • a particularly important feature of certain embodiments of the guanosine/GMP compositions disclosed herein is the ability of these compositions to produce gels having certain agents, e.g., antibodies, nucleic acids, ligands, particles, cells, etc., dispersed randomly throughout the gel.
  • agents e.g., antibodies, nucleic acids, ligands, particles, cells, etc.
  • This result is unexpected in light of certain guanosine-based gels which, when prepared with carbon nanotubes, produce gels having agents non-randomly dispersed, e.g., organized in rod-like structures that are part of the organized gel backbone.
  • agents which are randomly dispersed is the guanosine/GMP gels disclosed herein exhibit less aggregation, agglomeration and instability, and maintain activity longer, than equivalent agents in preparations of the prior art.
  • compositions comprising an agent, e.g., antibody, an ligand, a nucleic acid, a particle, dispersed in a guanosine/GMP gel.
  • the agent is randomly dispersed in the gel.
  • the concentration of the agent in the gel is at or above a concentration that results in aggregation, agglomeration, degradation, or decreased activity of the agent in a control solvent, e.g., water or a combination of water and an organic solvent.
  • the invention in other aspect provides methods for producing a composition comprising an agent dispersed in a guanosine/GMP gel.
  • the methods involve dispersing an agent in a gel comprising guanosine and/or GMP.
  • the dispersing comprises mechanically agitating, e.g., by shaking, vortexing, sonicating or passing through a small orifice, a composition comprising the agent and constituents of the guanosine/GMP gel.
  • the agitation is performed under conditions in which gelation of the composition is minimized.
  • conditions of the composition are altered to promote gelation of the composition.
  • the invention in other aspect provides methods for producing a composition that effects controlled-release of an agent in a subject.
  • the methods involve dispersing an agent in a guanosine/GMP gel that is tuned to release the agent from the gel into a surrounding medium at a predetermined rate.
  • the methods involve tuning a guanosine/GMP gel to release an agent from the gel into a surrounding medium at a predetermined rate, and dispersing the agent in a guanosine/GMP gel.
  • a guanosine/GMP gel is tuned by setting a parameter of the gel to a level that results in a gel that releases the agent at the predetermined rate.
  • the parameter is selected from: pH, viscosity, ionic strength, cation concentration, nucleoside concentration, ratio of guanosine to GMP, and agent concentration.
  • the invention in other aspect provides methods for administering an agent to a subject. Typically the subject is in need of a therapeutic and/or diagnostic intervention that the agent achieves or facilitates. In certain embodiments, the subject has or is suspected of having a disease or condition that is treatable by the agent. In other embodiments, the subject has or is suspected of having a disease or condition that is diagnosable by the agent, e.g., by detecting, e.g., by imaging, the agent in the subject.
  • the methods involve administering to the subject a composition comprising an effective amount of an antibody dispersed in a guanosine/GMP gel.
  • the administering may be achieved by any suitable means, e.g., the administering may be performed orally, intravenously, intrapleurally, intranasally, intramuscularly, subcutaneously, intraperitoneally, or as an aerosol.
  • the administering involves implanting a guanosine/GMP gel comprising the agent in the subject.
  • the agent is administered in a guanosine/GMP composition, e.g. , a viscous liquid, that forms a gel upon administration to the subject.
  • the agent is administered in a guanosine/GMP gel that forms a liquid, e.g. , a viscous liquid, upon administration to the subject.
  • the agent is administered in a guanosine/GMP composition that is tuned to release the agent at a predetermined rate in the subject.
  • a "subject” is a mammal, including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate.
  • Subjects can be house pets (e.g., dogs, cats), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), zoo animals (e.g., lions, giraffes, etc.), but are not so limited.
  • Preferred subjects are human subjects.
  • the human subject may be a pediatric, adult or a geriatric subject.
  • the human subject may be of either sex.
  • animal refers to humans as well as non- human animals, including, for example, mammals, birds, reptiles, amphibians, and fish.
  • the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig).
  • a non-human animal may be a transgenic animal.
  • the terms “approximately” or “about” in reference to a number are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).
  • biocompatible refers to substances that are not toxic to cells.
  • a substance is considered to be “biocompatible” if its addition to cells in vivo does not induce inflammation and/or other adverse effects in vivo.
  • a substance is considered to be “biocompatible” if its addition to cells in vitro or in vivo results in less than or equal to about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or less than about 5% cell death.
  • the "effective amount" of an agent refers to an amount sufficient to elicit the desired therapeutic or diagnostic response.
  • the effective amount of an agent of the invention may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, the diagnostic application and the patient.
  • the effective amount of a compound with anti -proliferative activity is the amount that results in a sufficient concentration at the site of the tumor to kill or inhibit the growth of tumor cells.
  • the effective amount of a compound used to treat infection is the amount needed to kill or prevent the growth of the organism(s) responsible for the infection.
  • the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within an organism.
  • in vivo refers to events that occur within an organism.
  • a "peptide, " "polypeptide,” or “protein” comprises a polymer of amino acid residues linked together by peptide (amide) bonds.
  • the term(s), as used herein, refers to proteins, polypeptides, and peptide of any size, structure, or function. Typically, a peptide or polypeptide will be at least three amino acids long.
  • a peptide or polypeptide may refer to an individual protein or a collection of proteins. Proteins preferably contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed.
  • amino acids in a peptide or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc.
  • a peptide or polypeptide may also be a single molecule or may be a multi-molecular complex.
  • a peptide or polypeptide may be just a fragment of a naturally occurring protein or peptide.
  • a peptide or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof.
  • nucleic acid refers to a polymer of covalently linked nucleotide bases.
  • a nucleic acid is of unspecified length, therefore polynucleotides, oligonucleotides, genomes, genes, open reading frames (ORFs), plasmids, probes, primers, linkers, spacers and adaptors are included within the definition.
  • a nucleic acid can be of biologic and/or synthetic origin. The nucleic acid may be in single-stranded or double-stranded form. The single strand may be in sense or anti-sense orientation. Also included within the definition are nucleic acids having modified nucleotides.
  • nucleic acid embraces DNA, RNA or PNA (peptide nucleic acid), or a combination thereof.
  • guanosine refers to a nucleoside comprising guanine covalently linked to a ribose (ribofuranose) ring.
  • ribose ribofuranose
  • guanosine embraces guanosine and guanosine derivatives having a chemical modification involving the guanine base, ribose, or both the guanine base and the ribose.
  • GMP guanosine mono-phosphate
  • GMP refers to a guanosine nucleoside comprising a phosphate group.
  • GMP comprises a phosphate group, a ribose (ribofuranose) ring, and a guanine base.
  • GMP embraces various GMP derivatives having one or more modifications typically involving the guanine base, ribose, or both the guanine base and the ribose.
  • the phosphate may be at the 2', 3', or 5' position of the ribose sugar, for example.
  • Cyclic GMPs e.g., guanosine-2',3'-cyclicphosphates, and guanosines comprising a phosphite group, rather than a phosphate group, are also embraced.
  • guanosine/GMP gel or "G-gel” refers to a guanosine-based gel which typically comprises guanosine and guanosine monophosphate.
  • guanosine/GMP composition refers to any guanosine-based composition, e.g., a liquid, a viscous liquid, a gel, etc., which typically comprises guanosine and guanosine monophosphate.
  • agents refers to peptides, polypeptides, proteins, other large hydrophilic molecules, small molecule compounds, organic or inorganic compounds, polysaccharides, lipids, nucleic acids, particles, e.g., microparticles or nanopaiticles, antibodies, ligands, or combinations thereof that may be dispersed in a guanosine/GMP composition.
  • therapeutic agents refers to peptides, polypeptides, proteins, other large hydrophilic molecules, small molecule compounds, organic or inorganic compounds, polysaccharides, lipids, or nucleic acids that, when provided in an effective amount, produce a desired therapeutic response in a subject or cell.
  • Therapeutic agents may be administered to a subject alone, e.g., released from a guanosine/GMP compositions, or conjugated with another agent e.g., an antibody, ligand or particle, e.g., a microparticle or nanoparticle.
  • diagnostic agents refers to peptides, polypeptides, proteins, other large hydrophilic molecules, small molecule compounds, organic or inorganic compounds, polysaccharides, lipids, or nucleic acids that facilitate a diagnostic readout, e.g., an imaging read-out, in a subject. Diagnostic agents may be administered to a subject or cell alone, e.g., released from a guanosine/GMP compositions, or conjugated with another agent, e.g., an antibody, ligand or particle, e.g., a microparticle or nanoparticle. Typically, diagnostic agents are imaging contrast agents or X-ray detection agents.
  • Diagnostic agents include but are not limited to imaging agents which include commercially available agents used in positron emission tomography (PET), computer assisted tomography (CAT), single photon emission computerized tomography, x-ray, fluoroscopy, and magnetic resonance imaging (MRI). Diagnostic agent may comprise a radioactive isotope.
  • imaging agents which include commercially available agents used in positron emission tomography (PET), computer assisted tomography (CAT), single photon emission computerized tomography, x-ray, fluoroscopy, and magnetic resonance imaging (MRI). Diagnostic agent may comprise a radioactive isotope.
  • antibody refers to an immunoglobulin protein molecule that has the ability to bind a target molecule or antigen.
  • antibody also embraces antibody fragments, such as Fab and scFv fragments, that maintain the ability to specifically bind a target molecule or antigen.
  • Ligand refers to any molecule which is capable of specifically binding to a receptor on or in a target cell.
  • Ligands may be natural or synthetic.
  • Ligands may be agonists, antagonists or exhibit properties of both agonists and antagonists, e.g. , may be agonist-antagonists.
  • administer refers to implanting, applying, absorbing, ingesting, injecting, or inhaling, the inventive compositions.
  • treat refers to partially or completely alleviating, inhibiting, ameliorating, and/or relieving the disease or condition from which the subject is suffering.
  • Figure 1 shows freshly prepared antibody dispersed in water. Images are 90 ⁇ m x 90 ⁇ m.
  • Figure 2 shows freshly prepared antibody dispersed in a guanosine/GMP gel (G-gel). Images are 90 ⁇ m x 90 ⁇ m.
  • Figure 3 shows antibody dispersed in water after 25 min storage in ice
  • Figure 4 shows antibody dispersed in water (left) and G-gel (right) after
  • Image is 30 ⁇ m x 30 ⁇ m.
  • Figure 5 shows diffusion of fluorescein (greenish-yellow color) from G- gel into water for a viscous gel (tube at left in both photographs) and a solution-like gel (tube at right in both photographs). The photographs and the graph show the fluorescence of the water phase for both gels after 24 hrs and after 2 weeks.
  • Figure 6 shows diffusion of rhodamine dye (red-orange) from G-gel of varying composition and pH into water.
  • FIG 7 shows Atomic Force Microscopy (AFM) images of SWNTs solubilized in a guanosine/GMP gel.
  • the individual SWNTs (thin, approximately vertical structures) show parallel alignment.
  • the top image is an expanded view of the bottom image.
  • the x-axis is 1 ⁇ m in total length in the bottom image.
  • Figure 8 shows suspension of six different samples of TiO 2 samples (1-3 mg/mL) in gel medium immediately following preparation (top) and after two weeks (bottom).
  • Figure 9 shows (left) TiO 2 samples 1 and 5 (1-3 mg/mL) in water after
  • FIG. 10 shows AFM images of TiO 2 in water and in G-gel. Solutions were sonicated for 10 min, dropped on a silicon surface, and air dried overnight.
  • Images are 1 ⁇ m x 1 ⁇ m.
  • Figure 11 shows ZnO nanoparticles in water (left) and get (right) after 2 days.
  • the vials were inverted immediately before being photographed to show the separation of ZnO precipitate from water (left) and the separation of a liquid ZnO dispersion from a more viscous ZnO suspension in the gel solution (right).
  • Figure 12 shows AFM images of ZnO in water and in G-gel. Solutions were sonicated for 10 min., dropped on silicon surface, and air dried overnight. Images are 1 ⁇ m x 1 ⁇ m.
  • Figure 13 shows indium tin oxide (ITO) nanoparticles in gel (left) and water (right) after 1 day.
  • ITO indium tin oxide
  • Figure 14 shows AFM image of ITO in G-gel. The solution was sonicated for 10 min., dropped on silicon surface, and air dried overnight. Image is 1 ⁇ m x 1 ⁇ m. ITO in water yielded a surface that was too rough for imaging.
  • Figure 16 shows digital photomicrographic images of fibroblast cells seeded in 6-well plates in two different G-gels. Guo to GMP molar ratios and KCl concentration of the G-GeIs are indicated. Images are shown for time points of 1 and 2 days following seeding.
  • Figure 17 A shows the photomicrographic results of survival assays on rat aortic smooth muscle cells in G-gels using confocal microscopy.
  • Ethidium homodimer were used to indicate the live and dead cells, respectively.
  • Calcien stains the cytoplasm of live cells. The location of Calcien staining was denoted by green pixels. Ethidium homodimer stains the nuclei of dead cells. The location of
  • FIG. 17B shows the photomicrographic results of survival assays on cells in G-gels after 1 week. As in Figure 17A, assays were performed in triplicate.
  • Figure 18A shows PLGA in G-gel (left) and buffer (right) 5 min after 10 seconds of vortexing.
  • the transparent crystals in bottom of the vials are polymer crystals.
  • Figure 18B shows PLGA in G-gel (left) and buffer (right) after sonication for >2 h.
  • Guanosine/GMP gels have been discovered to disperse and increase the stability of certain agents, that typically aggregate and/or are less stable in other aqueous solutions. G-gels have been found to be particularly useful in preparing high concentration formulations of such agents.
  • the agent is an antibody.
  • the agent is a ligand.
  • the agent is a cell.
  • the agent is a particle, e.g., a microparticle or nanoparticle.
  • the agent is a nucleic acid.
  • the agent is a targeting agent, e.g., an antibody or ligand, conjugated to a therapeutic agent, e.g., a cytotoxic compound, or a diagnostic e.g., an imaging agent.
  • a targeting agent e.g., an antibody or ligand
  • a therapeutic agent e.g., a cytotoxic compound
  • a diagnostic e.g., an imaging agent.
  • Some embodiments herein relate to the ability of the inventive guanosine/GMP compositions to produce gels having certain agents, e.g. , antibodies, nucleic acids, ligands, particles, etc., dispersed randomly throughout the gel.
  • agents which are randomly dispersed in these guanosine/GMP gels exhibit less aggregation, agglomeration and instability, and maintain activity longer, than equivalent agents in preparations of the prior art, e.g., in certain preparations where such agents are non-randomly dispersed.
  • guanosine/GMP gels are provided that exhibit distinctive temperature sensitive gelation properties and that are effective for a variety of storage and delivery applications.
  • guanosine/GMP gels are provided that are useful for high concentration antibody preparations.
  • guanosine/GMP gels are provided that are useful for nucleic acid preparations.
  • guanosine/GMP gels are provided that are useful for microparticle or nanoparticle preparations.
  • drug delivery compositions are provided that are capable of delivering a drug, e.g., an antibody, over an extended period of time at a predetermined rate.
  • guanosine/GMP gels are provided that are biocompatible and that are capable of releasing an agent over an extended period of time at a predetermined rate.
  • methods are provided for controlled release of an agent in a subject from biocompatible guanosine/GMP gels.
  • Gels formed by individual guanosine derived compounds have been extensively studied (Gellert, M., et al., Proc. Natl. Acad. Sci. 1962, 48, 2013; Sasisekharan, V., and Zimmerman, S.; Davies, D.R. J. MoI. Biol. 1975, 92, 171; Proni, G., et al., Chem. Eur. J. 2000, 6, 3249; Walmsley, J.A. and Burnett, J.F.
  • individual guanosine derived compounds e.g., guanosine, L-guanosine, ara-guanosine, guanosine 5 '-monophosphate, etc.
  • G-quartets planar tetrameric structures
  • the basic building block is the G-quartet formed by Hoogsteen hydrogen bonding between each of four guanines and its two nearest neighbors.
  • An exemplary G-quartet is shown below. As the monomer concentration increases, the G-quartets can aggregate into columnar stacks through ⁇ - ⁇ interactions or, in the case of 5 '-guanosine monophosphate (GMP), into continuous, hydrogen-bonded helices.
  • GMP 5 '-guanosine monophosphate
  • the guanosine/GMP gels disclosed herein comprise guanosine and GMP compounds.
  • the guanosine is selected from the group consisting of: guanosine (Guo), L-guanosine, D-guanosine, ara-guanosine (guanine 9- ⁇ -D-arabinofuranoside), xylo-guanosine, guanosine-2 ',3'- isoproplidene, guanosine-5 '-sulfate, guanosine-5 '-chloride, N-methyl-guanosine, 8- bromo-guanosine, 8-iodo-guanosine, 8-bromo-D-guanosine, 8,2'-anhydro-guanosine, and isoguanosine.
  • the GMP is selected from the group consisting of: guanosine -2 '-monophosphate, guanosine-3' -monophosphate, guanosine- 5 '-monophosphate, guanosine-3 '(2')- monophosphate, guanosine-2', 3 '-cyclic phosphate, guanosine-2', 3'-cyclic phosphite, D-guanosine-5 '-monophosphate, 8- bromo-guanosine-5' -monophosphate, and 8-bromo-guanosine-3'-monophosphate.
  • guanosine and GMPs which may be used in the guanosine/GMP gels disclosed herein, will be apparent to the skilled artisan (See, e.g., Guschlbauer W, et al., Four- Stranded Nucleic Acid Structures 25 Years Later: From Guanosine Gels to Telomeric DNA, Journal of Biomolecular Structure and Dynamics, Vol. 8(3), 1990, the contents of which are incorporated herein by reference.)
  • the G-gel is made up of guanosine and guanosine-5 '-monophosphate as the only guanosine and GMP derivatives, respectively.
  • the G-gel may contain another guanosine or GMP derivative.
  • the G-gel may contain only guanosines.
  • the G-gel may contain only GMPs.
  • the G-gel does not contain a guanosine-5'-hydrazide.
  • the present invention relates to the use of a reversible, biocompatible guanosine/GMP gels (G-gels) to stabilize certain agents in non- aggregate form at a high concentration.
  • G-gels a reversible, biocompatible guanosine/GMP gels
  • These guanosine/GMP gels are aqueous mixtures of guanosine compounds of different solubilities, such as the relatively insoluble guanosine (Guo) and the highly soluble guanosine 5 '-monophosphate (GMP).
  • the properties of the inventive guanosine/GMP gels are highly tunable as a function of the total concentration of guanosine compounds, the ratio of the two different compounds, pH, ionic strength, and cation content.
  • the gels are thermodissociative (gel with decreasing temperature) while in others the gels are thermoassociative (liquid at lower temperatures and a gel at room or body temperature). Since the G-gels comprise simple compounds that occur naturally in humans, the G- gels are biocompatible and well suited to administration to humans and other animals.
  • Guanosine/GMP gels disclosed herein may be formed by mixing soluble guanosine 5 '-monophosphate (GMP) with insoluble guanosine (Guo). In such guanosine/GMP-gels, hydrophobic Guo facilitates the self-association of GMP while the hydrophilic GMP helps solubilize Guo in the aqueous solution.
  • the ratio of guanosine to GMP may be at a variety of different ranges.
  • the ratio of guanosine to GMP may be from about 0.01 to about 4, from about 0.05 to about 0.25.
  • the ratio of guanosine to GMP may be from about 0.01 to about 0.1, from about 0.02 to about 0.2, from about 0.03 to about 0.3, from about 0.04 to about 0.4, from about 0.05 to about 0.5, from about 0.06 to about 0.6, from about 0.07 to about 0.7, from about 0.08 to about 0.8, from about 0.09 to about 0.9, from about 0.1 to about 1, from about 0.2 to about 2, from about 0.3 to about 3, or from about 0.4 to about 4.
  • the ratio of guanosine to GMP is about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.9, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.2, about 1.4, about 1.6, about 1.8, about 2, about 4, or more.
  • the mole fraction of GMP (X GMP ) in a gel composition may be formulated at a variety of levels.
  • the mole fraction may be about 0.1 , about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or more.
  • the mole fraction of GMP ranges from about 0.4 to 0.5, from about 0.5 to about 0.6, from about 0.6 to about 0.7, from about 0.7 to about 0.8, or from about 0.8 to about 0.9.
  • the GMP in a gel composition may be formulated at a variety of concentrations.
  • the GMP concentration may range from up to about 1 mM to about 500 mM or from about 50 mM to about 300 mM or more.
  • the GMP concentration is up to about 10 mM, from about 10 mM to about 20 mM, from about 20 mM to about 30 mM, from about 30 mM to about 40 mM, from about 40 mM to about 50 mM, from about 50 mM to about 60 mM, from about 6OmM to about 70 mM, from about 70 mM to about 80 mM, from about 80 mM to about 90 mM, from about 90 mM to about 100 mM, from about 100 mM to about 110 mM, from about 110 mM to about 120 mM, from about 120 mM to about 130 mM, from about 130 mM to about 140 mM, from about 140 mM, from about 140 m
  • the GMP concentration is about 10 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500 mM, or more.
  • guanosine in a gel composition may be formulated at a variety of concentrations.
  • the guanosine concentration may range from up to about 0.1 mM to about 200 mM, from about 1 mM to about 100 mM, or from about 10 mM to about 70 mM.
  • the guanosine concentration is up to about 10 mM, from about 10 mM to about 20 mM, from about 20 mM to about 30 mM, from about 30 mM to about 40 mM, from about 40 mM to about 50 mM, from about 50 mM to about 60 mM, from about 60 mM to about 70 mM, from about 70 mM to about 80 mM, from about 80 mM to about 90 mM, from about 90 mM to about 100 mM, from about 100 mM to about 110 mM, from about 110 mM to about 120 mM, from about 120 mM to about 130 mM, from about 130 mM to about 140 mM, from about 140 mM to about 150 mM, from about 150 mM to about 160 mM, from about 160 mM to about 170 mM, from about 170 mM to about 180 mM, from about 180 mM to about 190
  • the guanosine concentration is about 0.01 mM, about 0.1 mM, about 0.5 mM, about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 200 mM, or more.
  • the gels of the present invention are formed by self- association of guanosine and guanosine 5 '-monophosphate that are solution at low temperatures and then become a firm gel at higher temperatures before melting at even higher temperatures.
  • low temperatures are below room temperature and are between 2-20 °C while room temperature is defined as between 20-27 °C and high temperatures are defined as those above room temperature, particularly above 35 °C, above 45 °C, above 55 °C, above 65 °C or above 100 0 C. It is noted that the actual transition temperatures will vary depending upon the specific gel composition, but typically the solution phase exists below room temperature, the gel exits at and above room temperature, and the higher temperature melting occurs above 40-50 0 C.
  • guanosine/GMP gels may form at up to about 0 °°C.
  • guanosine/GMP gels may form within a range of from 0 0 C to about 5 0 C.
  • guanosine/GMP gels may form within a range of from 15 °C to about 25 °C.
  • guanosine/GMP gels may form within a range of from 35 0 C to about 40 °C or more.
  • Gelation at any particular temperature, constituent concentration, ratio, etc. may also depend on pH.
  • acidic pH may favor gelation for some formulations
  • a basic pH may favor gelation for other formulations.
  • the pH of a guanosine/GMP gel composition is up to about 6.5.
  • the pH of a guanosine/GMP gel composition ranges from about 6.5 and about 7.5.
  • the pH of a guanosine/GMP gel composition is above 7.5.
  • the pH of a guanosine/GMP gel composition may range from about 2.0 to about 2.5, from about 2.5 to about 3.0, from about 3.0 to about 3.5, from about 3.5 to about 4.0, from about 4.0 to about 4.5, from about 4.5 to about 5.0, from about 5.0 to about 5.5, from about 5.5 to about 6.0, from about 6.0 to about 6.5, from about 6.5 to about 7.0, from about 7.0 to about 7.5, from about 7.5 to about 8.0, from about 8.0 to about 8.5, from about 8.5 to about 9.0, from about 9.0 to about 9.5, from about 9.5 to about 10.0, from about 10.0 to about 10.5, from about 10.5 to about 1 1.0, from about 11.0 to about 11.5, or from about 11.5 to about 12.
  • the pH of a guanosine/GMP gel composition ranges from about 6.0 to about 6.2, from about 6.2 to about 6.4, from about 6.4 to about 6.6, from about 6.6 to about 6.8, from about 6.8 to about 7.0, from about 7.0 to about 7.2, from about 7.2 to about 7.4, from about 7.4 to about 7.6, from about 7.6 to about 7.8, or from about 7.8 to about 8.0.
  • Guanosine/GMP gel compositions in particular, comprise one or more cations.
  • Exemplary cations include, but are not limited to, Li + , Na + , K + , Rb + , Cs + , NH 4 + , Ag + , Ti + , Mg + *, Ca + ⁇ Sr + *.
  • N(CH 3 ) 4+ and Ba + ⁇ Cations may be provided at a variety of concentrations including for example up to about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 300
  • the concentration of the cation ranges from about 10 mM to about 30 mM, from about 30 mM to about 60 mM, from about 60 mM to about 90 mM, from about 90 mM to about 120 mM, from about 120 mM to about 150 mM, from about 150 mM to about 180 mM, from about 180 mM to about 210 mM, from about 210 mM to about 240 mM, from about 240 mM to about 270 mM, from about 270 mM to about 300 mM, from about 350 mM to about 400 mM, or from about 400 to about 500 mM.
  • Guanosine/GMP gel compositions comprise one or more anions.
  • exemplary anions include, but are not limited to, halide ions including F, Cl, Br and I and non-halide anions including for example NO 2 , NO 3 , OCH 3 , CH 3 COO, OH, O, CO 3 , SO 4 , HSO 4 , PO 4 , H 2 PO 4 and HPO 4.
  • Anions may be provided at a variety of concentrations including for example up to about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 1 10 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, or more.
  • the concentration of the anion ranges from about 10 mM to about 30 mM, from about 30 mM to about 60 mM, from about 60 mM to about 90 mM, from about 90 mM to about 120 mM, from about 120 mM to about 150 mM, from about 150 mM to about 180 mM, from about 180 mM to about 210 mM, from about 210 mM to about 240 mM, from about 240 mM to about 270 mM, from about 270 mM to about 300 mM, from about 350 mM to about 400 mM, or from about 400 to about 500 mM.
  • a guanosine/GMP gel may comprise ammonium salt(s), e.g., (NH 4 ) 2 SO 4 , (NH 4 ) 3 PC> 4 , sodium salt(s), e.g., Na 3 PO 4 , NaCl, and/or a potassium salt(s), e.g., KCl.
  • the guanosine/GMP gel comprises about 50 mM to about 200 mM of Na 3 PO 4 and about 50 mM to about 200 mM of KCl.
  • the guanosine/GMP gels may further comprise one or more buffering agents.
  • Formulations of guanosine/GMP gels may be selected based on the desired properties of the gel, e.g., the predetermined rate of release of an agent, the temperature of gelation, etc.
  • the skilled artisan will be capable of selecting appropriate formulations to achieve a gel composition with a desired functional characteristic.
  • Figure 15 (reproduced from Yu Y, et al., Tunable Thermoassociation of Binary Guanosine Gels, J. Phys.
  • Guanosine/GMP gels comprising Therapeutic and Diagnostic Agents
  • aspects of the invention provide guanosine/GMP gels that exhibit distinctive temperature sensitive gelation properties and that are effective for a variety of molecular storage and therapeutic and diagnostic delivery applications.
  • the guanosine/GMP gels disclosed herein are useful for high concentration antibody preparations.
  • the guanosine/GMP gels disclosed herein are also useful for nucleic acids, e.g., siRNA, microRNA, DNA.
  • Guanosine/GMP gels may also be used for microparticle, nanoparticle, or other particles, e.g., particles conjugated with, or encapsulating, therapeutic or diagnostic agents.
  • the guanosine/GMP gels disclosed herein e.g., gels comprising guanosine and guanosine 5 '-monophosphate (GMP)
  • GMP guanosine/guanosine 5 '-monophosphate
  • the agent is randomly dispersed in the guanosine/GMP gels.
  • a concentration of an agent in the gel that is at or above a concentration that results in aggregation, agglomeration, degradation, or decreased activity of the agent in a conventional formulation.
  • Any one of a number of techniques known in the art may be used to assess aggregation, agglomeration, degradation, or activity of an agent stored in a gel preparation of the invention. Often, this assessment involves an evaluation of aggregation, agglomeration, degradation, or activity of an equivalent agent stored in a control solvent.
  • control solvent is an aqueous solution, such as, for example, water or a mixture of water and an organic solvent, such as, for example, methanol, ethanol, isopropanol, n- propanol, tert-butyl alcohol, n-butyl alcohol, ethyl acetate, and acetonitrile.
  • organic solvent such as, for example, methanol, ethanol, isopropanol, n- propanol, tert-butyl alcohol, n-butyl alcohol, ethyl acetate, and acetonitrile.
  • This solvent list in not limiting and other appropriate solvents may be used.
  • antibodies including fragments thereof
  • a broad range of concentrations of the antibody may be achieved in the guanosine/GMP gel.
  • antibodies may be maintained in guanosine/GMP gels at a concentration ranging from about 0.1 mg/ml to about 100 mg/ml or more, from about 1 mg/ml to about 25 mg/ml, or from about 5 mg/ml to about 15 mg/ml.
  • the concentration of the antibody in guanosine/GMP gels is from about 0.1 mg/ml to about 10 mg/ml, from about 10 mg/ml to about 20 mg/ml, from about 20 mg/ml to about 30 mg/ml, from about 30 mg/ml to about 40 mg/ml, from about 40 mg/ml to about 50 mg/ml, from about 50 mg/ml to about 60 mg/ml, from about 60 mg/ml to about 70 mg/ml, from about 70 mg/ml to about ⁇ O mg/ml, from about 80 mg/ml to about 90 mg/ml, from about 90 mg/ml to about 100 mg/ml, from about 100 mg/ml to about 110 mg/ml, from about 110 mg/ml to about 120 mg/ml, from about 120 mg/ml to about 130 mg/ml, from about 130 mg/ml to about 140 mg/ml, from about 140 mg/ml to about 150 mg/ml, from about 150 mg/ml, from about
  • the concentration of the antibody in guanosine/GMP gels is about 0.1 mg/ml, about 1 mg/ml, about 10 mg/ml, about 20 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 110 mg/ml, about 120 mg/ml, about 130 mg/ml, about 140 mg/ml, about 150 mg/ml, about 160 mg/ml, about 170 mg/ml, about 180 mg/ml, about 190 mg/ml, about 200 mg/ml, about 250 mg/ml, or more. These concentrations are not intended to be limiting and other appropriate ranges are envisioned.
  • compositions that are capable of delivering an agent, e.g., an antibody, over an extended period of time at a predetermined rate to a subject from a biocompatible guanosine/GMP gel.
  • an antibody may serve as an agent, e.g., therapeutic or diagnostic agent, e.g., an antibody that binds specifically to and inhibits activity of a disease associated molecule.
  • an antibody refers to antibodies, including for example polyclonal, monoclonal, chimeric, and humanized antibodies, and fragments of antibodies that specifically bind to antigens.
  • the antibody is a monoclonal antibody.
  • the antibody is a polyclonal antibody.
  • the antibody is a humanized antibody.
  • Antibodies may comprise an immunoglobulin of any class, e.g., IgG, IgM, IgA, IgD, IgE.
  • an immunoglobulin of any class e.g., IgG, IgM, IgA, IgD, IgE.
  • an antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody.
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule.
  • Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd.
  • the Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
  • CDRs complementarity determining regions
  • FRs framework regions
  • CDRl through CDR3 complementarity determining regions
  • Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti- mouse antibody (HAMA) responses when administered to humans.
  • HAMA human anti- mouse antibody
  • the present invention also provides for F(ab')2, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDRl and/or CDR2 regions have been replaced by homologous human or non-human sequences.
  • Therapeutic antibodies may bind to, and inhibit the activity of, a protein of a variety of disease relevant signaling pathways, including for example Integrin, Androgen Receptor, B Cell Receptor, EGFRl, IL-2, IL-4, IL-6, Kit, Notch, T Cell Receptor, TGF ⁇ , TNF- ⁇ , or WNT.
  • Abciximab for example, is a platelet aggregation inhibitor that targets the glycoprotein Ilb/IIIa receptor on the platelet membrane and is mainly used during and after coronary artery procedures like angioplasty to prevent platelets from sticking together and causing thrombus formation within the coronary artery.
  • Adalimumab is another example that binds to TNF ⁇ , preventing it from activating TNF receptors and is useful as an immunosuppressant for treating a variety of inflammatory conditions, including for example Crohn's disease.
  • the antibody is selected from the group consisting of: Abciximab, Adalimumab, Alemtuzumab, Basiliximab, Bevacizumab, Cetuximab, Daclizumab, Eculizumab, Efalizumab, Gemtuzumab, Ibritumomab tiuxetan, Infliximab, Muromonab-CD3, Natalizumab, Omalizumab, Palivizumab, Panitumumab, Ranibizumab, Rituximab, Tositumomab, and Trastuzumab.
  • the antibody is an antibody that binds specifically to an antigen of the foregoing list of antibodies.
  • the antibody binds to a cell surface receptor, including for example a cell surface receptor selected from the group consisting of insulin receptor (insulin), insulin-like growth factor receptor (including both IGF-I and IGF-2), growth hormone receptor, glucose transporters (particularly GLUT 4 receptor), transferring receptor (transferring), epidermal growth factor receptor (EGF), low density lipoprotein receptor, high density lipoprotein receptor, leptin receptor, estrogen receptor (estrogen); interleukin receptors including IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, 1L-9, IL-11, IL-12, IL-13, IL-15, and IL-17 receptors, human growth hormone receptor, vascular endothelial growth factor (“VEGF”) receptor, platelet- derived growth factor (“PDGF”) receptor, transforming growth factor receptor (including TGF
  • VEGF vascular endotheli
  • the antibody binds to a G-protein coupled receptor, including for example a G-protein coupled receptor selected from the group consisting of: ⁇ l A-adrenergic receptor, ⁇ lB-adrenergic receptor, ⁇ 2-adrenergic receptor, ⁇ 2B-adrenergic receptor, ⁇ l -adrenergic receptor, ⁇ 2-adrenergic receptor, ⁇ 3- adrenergic receptor, ml acetylcholine receptor (AChR), m2 AChR, m3 AChR, m4 AChR, m5 AChR, Dl dopamine receptor, D2 dopamine receptor, D3 dopamine receptor, D4 dopamine receptor, D5 dopamine receptor, Al adenosine receptor, A2b adenosine receptor, 5-HTla, 5-HTlb, 5HTl-like, 5-HTld, 5HTld-like, 5HTId beta, substance K
  • Antibodies may also be conjugated to other therapeutic or diagnostic agents, e.g., cytotoxic compound, toxins, small molecules, nucleic acids, or radioisotopes. Such antibodies may be useful for targeting certain therapeutic agents to specific cell types, e.g., cancer cells.
  • cytotoxic compounds which may be conjugated to a therapeutic antibody include for example, calicheamicin, esperamicin, methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin C, cisplatinum, etopside, bleomycin and 5-fluorouracil. Still other cytotoxic compounds are disclosed herein and will be apparent to the skilled artisan.
  • a ligand e.g., a natural or synthetic ligand
  • an agent e.g., a therapeutic or diagnostic agent.
  • the ligand is all or a portion (e.g. a binding portion) of a ligand for a cell surface receptor.
  • Suitable ligands include, but are not limited to, all or a functional portion of the ligands that bind to a cell surface receptor selected from the group consisting of a G-protein-coupled receptor, insulin receptor (insulin), insulin-like growth factor receptor (including both IGF-I and IGF -2), growth hormone receptor, glucose transporters (particularly GLUT 4 receptor), transferring receptor (transferring), epidermal growth factor receptor (EGF), low density lipoprotein receptor, high density lipoprotein receptor, leptin receptor, estrogen receptor (estrogen); interleukin receptors including IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 11, IL-12, IL-13, IL-15, and IL-17 receptors, human growth hormone receptor, vascular endothelial growth factor (“VEGF”) receptor, platelet-derived growth factor (“PDGF”) receptor, transforming growth factor receptor (including TGF- ⁇ and TGF- ⁇ ), erythro
  • Hormone ligands are also envisioned. Hormones include both steroid hormones and proteinaceous hormones, including, but not limited to, epinephrine, thyroxine, oxytocin, insulin, thyroid- stimulating hormone, calcitonin, chorionic gonadotropin, cortictropin, follicle- stimulating hormone, glucagon, leuteinizing hormone, lipotropin, melanocyte- stimutating hormone, norepinephrine, parathyroid hormone, thyroid-stimulating hormone (TSH), vasopressin, enkephalins, seratonin, estradiol, progesterone, testosterone, cortisone, and glucocorticoids and the hormones listed above.
  • Receptor ligands include ligands that bind to receptors such as cell surface receptors, which include hormones, lipids, proteins, glycoproteins, signal transducers, growth factors, cytokines, and others.
  • Ligands may also be conjugated to other therapeutic or diagnostic agents, e.g., cytotoxic compound, toxins, small molecules, nucleic acids, or radioisotopes. Such antibodies may be useful for targeting certain therapeutic agents to specific cell types, e.g., cancer cells.
  • a particle may serve as an agent, e.g., therapeutic or diagnostic agent.
  • the term “particle” refers to any particle, in terms of composition, shape, and size, that is suitable for delivery of an agent to a subject or cell.
  • a "microparticle” is a particle having an average diameter on the order of micrometers (e.g., between about 1 micrometer and about 1 mm), while a “nanoparticle” is a particle having an average diameter on the order of nanometers (e.g., between about 1 nm and about 1 micrometer).
  • Particles may have any shape or size.
  • particles may have an average diameter of less than about 5 mm or 2 mm, or less than about 1 mm, or less than about 500 microns, less than about 200 microns, less than about 100 microns, less than about 60 microns, less than about 50 microns, less than about 40 microns, less than about 30 microns, less than about 25 microns, less than about 10 microns, less than about 3 microns, less than about 1 micron, less than about 300 nm, less than about 100 nm, less than about 30 nm, or less than about 10 nm.
  • the particles may be spherical or non-spherical.
  • the average diameter of a non-spherical particle is the diameter of a perfect sphere having the same volume as the non-spherical particle.
  • the particles may be formed of any suitable material, depending on the application.
  • the particles may comprise a glass, and/or a polymer such as polyethylene, polystyrene, silicone, polyfluoroethylene, polyacrylic acid, a polyamide (e.g., nylon), polycarbonate, polysulfone, polyurethane, polybutadiene, polybutylene, polyethersulfone, polyetherimide, polyphenylene oxide, polymethylpentene, polyvinylchloride, polyvinylidene chloride, polyphthalamide, polyphenylene sulfide, polyester, polyetheretherketone, polyimide, polymethylmethacylate and/or polypropylene.
  • a polymer such as polyethylene, polystyrene, silicone, polyfluoroethylene, polyacrylic acid, a polyamide (e.g.
  • the particles may comprise a ceramic such as tricalcium phosphate, hydroxyapatite, fluorapatite, aluminum oxide, or zirconium oxide.
  • the particles may be formed from biocompatible and/or biodegradable polymers such as polylactic and/or polyglycolic acids, polyanhydride, polycaprolactone, polyethylene oxide, polybutylene terephthalate, starch, cellulose, chitosan, proteins, carbohydrates, and/or combinations of these.
  • the particles may comprise a hydrogel, such as agarose, collagen, fibrin.
  • the particles may include a magnetically susceptible material in some cases, e.g. , a material displaying paramagnetism or ferromagnetism.
  • the particles may include iron, iron oxide, magnetite, hematite, or some other compound containing iron.
  • the particles can include a conductive material (e.g., a metal such as titanium, copper, platinum, silver, gold, tantalum, palladium, rhodium, etc.), or a semiconductive material (e.g., silicon, germanium, CdSe, CdS, etc.).
  • Other particles include ZnS, ZnO, ITO, TiO 2 , AgI, AgBr, HgI 2 , PbS, PbSe, ZnTe, CdTe, In 2 S 3 , In 2 Se 3 , Cd 3 P 2 , Cd 3 As 2 , InAs, or GaAs.
  • the particles are conjugated to a therapeutic agent, including for example peptide, polypeptide, protein drugs, other large hydrophilic molecules, small molecule compounds, and nucleic acids.
  • a therapeutic agent including for example peptide, polypeptide, protein drugs, other large hydrophilic molecules, small molecule compounds, and nucleic acids.
  • microparticles or nanoparticles encapsulate a therapeutic agent, including for example peptide, polypeptide, protein drugs, other large hydrophilic molecules, small molecule compounds, and nucleic acids.
  • the particle is conjugated to a targeting agent, e.g., an antibody or ligand.
  • Agents may also be nucleic acids.
  • Nucleic acids include, but are not limited to, RNA, DNA, peptide nucleic acids (PNA), and combinations thereof.
  • Nucleic acids include expression vectors for delivering exogenous gene products, e.g., cDNA, non-protein coding RNAs, and short interfering nucleic acids (siNA) that include, for example: microRNA (miRNA), short interfering RNA (siRNA), double- stranded RNA (dsRNA), and short hairpin RNA (shRNA) molecules, for example.
  • miRNA microRNA
  • siRNA short interfering RNA
  • dsRNA double- stranded RNA
  • shRNA short hairpin RNA
  • siNA of the instant invention can be chemically synthesized, expressed from a vector or enzymatically synthesized.
  • the disclosure also features various chemically- modified synthetic short interfering nucleic acid molecules capable of modulating gene expression or activity in cells by RNA interference (RNAi).
  • RNAi RNA interference
  • the use of chemically- modified siNA improves various properties of native siNA molecules through, for example, increased resistance to nuclease degradation in vivo and/or through improved cellular uptake.
  • siNA having multiple chemical modifications may retain RNAi activity.
  • the siNA molecules of the instant invention provide useful reagents and methods for a variety of therapeutic applications.
  • oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'amino, 2'-C-allyl, 2'-flouro, 2'-O-methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry , 35, 14090).
  • nuclease resistant groups for example, 2'amino, 2'-C-allyl, 2'-flouro, 2'-O-methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry , 35,
  • nucleic acids known in the art may also serve as therapeutic agents in the instant invention.
  • triple helix approaches may be used to mediate sequence-specific gene suppression.
  • Triple helix forming oligonucleotides have been found in some cases to bind in a sequence-specific manner (Postel et al., Proc. Natl. Acad. Sci. U.S.A. 88(18):8227-31, 1991; Duval-Valentin et al., Proc. Natl. Acad. Sci. U.S.A. 89(2):504-8, 1992; Hardenbol and Van Dyke Proc. Natl. Acad. Sci. U.S.A.
  • RNA aptamers may also be used to, e.g., inhibit the activity of target a protein of interest.
  • Nucleic acid molecules of the invention may be provided in any one of a number of different vectors.
  • a "vector” may be any of a number of nucleic acid molecules into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell.
  • Vectors are typically composed of DNA although RNA vectors are also available.
  • Vectors include, but are not limited to, plasmids, phagemids and virus genomes or portions thereof.
  • An expression vector is one into which a desired sequence may be inserted, e.g., by restriction and ligation such that it is operably joined to regulatory sequences ⁇ e.g., a promoter sequence, enhancer sequence) and may be expressed as an RNA transcript.
  • Vectors may further contain one or more marker sequences suitable for use in the identification of cells that have or have not been transformed or transfected with the vector.
  • Markers include, for example, genes encoding proteins that increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes that encode enzymes whose activities are detectable by standard assays known in the art ⁇ e.g., ⁇ -galactosidase or alkaline phosphatase), and genes that visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques ⁇ e.g., green fluorescent protein).
  • a coding sequence and regulatory sequences are said to be "operably” joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences.
  • two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.
  • a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.
  • a coding sequence need not encode a protein but may instead, for example, encode a functional RNA such as an shRNA.
  • the precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5 '-non-transcribed and 5 '-non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.
  • Such 5 '-non-transcribed regulatory sequences will include a promoter region that includes a promoter sequence for transcriptional control of the operably joined gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired.
  • the vectors of the invention may optionally include 5' leader or signal sequences.
  • the choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.
  • One of skill in the art will be aware of appropriate regulatory sequences for expression of interfering RNA, e.g., shRNA, miRNA, etc.
  • an siNA is an shRNA, shRNA-mir, or microRNA molecule encoded by and expressed from a plasmid-based expression vector.
  • a nucleic acid is a transgene incorporated in a plasmid-based expression vector that encodes a small-interfering nucleic acid.
  • Such transgenes and expression vectors can employ either polymerase II or polymerase III promoters to drive expression of these shRNAs and result in functional siRNAs in cells. The former polymerase permits the use of classic protein expression strategies, including inducible and tissue-specific expression systems.
  • transgenes and expression vectors are controlled by tissue specific promoters.
  • transgenes and expression vectors are controlled by inducible promoters, such as tetracycline inducible expression systems.
  • a small interfering nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • the recombinant mammalian expression vector may be capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the myosin heavy chain promoter, albumin promoter, lymphoid-specific promoters, neuron specific promoters, pancreas specific promoters, and mammary gland specific promoters.
  • a virus vector for delivering a nucleic acid molecule is selected from the group consisting of adenoviruses, adeno-associated viruses, poxviruses including vaccinia viruses and attenuated poxviruses, Semliki Forest virus, Venezuelan equine encephalitis virus, retroviruses, Sindbis virus, and Ty virus-like particle.
  • replication-defective adenoviruses e.g., Xiang et al., Virology 219:220-227, 1996; Eloit et al., J. Virol. 7:5375-5381, 1997; Chengalvala et al., Vaccine 15:335-339,
  • Venzuelan equine encephalitis virus (Davis et al., J. Virol. 70:3781-3787, 1996), Sindbis virus (Pugachev et al., Virology 212:587-594, 1995), lentiviral vectors (Naldini L, et al., Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11382-8) and Ty virus-like particle (Allsopp et al., Eur. J. Immunol 26: 1951-1959, 1996).
  • Another virus useful for certain applications is the adeno-associated virus, a double-stranded DNA virus.
  • the adeno-associated virus is capable of infecting a wide range of cell types and species and can be engineered to be replication-deficient. It further has advantages, such as heat and lipid solvent stability, high transduction frequencies in cells of diverse lineages, including hematopoietic cells, and lack of superinfection inhibition thus allowing multiple series of transductions.
  • the adeno- associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression.
  • wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno-associated virus can also function in an extrachromosomal fashion.
  • Non-cytopathic viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest.
  • Non-cytopathic viruses include certain retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA.
  • the retroviruses are replication- deficient (i.e., capable of directing synthesis of the desired transcripts, but incapable of manufacturing an infectious particle).
  • retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • a cell e.g., a mammalian cell, e.g., a human cell
  • a cell may serve as an agent, e.g., a therapeutic agent.
  • the cells may be any mammalian cells.
  • the cells may be any human cells.
  • the cells may be selected from the group consisting of Lymphocytes, B cells, T cells, cytotoxic T cells, natural killer T cells, regulatory T cells, T helper cells, myeloid cells, granulocytes, basophil granulocytes, eosinophil granulocytes, neutrophil granulocytes, hypersegmented neutrophils, monocytes, macrophages, reticulocytes, platelets, mast cells, thrombocytes, megakaryocytes, dendritic cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, oxyphil cells, adrenal cells, chromaffin cells, pineal cells, pinealocytes, glial cells, glioblasts, astrocytes, oligodendrocytes, microglial cells, magnocellular neurosecretory cells, stellate cells, boettcher cells; pituitary cells, gonadotropes, corticotropes, thyrot
  • the cells may be of mesenchymal, ectodermal, and endodermal origin.
  • the cells may be selected from the group consisting of cord-blood cells, stem cells, embryonic stem cells, adult stem cells, progenitor cells, induced progenitor cells, autologous cells, isograft cells, allograft cells, xenograft cells, and genetically engineered cells.
  • agents disclosed herein may be dispersed in the guanosine/GMP gel compositions alone or, preferably, conjugated to another agent, e.g., an antibody, a ligand, a nucleic acid, or a particle, e.g. microparticle or nanoparticle.
  • therapeutic agents having molecular weights less than 100,000 Da, more preferably less than 20,000 Da, and most preferably less than 2,000 Da, advantageously may be used in the compositions and methods of the present invention, e.g., alone or conjugated to particles or antibodies.
  • therapeutic agents include, for example, physiologically active materials or medicinal drugs, such as agents affecting the central nervous system, anti-allergic agents, cardiovascular agents, agents affecting respiratory organs, agents affecting digestive organs, hormone preparations, agents affecting metabolism, cytotoxic agents, antibiotic preparations, chemotherapeutics, antimicrobials, local anesthetics, antihistaminics, antiphlogistics, astringents, vitamins, antifungal agents, peripheral nervous anesthetics, vasodilators, crude drug essences, tinctures, crude drug powders, hypotensive agents, nucleic acids or immunosuppressants.
  • Antibodies e.g., antibodies conjugated with other therapeutic agents, e.g., a cytotoxic compound, a radioisotope, or a nucleic acid, may also be considered therapeutic agents.
  • Cytokines suitable for use in the compositions and methods of the present invention include, for example, interferons (IFNs), tumor necrosis factors (TNFs), interleukins, colony stimulating factors (CSFs), growth factors such as osteogenic factor extract (OFE), epidermal growth factor (EGF), transforming growth factor (TGF) alpha, TGF- ⁇ (including any combination of TGF- ⁇ s), TGF- ⁇ l, TGF- ⁇ 2, platelet derived growth factor (PDGF-AA, PDGF-AB, PDGF-BB), acidic fibroblast growth factor (FGF), basic FGF, connective tissue activating peptides (CTAP), ⁇ -thromboglobulin, insulin-like growth factors, erythropoietin (EPO), nerve growth factor (NGF), bone morphogenic protein (BMP), osteogenic factors, and others.
  • IFNs interferons
  • TNFs tumor necrosis factors
  • CSFs colony stimulating factors
  • OFF colony stimulating factors
  • therapeutic agents which may be used in the methods and compositions disclosed herein include, for example, oxytocin, vasopressin, adrenocorticotrophic hormone (ACTH), transforming growth factor antagonists, prolactin, luliberin or luteinizing hormone releasing hormone (LH-RH), LH-RH agonists or antagonists, growth hormone, growth hormone releasing factor, insulin, somatostatin, bombesin antagonists, glucagon, interferon, gastrin, tetragastrin, pentagastrin, urogastrone, secretin, calcitonin, enkephalins, endomorphins, angiotensins, renin, bradykinin, bacitracins, polymyzins, colistins, tyrocidin, gramicidines, monoclonal antibodies, soluble vaccines, and synthetic analogues, modifications and pharmaceutically-active fragments thereof.
  • ACTH adrenoc
  • Polypeptides can be natural or synthetic.
  • suitable therapeutic agents for use in the invention include antiinflammatory agents such as hydrocortisone and prednisone; antibacterial agents such as penicillin, cephalosporins, and bacitracin; antiparasitic agents such as quinacrine, and chloroquine; antifungal agents such as nystatin, and gentamicin; antiviral agents such as acyclovir, ribavirin, and interferons; antineoplastic agents such as methotrexate, 5-fluorouracil, and adriamycin, and tumor necrosis factor; analgesic agents such as salicylic acid, acetaminophen, ibuprofen, flurbiprofen, and morphine; local anesthetics such as lidocaine, bupivacaine, and benzocaine; vaccines such as hepatitis, influenza, measles, rubella, tetanus, poli
  • cytotoxic compounds that may be suitable as therapeutic agents in the methods and compositions disclosed herein include, but are not limited to, the following: Antineoplastic agents such as: Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Adriamycin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Buniodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubi
  • antibiotics that may be suitable as therapeutic agents in the methods and compositions disclosed herein include, but are not limited to, the following: Aminoglycosides, such as Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Streptomycin, Tobramycin, Paromomycin; Ansamycins such as Geldanamycin and Herbimycin; the Carbacephem, Loracarbef; Carbapenems such as Ertapenem, Doripenem, Imipenem, and Meropenem; Cephalosporins, such as Cefadroxil, Cefazolin, Cefalotin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceft
  • radioactive molecules may be used as therapeutic and/or diagnostic agents in the compositions and methods disclosed herein.
  • exemplary radioactive agents for use in the gels include but are not limited to Fibrinogen I 125; Fludeoxyglucose F18; Fluorodopa F 18; Insulin I 125; Insulin 1 131; Iobenguane I 123; Iodipamide Sodium I 131; Iodoantipyrine 1 131; Iodocholesterol 1 131; Iodohippurate Sodium I 123; Iodohippurate Sodium I 125; Iodohippurate Sodium I 131 ; Iodopyracet I 125; Iodopyracet 1 131 ; Iofetamine Hydrochloride I 123; Iomethin I 125; Iomethin I 131; Iothalamate Sodium I 125; Iothalamate Sodium I 131 ; Iotyrosine 1 131 ;
  • Radioisotopes may also be conjugated to agents, e.g., antibodies, to create therapeutic and diagnostic agents, e.g., radiolabel antibody cancer therapies. Radioisotopes may be selected from the group consisting of: 225 Ac, 11 At, 21 Bi, ' Bi,
  • contrast agents in MRI include but are not limited to the gadolinium chelates currently available, such as diethylene triamine pentacetic acid (DTPA) and gadopentotate dimeglumine, as well as iron, magnesium, manganese, copper and chromium.
  • gadolinium chelates currently available, such as diethylene triamine pentacetic acid (DTPA) and gadopentotate dimeglumine, as well as iron, magnesium, manganese, copper and chromium.
  • Examples of materials useful for CAT and x-rays include iodine based materials for intravenous administration, such as ionic monomers typified by diatrizoate and iothalamate, non-ionic monomers such as iopamidol, isohexyl, and ioversol, non-ionic dimers, such as iotrol and iodixanol, and ionic dimers, for example, ioxagalte.
  • iodine based materials for intravenous administration such as ionic monomers typified by diatrizoate and iothalamate, non-ionic monomers such as iopamidol, isohexyl, and ioversol, non-ionic dimers, such as iotrol and iodixanol, and ionic dimers, for example, ioxagalte.
  • An agent may be dispersed in a guanosine/GMP gel in a variety of ways.
  • an agent is first suspended or diluted in an aqueous solution comprising guanosine and GMP, which may also comprise appropriate salts and buffering agents.
  • guanosine and/or GMP may be suspended or diluted in an aqueous solution comprising an agent.
  • the suspension or dilution is typically performed under conditions, e.g., at a temperature, ionic strength, and pH, in which gelation of the solution is minimized, e.g., in which the solution remains a liquid.
  • the agent Prior to addition to the aqueous solution, the agent may be in a solid form, e.g., as a lyophilized solid, or in a liquid form, e.g., in an aqueous solution.
  • the solution is maintained under conditions, e.g., at a temperature, ionic strength, and pH, in which gelation of the solution is minimized, and is mechanically agitated to disperse the agent throughout the solution.
  • the mechanical agitation may be accomplished by any one of a variety of methods disclosed herein and known in the art.
  • mechanical agitation may be accomplished by shaking, vortexing, sonicating or passing through a small orifice the composition comprising the agent, guanosine, and GMP.
  • the conditions of the composition comprising the agent, guanosine, and GMP are altered to facilitate gelation of the guanosine and GMP.
  • gelation may be induced by adjusting the pH of the composition, e.g., by decreasing the pH.
  • Gelation may also be induced by adjusting the temperature of the composition, e.g., by decreasing the temperature.
  • the agitation is performed at a temperature in a range of about 15 °C to about 25 °C and gelation is brought about following agitation by cooling the composition to a temperature below about 15 °C, e.g., to a range of about 0 °C to about 10 0 C.
  • Gelation may also be induced by adjusting the ionic strength of the composition, e.g., by addition of salts. For example, the addition of potassium cations is known to promote G-quartet formation.
  • guanosine/GMP gels it is often desirable to control the release rate of certain molecules (e.g., diagnostic agents or therapeutic agents) that are dispersed within the inventive guanosine/GMP gels disclosed herein, e.g., for delivery of the molecules or therapeutic agents to a subject. It may be desirable for this release rate to occur over several days, weeks, or even months. Also, it may be desirable to have a release rate that is controlled and gradual over this time period, with zero order kinetics or time independent release being most preferred and desirable. [00123] Accordingly, methods are provide for tuning the guanosine/GMP gels to achieve a predetermined release rate, which is typically a rate desirable for a particular application, e.g., for bringing about desired therapeutic or diagnostic result.
  • a predetermined release rate which is typically a rate desirable for a particular application, e.g., for bringing about desired therapeutic or diagnostic result.
  • Tuning of guanosine/GMP gels typically comprises setting a parameter of the gel to a level that results in a gel that releases the agent at the predetermined rate.
  • parameters include, for example, pH, viscosity, ionic strength, cation concentration, nucleoside concentration, ratio of guanosine to GMP, and agent concentration.
  • the methods typically include an evaluation or determination of the level of particular parameter that results in a gel that releases the agent at the predetermined rate.
  • the examples section provides exemplary tuning methods, e.g., based on pH levels. Other tuning methods can be achieved using methods well known in the art.
  • the level of particular parameter that results in a gel that releases an agent e.g., an antibody, a ligand, a nucleic acid, a particle, at the predetermined rate may be determined by preparing a gel at a first test level of the parameter; determining a rate of release of an agent from the gel at the first test level; preparing a gel at a second test level of the parameter; determining a rate of release of an agent from the gel at the second test level; and evaluating the rates of release of the agent at the first and second test levels to establish a level of the parameter that results in a gel that releases the agent at the predetermined rate.
  • an agent e.g., an antibody, a ligand, a nucleic acid, a particle
  • the methods are not limited to evaluating only two test levels and that any number of test levels may be evaluated to establish appropriate parameter levels for tuning gels.
  • the rate of release of the agent from the gel may be determined by maintaining the gel in a medium, e.g., a aqueous solution, a buffer solution, a tissue, in vivo or ex vivo, for a predetermined time, and measuring the concentration of the agent in the medium on one or more occasions during the predetermined time.
  • the predetermined rate is typically a rate of release of an agent from the gel into a medium that maintains, for a predetermined time, a concentration of the agent, in the medium, that is at or above a concentration of the agent in a subject which effects a desired therapeutic response in the subject.
  • the predetermined rate is typically a rate of release of an agent from the gel into a medium that maintains, for a predetermined time, a concentration of the agent, in the medium, that is at or above a concentration of the agent in a subject which facilitates a desired diagnostic read-out, e.g., an imaging read-out, in the subject.
  • the predetermined rate may be 50% of the agent in the gel being released from the gel within up to about 1 day. In other applications, the predetermined rate may be 50% of the agent in the gel being released from the gel within a range of about 1 day to about 30 days. In other applications, the predetermined rate may be 50% of the agent in the gel being released from the gel within a range of about 1 week to about 26 weeks. In still other applications, the predetermined rate may be 50% of the agent in the gel being released from the gel within a range of up to about 1 month to about 12 months.
  • a "therapeutically effective amount” is an amount of a compound or composition that produces a desired therapeutically beneficial result in a subject.
  • a therapeutically effective amount can refer to any compounds, e.g., therapeutic agents, or compositions described herein.
  • the therapeutically effective amount of the active agent to be included in pharmaceutical compositions depends, in each case, upon several factors, e.g., the type, size and condition of the patient to be treated, the intended mode of administration, the capacity of the patient to incorporate the intended dosage form, etc.
  • an amount of active agent is included in each dosage form to provide from about 0.1 to about 250 mg/kg, and preferably from about 0.1 to about 100 mg/kg.
  • compositions comprise compounds or compositions that have therapeutic utility, and a pharmaceutically acceptable carrier, i.e., that facilitate delivery of compounds or compositions, in a therapeutically effective amount.
  • the disclosure in other embodiments provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • container(s) can be various written materials (written information) such as instructions (indicia) for use, or a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • written information such as instructions (indicia) for use
  • a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • compositions of this invention are said to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by a recipient patient.
  • Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier.
  • suitable carriers are well-known in the art. See, for example, Remingon's Pharmaceutical Sciences, 18th Ed. (1990). [00130] It will be understood by those skilled in the art that any mode of administration, vehicle or carrier conventionally employed and which is inert with respect to the active agent may be utilized for preparing and administering the pharmaceutical compositions of the present invention.
  • the pharmaceutical compositions of the present invention preferably contain a pharmaceutically acceptable carrier or excipient suitable for rendering the compound or mixture administrable orally as a tablet, capsule or pill, or parenterally, intravenously, intradermally, intramuscularly or subcutaneously, or transdermally.
  • the active ingredients may be admixed or compounded with any conventional, pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical compositions may also comprise suitable buffering agents.
  • the pharmaceutical composition of the invention are also produced under sterile conditions using Good Manufacturing Practices (GMP).
  • compositions disclosed herein may also be formulated with pharmaceutically acceptable salts.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.
  • compositions disclosed herein may be administered by any suitable means such as orally, intranasally, subcutaneously, intramuscularly, intravenously, intra-arterially, parenterally, intraperitoneally, intrathecally, intratracheally, ocularly, sublingually, vaginally, rectally, dermally, or as an aerosol.
  • compounds of the invention may, for example, be inhaled, ingested or administered by systemic routes.
  • parenteral includes subcutaneous, intravenous, intramuscular, intraperitoneal, and intrasternal injection, or infusion techniques.
  • the particular mode selected will depend, of course, upon the particular composition selected, the particular condition being treated and the dosage required for therapeutic efficacy.
  • the methods, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces acceptable levels of efficacy without causing clinically unacceptable adverse effects.
  • the guanosine/GMP gel compositions may also be implanted, e.g., within, partially or completely, or adjacent to a target tissue, e.g., a diseased tissue, to effect local delivery of a therapeutic or diagnostic agent to the tissue.
  • Guanosine (Guo) and guanosine 5 '-monophosphate (GMP) were purchased in solid form from Sigma Aldrich.
  • lyophilized anti-goat IgG antibody powder produced from rabbit containing sodium chloride and sodium phosphate buffer crystals at pH 7.2 was applied.
  • the sample was 1.7 mg lyophilized antibody powder (containing 0.6 mg protein and 1.1 mg Na 3 PO 4 ZNaCl buffer crystal) diluted into 50 ⁇ L guanosine/GMP gel composed of 0.25 M GMP and 0.020 M Guo, which gave a final antibody concentration of 12 mg/mL and buffer concentration of 0.10 M Na 3 PO 4 , 0.15 M KCl.
  • the control was the same amount of lyophilized antibody powder diluted into 50 ⁇ L HPLC-grade water, which gave the same final antibody and buffer concentrations.
  • the same media can be used to stabilize and deliver other proteins such as enzymes, ligands, nucleic acids and receptors, and for peptide release as well.
  • IgG antibody labeled with atto-540 fluorescence dye was added into each vial and vortexed for 15 seconds to ensure even distribution. This served as a secondary antibody to the anti-goat IgG antibody powder produced from rabbit and provided a fluorescent signal.
  • Antibody aggregation was tested using a Zeiss LSM 510 Meta laser scanning confocal microscope equipped with a Meta detector that can capture emissions ranging from 411-754 run, in 10.7 nm bins across 32 channels.
  • the XlOO oil objective was used and the 518 F oil was applied between the sample slice and objective to achieve the best light transmission.
  • the 1 mW 543 nm green line from a HeNe laser was applied as excitation with an output at around 90% and the detector was set with gain at around 700.
  • Figures 1 shows two randomly selected, representative spots of freshly prepared antibody in water.
  • Figure 2 shows two randomly selected, representative spots of fresh prepared antibody in the G-gel.
  • Figure 3 shows the antibody in water after 25 min storage in ice and
  • Figure 3 shows antibody in G-gel after 30 min storage in ice.
  • Figure 4 shows images of a different sample of antibody dispersed in H 2 O (left) and G-gel (right) after 3 h storage in the refrigerator. The preparation and microscopy were performed identically to the samples used in Figures 1-3.
  • Example 2 Extended Release Delivery
  • the guanosine/GMP gels are able to be tuned for timed release of pharmaceutical compounds.
  • we made solutions of the fluorescent dye fluorescein in different G-gel formulations added a layer of water on top of the gel, and observed the diffusion of the fluorescein dye from the gel into the water.
  • fluorescein is released much faster from the low viscosity G-gel than high viscosity G-gel, demonstrating the tunability of the G-gel to specify the rate of release.
  • a second set of experiments we tested the effects of different G-gel conditions including pH and G-gel composition on diffusion of a different fluorescent dye, Rhodamine, from the G-gel into the water layer.
  • both pH and composition are important determinants of diffusion rate and they can be combined to tailor delivery for specific conditions for drug delivery. For example, diffusion tends to be slow at acidic pH and rapid at basic pH, especially at the lower Guo content. If a drug in this gel were ingested orally, it would not diffuse in the acidic environment of the stomach but would be released once it reached the higher pH environment of the intestines.
  • G-gel composition could be optimized for different administration routes as well, for example, to control the rate of release of a drug that is delivered intravenously into the near-neutral pH of blood.
  • Example 3 Nanoparticle Dispersion in G-gelsfor " Nanopharmaceuticals”
  • Inorganic nanoparticles have low solubility in aqueous solution and high tendency to agglomerate.
  • Existing methods generally involve surface treatment to stabilize the particles in aqueous solution.
  • Drawbacks are that these approaches are time consuming, they add to the production cost, the processes may not be scalable, and the modifications may limit the product applications.
  • the G-gels discussed in this paper are capable of dispersing individual, unmodified, inorganic nanoparticles such as TiO 2 , ITO, and ZnO in suspensions that are stable for weeks to years. The mechanism is not yet understood but appears to be a random encapsulation of the particles by the gel.
  • Inorganic nanoparticles could be used to fine-tune the G-gel media for protein dispersion and drug delivery. Further, the nanoparticles could be used to form nanostructured proteins and drugs within the G-gel media to enable delivery on a nano- scale. The facile, individual dispersion of nanoparticles in G-gels will provide a simple and scalable means for development and implementation of "nanopharmaceuticals”. [00144] Without being bound by a particular theory, the mechanism of solubilization appears to be encapsulation of the particles by the gel through nonspecific interactions.
  • the nanoparticles show no evidence of alignment or other non-random organization; this indicates a mechanism of solubilization and dispersion that is different than that observed in the carbon nanotube containing gels noted above.
  • the nanoparticles are randomly dispersed in the G-gel without actually becoming a part of the G-gel architecture itself. The nanoparticles are merely encapsulated in random sites in the bulk G-gel medium and are therefore more readily released from the gel simply by changing the properties of the bulk solution such as pH.
  • ITO Indium tin Oxide
  • TiO 2 is the mostly used white pigment and has applications in wide-ranging areas including paints and coatings, sunscreens and solar cells.
  • ITO nanoparticles can be utilized to prepare transparent conductive film and are widely used in touch screens.
  • Quantum dots have numerous applications in chemical and biological imaging and analysis as well as nanostructured devices. Inorganic nanoparticles are difficult to handle due to their low solubility in aqueous solution and high tendency to agglomerate.
  • Existing methods generally involve surface treatment to stabilize the particles in aqueous solution.
  • the result is a medium in which the carbon nanotubes are selectively aligned in the gel.
  • the nanoparticles are spherical and show no evidence of alignment or other organization, indicating a different mechanism of solubilization and dispersion.
  • the gel medium is nontoxic and biocompatible. It is formed by simple, commercial compounds in aqueous solution.
  • the properties such as viscosity and thermal stability of the nanoparticle suspensions can be tuned by varying the composition of the gel medium. For example, the temperature response of the suspensions can be tuned so that they transition from liquid to gel with increasing or decreasing temperature to facilitate handline, formation of homogeneous dispersions, injectability, etc.
  • the rate of gelation can be controlled as well using thermal, pH or cation gradients.
  • nanoparticle dispersion The following are specific examples of nanoparticle dispersion:
  • Figure 8 shows dispersion of 6 different samples of TiO 2 (mean diameter approximately 6-8 run) in the aqueous gel medium immediately following 30 s sonication at 0 ° C and the same coated with ZnO 2 and/or Al 2 O 3 .
  • the results demonstrate the long-term stability of the aqueous suspensions.
  • Figure 9 shows suspensions of TiO 2 samples 1 and 5 in water prepared using the same protocol as for the gel suspensions. The photograph was taken 3 days after sample preparation. The TiO 2 begins to precipitate within 12 hours following sonication.
  • Figure 9 also shows a side-by-side comparison OfTiO 2 sample 1 in water and in the gel 3 days following preparation. The TiO 2 has precipitated out of the water but remains dispersed in the gel.
  • Figure 10 shows AFM images Of TiO 2 that was sonicated for 10 min in water and in the gel, dropped on a silicon substrate and air dried overnight.
  • the TiO 2 exists as large aggregates in water but is highly dispersed as small (individual) particles in the gel.
  • Figure 11 shows untreated ZnO nanoparticles prepared in water and in gel after 2 days.
  • the vials were inverted for the photograph in order to show phase separation in both vials.
  • the solution at the bottom of the inverted vial contains essentially no nanoparticles, which remained instead as a precipitate at the top of the inverted vial and coating the surrounding vial wall.
  • gel there is a highly viscous phase at the top of the inverted vial that contains nanoparticles as well.
  • Figure 12 shows AFM images of ZnO in water and in gel, prepared as described above for AFM images of TiO 2 . Again, the particles show heavy aggregation and clumping in water but are much more individually dispersed in the gel.
  • Figure 13 shows ITO (approximately 50 nm mean diameter) prepared in water and in gel after 1 day. The nanoparticles have begun to precipitate from the water solution but remain dispersed in the gel.
  • Figure 14 shows an AFM image of the ITO in gel. The particles are well-dispersed and discrete. The ITO in water was too clumped and rough to be imaged by AFM.
  • Example 5 Binary Guanosine Gels For Encapsulation Of Living Cells
  • Certain in vivo applications involve cells being encapsulated in a G-GeI in a form that establishes a boundary between the cells and a host organism. Encapsulation may sequester the living cells and protect them from degradation and immunological attack by the host organism while sustaining them during the course of their desired function.
  • a cell encapsulating G-gel will be prepared with consideration of the following attributes:
  • Selective permeability e.g., allowing exchange of small ions and molecules (oxygen, nutrients, electrolytes, hormones, wastes) with the external surroundings while preventing transfer of antigenic macromolecules to the host and immunogenic responders such as antibodies and T-cells from the host
  • capsules may be scaled up in size without losing mechanical stability and selective permeability.
  • G-gels Reversible guanosine gels as biomaterials for encapsulation of living cells are disclosed.
  • G-gels are self-assembled networks of hydrogen-bonded guanine tetrads formed by guanosine nucleosides and nucleotides. Their organization and viscosity are dependent upon monomer concentration, temperature, pH and cation content, providing multiple variables for controlling and modulating the properties of the gels.
  • thermoassociative G-gels formed by mixtures of guanosine and guanosine monophosphate in certain proportions that have the potential to overcome certain challenges that have limited the success of other materials for cell encapsulation:
  • Cells can be readily and homogeneously incorporated into the G-gels simply by adding them to refrigerated solutions and bringing the resulting mixtures to room or physiological temperature.
  • the negatively charged surface of the G-gels will inhibit adhesion and adsorption of host cells and proteins.
  • G-gels are formed by small molecules that occur naturally in the body and may therefore be more easily eliminated from the body and less likely to trigger immune responses.
  • the G-gel scaffold offers higher regularity that may provide better control of "pore” size and cell distribution compared to randomly cross-linked polymers.
  • the G-gels are formed from simple compounds that are readily available at high purity and relatively low cost, unlike gels formed from alginates and other biomaterials derived from natural sources that may require purification, adding to their cost, and are difficult to standardize, to standardize.
  • G-gels are stable at neutral pH and their stability increases with decreasing pH, which makes them excellent candidates for certain uses in the gastrointestinal tract.
  • G-gels are highly responsive to temperature and pH, as well as specific cations such as K+, providing several independent variables for manipulation of gelation.
  • thermoassociative G-gels incorporate and sustain living cells.
  • the fibroblast cells first were cultured on standard tissue culture plastic in 50/50 Dulbecco's Modified Eagle Medium (DMEMf) with 10% fetal bovine serum, 10 U/ml Penicillin and 100 ⁇ g/ml Streptomycin, and 2 mM L-glutamine at pH 7.2., and were passaged at confluence.
  • DMEMf Dulbecco's Modified Eagle Medium
  • Example 6 Evaluation of PLGA in G-gels
  • Poly(DL-lactide-co-glycolide) (PLGA) (average Mw 5,000-15,000, lactide:glycolide (50:50), Sigma- Aldrich) was dissolved at a concentration of 1 mg/mL in G-gel (0.25 M GMP, 0.02 M Guo), and 0.04 M phosphate buffer (pH 7.4) as a control.
  • the solutions were gently vortexed for ⁇ 10 s and monitored for precipitation for 1 h post solubilization. Initially, no precipitation of polymer was observed in G-gel or control buffer, however, precipitation was apparent within 5 min post solubilization.
  • Figure 18A shows precipitation in both G-gel (left) and buffer (right) 5 min after solubilization.
  • the precipitation of polymer crystals indicated poor dispersability of PLGA in G-gels.

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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The invention relates in some aspects to guanosine/GMP gel compositions for delivering antibodies, nucleic acids, and particles. In other aspects the invention relates to guanosine/GMP gel compositions and methods of use thereof for controlled-release of antibody, nucleic acid, and micro- and nano-particles.

Description

GUANOSINE/GMP GELS AND USES THEREOF
RELATED APPLICATIONS
[0001] The present application claims priority under 35 U.S.C. § 1 19(e) to U.S. provisional application, U.S.S.N. 61/216,183, filed May 14, 2009, and U.S. provisional application, U.S.S.N. 61/216,510, filed May 15, 2009, both of which are incorporated herein by reference in their entireties.
FIELD OF INVENTION
[0002] The invention relates to guanosine/guanosine monophosphate-based compositions for the delivery of antibodies and other proteins, cells, nucleic acids, particles, and other agents, including controlled-release of the agent.
BACKGROUND
[0003] According to recent reports there are nearly 200 monoclonal antibodies in human clinical trials as therapeutic agents [1-2]. The therapeutic effectiveness of antibodies often requires that they exist in monomeric form, since aggregation reduces the therapeutic effectiveness and the presence of aggregates may lead to allergic reactions, hypersensitivity, and anaphylaxis [1-3]. Aggregation may also complicate administration due to clogging of intravenous lines and filters [2-3]. Since therapeutic antibodies typically are formulated at high antibody concentrations, there is a pressing need for new formulations that will stabilize antibodies and reduce aggregation in these high-concentration therapeutics. Similarly, there is a need for new formulations for maintaining other agents, including nucleic acids and particles, e.g., microparticles and nanoparticles, at high concentrations for storage and related delivery applications.
SUMMARY OF THE INVENTION
[0004] Aspects of the invention are based in part on the discovery of guanosine/guanosine monophosphate (GMP) gels that are useful for dispersing certain molecules (e.g., proteins, nucleic acids) and other agents (e.g., microparticles, nanoparticles) at high concentrations, e.g., for drug delivery. Accordingly, in some embodiments new approaches are provided for overcoming challenges associated with maintaining these agents at high concentrations, namely aggregation, agglomeration, decreased activity, and/or instability. In some embodiments, guanosine/GMP gels are provided that exhibit distinctive temperature-sensitive gelation properties and that are effective for a variety of storage and delivery applications. In certain embodiments, guanosine/GMP gels are provided that are useful for antibody preparations, particularly high concentration antibody preparations. In other embodiments, guanosine/GMP gels are provided that are useful for nucleic acid preparations. In other embodiments, guanosine/GMP gels are provided that are useful for microparticle or nanoparticle preparations. In other embodiments, guanosine/GMP gels are provided that are useful for cell encapsulation preparations.
[0005] Other aspects of the invention are based in part on the discovery of guanosine/GMP gels that are capable of releasing agents over an extended period of time at a predetermined rate. Accordingly, in some embodiments, pharmaceutical compositions are provided that are capable of delivering an agent, e.g., an antibody, over an extended period of time at a predetermined rate to a surrounding medium. In some embodiments, guanosine/GMP gels, which comprise guanosine and guanosine monophosphate, are provided that are biocompatible and that are capable of releasing agents over an extended period of time at a predetermined rate. In other embodiments, methods are provided for controlled release of agents in a subject from biocompatible guanosine/GMP gels.
[0006] A particularly important feature of certain embodiments of the guanosine/GMP compositions disclosed herein is the ability of these compositions to produce gels having certain agents, e.g., antibodies, nucleic acids, ligands, particles, cells, etc., dispersed randomly throughout the gel. This result is unexpected in light of certain guanosine-based gels which, when prepared with carbon nanotubes, produce gels having agents non-randomly dispersed, e.g., organized in rod-like structures that are part of the organized gel backbone. Furthermore, in some embodiments, agents which are randomly dispersed is the guanosine/GMP gels disclosed herein exhibit less aggregation, agglomeration and instability, and maintain activity longer, than equivalent agents in preparations of the prior art.
[0007] The invention in some aspect provides compositions comprising an agent, e.g., antibody, an ligand, a nucleic acid, a particle, dispersed in a guanosine/GMP gel. In particular embodiments, the agent is randomly dispersed in the gel. In certain embodiments, the concentration of the agent in the gel, is at or above a concentration that results in aggregation, agglomeration, degradation, or decreased activity of the agent in a control solvent, e.g., water or a combination of water and an organic solvent.
[0008] The invention in other aspect provides methods for producing a composition comprising an agent dispersed in a guanosine/GMP gel. In some embodiments, the methods involve dispersing an agent in a gel comprising guanosine and/or GMP. In some embodiments, the dispersing comprises mechanically agitating, e.g., by shaking, vortexing, sonicating or passing through a small orifice, a composition comprising the agent and constituents of the guanosine/GMP gel. In some embodiments, the agitation is performed under conditions in which gelation of the composition is minimized. In certain embodiments, following the agitation, conditions of the composition are altered to promote gelation of the composition. [0009] The invention in other aspect provides methods for producing a composition that effects controlled-release of an agent in a subject. In some embodiments, the methods involve dispersing an agent in a guanosine/GMP gel that is tuned to release the agent from the gel into a surrounding medium at a predetermined rate. In some embodiments, the methods involve tuning a guanosine/GMP gel to release an agent from the gel into a surrounding medium at a predetermined rate, and dispersing the agent in a guanosine/GMP gel. In particular embodiments, a guanosine/GMP gel is tuned by setting a parameter of the gel to a level that results in a gel that releases the agent at the predetermined rate. In certain embodiments, the parameter is selected from: pH, viscosity, ionic strength, cation concentration, nucleoside concentration, ratio of guanosine to GMP, and agent concentration. [0010] The invention in other aspect provides methods for administering an agent to a subject. Typically the subject is in need of a therapeutic and/or diagnostic intervention that the agent achieves or facilitates. In certain embodiments, the subject has or is suspected of having a disease or condition that is treatable by the agent. In other embodiments, the subject has or is suspected of having a disease or condition that is diagnosable by the agent, e.g., by detecting, e.g., by imaging, the agent in the subject. In some embodiments, the methods involve administering to the subject a composition comprising an effective amount of an antibody dispersed in a guanosine/GMP gel. The administering may be achieved by any suitable means, e.g., the administering may be performed orally, intravenously, intrapleurally, intranasally, intramuscularly, subcutaneously, intraperitoneally, or as an aerosol. In particular embodiments, the administering involves implanting a guanosine/GMP gel comprising the agent in the subject. In some embodiments, the agent is administered in a guanosine/GMP composition, e.g. , a viscous liquid, that forms a gel upon administration to the subject. In other embodiments, the agent is administered in a guanosine/GMP gel that forms a liquid, e.g. , a viscous liquid, upon administration to the subject. In particular embodiments, the agent is administered in a guanosine/GMP composition that is tuned to release the agent at a predetermined rate in the subject.
DEFINITIONS
[0011] As used herein, a "subject" is a mammal, including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate. Subjects can be house pets (e.g., dogs, cats), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), zoo animals (e.g., lions, giraffes, etc.), but are not so limited. Preferred subjects are human subjects. The human subject may be a pediatric, adult or a geriatric subject. The human subject may be of either sex.
[0012] The term "animal", as used herein, refers to humans as well as non- human animals, including, for example, mammals, birds, reptiles, amphibians, and fish. Preferably, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig). A non-human animal may be a transgenic animal.
[0013] As used herein, the terms "approximately" or "about" in reference to a number are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).
[0014] As used herein, the term "biocompatible" refers to substances that are not toxic to cells. In some embodiments, a substance is considered to be "biocompatible" if its addition to cells in vivo does not induce inflammation and/or other adverse effects in vivo. In some embodiments, a substance is considered to be "biocompatible" if its addition to cells in vitro or in vivo results in less than or equal to about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or less than about 5% cell death. [0015] As used herein, the "effective amount" of an agent refers to an amount sufficient to elicit the desired therapeutic or diagnostic response. As will be appreciated by those of ordinary skill in this art, the effective amount of an agent of the invention may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, the diagnostic application and the patient. For example, the effective amount of a compound with anti -proliferative activity is the amount that results in a sufficient concentration at the site of the tumor to kill or inhibit the growth of tumor cells. The effective amount of a compound used to treat infection is the amount needed to kill or prevent the growth of the organism(s) responsible for the infection. [0016] As used herein, the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within an organism.
[0017] As used herein, the term "in vivo" refers to events that occur within an organism.
[0018] A "peptide, " "polypeptide," or "protein" comprises a polymer of amino acid residues linked together by peptide (amide) bonds. The term(s), as used herein, refers to proteins, polypeptides, and peptide of any size, structure, or function. Typically, a peptide or polypeptide will be at least three amino acids long. A peptide or polypeptide may refer to an individual protein or a collection of proteins. Proteins preferably contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in a peptide or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A peptide or polypeptide may also be a single molecule or may be a multi-molecular complex. A peptide or polypeptide may be just a fragment of a naturally occurring protein or peptide. A peptide or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. [0019] As used herein, the term "nucleic acid" refers to a polymer of covalently linked nucleotide bases. A nucleic acid is of unspecified length, therefore polynucleotides, oligonucleotides, genomes, genes, open reading frames (ORFs), plasmids, probes, primers, linkers, spacers and adaptors are included within the definition. A nucleic acid can be of biologic and/or synthetic origin. The nucleic acid may be in single-stranded or double-stranded form. The single strand may be in sense or anti-sense orientation. Also included within the definition are nucleic acids having modified nucleotides. Modifications in the bases of the nucleic acid may be made, and bases such as Inosine may be incorporated. Other modifications may involve, for example, modifications of the backbone. The term nucleic acid embraces DNA, RNA or PNA (peptide nucleic acid), or a combination thereof.
[0020] The term "guanosine" ("Guo"), as used herein, refers to a nucleoside comprising guanine covalently linked to a ribose (ribofuranose) ring. The term guanosine embraces guanosine and guanosine derivatives having a chemical modification involving the guanine base, ribose, or both the guanine base and the ribose.
[0021] The term "guanosine mono-phosphate" ("GMP"), as used herein, refers to a guanosine nucleoside comprising a phosphate group. Accordingly, GMP comprises a phosphate group, a ribose (ribofuranose) ring, and a guanine base. The term GMP embraces various GMP derivatives having one or more modifications typically involving the guanine base, ribose, or both the guanine base and the ribose. The phosphate may be at the 2', 3', or 5' position of the ribose sugar, for example. Cyclic GMPs, e.g., guanosine-2',3'-cyclicphosphates, and guanosines comprising a phosphite group, rather than a phosphate group, are also embraced. [0022] The term "guanosine/GMP gel" or "G-gel" refers to a guanosine-based gel which typically comprises guanosine and guanosine monophosphate. [0023] The term "guanosine/GMP composition" refers to any guanosine-based composition, e.g., a liquid, a viscous liquid, a gel, etc., which typically comprises guanosine and guanosine monophosphate.
[0024] The term "agents" refers to peptides, polypeptides, proteins, other large hydrophilic molecules, small molecule compounds, organic or inorganic compounds, polysaccharides, lipids, nucleic acids, particles, e.g., microparticles or nanopaiticles, antibodies, ligands, or combinations thereof that may be dispersed in a guanosine/GMP composition.
[0025] The term "therapeutic agents" refers to peptides, polypeptides, proteins, other large hydrophilic molecules, small molecule compounds, organic or inorganic compounds, polysaccharides, lipids, or nucleic acids that, when provided in an effective amount, produce a desired therapeutic response in a subject or cell. Therapeutic agents may be administered to a subject alone, e.g., released from a guanosine/GMP compositions, or conjugated with another agent e.g., an antibody, ligand or particle, e.g., a microparticle or nanoparticle.
[0026] The term "diagnostic agents" refers to peptides, polypeptides, proteins, other large hydrophilic molecules, small molecule compounds, organic or inorganic compounds, polysaccharides, lipids, or nucleic acids that facilitate a diagnostic readout, e.g., an imaging read-out, in a subject. Diagnostic agents may be administered to a subject or cell alone, e.g., released from a guanosine/GMP compositions, or conjugated with another agent, e.g., an antibody, ligand or particle, e.g., a microparticle or nanoparticle. Typically, diagnostic agents are imaging contrast agents or X-ray detection agents. Diagnostic agents include but are not limited to imaging agents which include commercially available agents used in positron emission tomography (PET), computer assisted tomography (CAT), single photon emission computerized tomography, x-ray, fluoroscopy, and magnetic resonance imaging (MRI). Diagnostic agent may comprise a radioactive isotope.
[0027] As used herein, the term "antibody" refers to an immunoglobulin protein molecule that has the ability to bind a target molecule or antigen. The term "antibody" also embraces antibody fragments, such as Fab and scFv fragments, that maintain the ability to specifically bind a target molecule or antigen.
[0028] The term "ligand", as used herein, refers to any molecule which is capable of specifically binding to a receptor on or in a target cell. Ligands may be natural or synthetic. Ligands may be agonists, antagonists or exhibit properties of both agonists and antagonists, e.g. , may be agonist-antagonists.
[0029] The terms "administer," "administering," or "administration," as used herein refers to implanting, applying, absorbing, ingesting, injecting, or inhaling, the inventive compositions. [0030] The terms "treat" or "treating," as used herein, refers to partially or completely alleviating, inhibiting, ameliorating, and/or relieving the disease or condition from which the subject is suffering.
BRIEF DESCRIPTION OF DRAWINGS
[0031] Figure 1 shows freshly prepared antibody dispersed in water. Images are 90 μm x 90 μm.
[0032] Figure 2 shows freshly prepared antibody dispersed in a guanosine/GMP gel (G-gel). Images are 90 μm x 90 μm.
[0033] Figure 3 shows antibody dispersed in water after 25 min storage in ice
(left); antibody dispersed in G-gel after 30 min storage in ice (right). Images are 90 μm x 90 μm.
[0034] Figure 4 shows antibody dispersed in water (left) and G-gel (right) after
3 hours storage in refrigerator. Image is 30 μm x 30 μm.
[0035] Figure 5 shows diffusion of fluorescein (greenish-yellow color) from G- gel into water for a viscous gel (tube at left in both photographs) and a solution-like gel (tube at right in both photographs). The photographs and the graph show the fluorescence of the water phase for both gels after 24 hrs and after 2 weeks. [0036] Figure 6 shows diffusion of rhodamine dye (red-orange) from G-gel of varying composition and pH into water. Tubes from left to right (GMP/Guanosine (Guo), pH); 0.2/0.01, pH 4; 0.2/0.03, pH 4; 0.2/0.05, pH 4; 0.2/0.01, pH 7; 0.2/0.03, pH 7; 0.2/0.05, pH 7; 0.2/0.01, pH 10; 0.2/0.03, pH 10; 0.2/0.05, pH 10. Days (top to bottom) are shown as 0, 1, 3, 4, and 5.
[0037] Figure 7 shows Atomic Force Microscopy (AFM) images of SWNTs solubilized in a guanosine/GMP gel. The individual SWNTs (thin, approximately vertical structures) show parallel alignment. The top image is an expanded view of the bottom image. The x-axis is 1 μm in total length in the bottom image. [0038] Figure 8 shows suspension of six different samples of TiO2 samples (1-3 mg/mL) in gel medium immediately following preparation (top) and after two weeks (bottom).
[0039] Figure 9 shows (left) TiO2 samples 1 and 5 (1-3 mg/mL) in water after
12 hours. (Right) TiO2 sample 1 in water (left) and gel (right) after 3 days. [0040] Figure 10 shows AFM images of TiO2 in water and in G-gel. Solutions were sonicated for 10 min, dropped on a silicon surface, and air dried overnight.
Images are 1 μm x 1 μm.
[0041] Figure 11 shows ZnO nanoparticles in water (left) and get (right) after 2 days. The vials were inverted immediately before being photographed to show the separation of ZnO precipitate from water (left) and the separation of a liquid ZnO dispersion from a more viscous ZnO suspension in the gel solution (right).
[0042] Figure 12 shows AFM images of ZnO in water and in G-gel. Solutions were sonicated for 10 min., dropped on silicon surface, and air dried overnight. Images are 1 μm x 1 μm.
[0043] Figure 13 shows indium tin oxide (ITO) nanoparticles in gel (left) and water (right) after 1 day.
[0044] Figure 14 shows AFM image of ITO in G-gel. The solution was sonicated for 10 min., dropped on silicon surface, and air dried overnight. Image is 1 μm x 1 μm. ITO in water yielded a surface that was too rough for imaging.
[0045] Figure 15 shows classification of guanosine/GMP compositions as liquid (open circle), viscous (open triangle), or gel (solid square) as a function of GMP and Guo concentration at 5 0C (top), 25 0C (center), and 37 0C (bottom). Diagonal lines correspond to XGMP = 0.83 (where XGMP ΪS the GMP mole fraction).
[0046] Figure 16 shows digital photomicrographic images of fibroblast cells seeded in 6-well plates in two different G-gels. Guo to GMP molar ratios and KCl concentration of the G-GeIs are indicated. Images are shown for time points of 1 and 2 days following seeding.
[0047] Figure 17 A shows the photomicrographic results of survival assays on rat aortic smooth muscle cells in G-gels using confocal microscopy. Calcien and
Ethidium homodimer were used to indicate the live and dead cells, respectively.
Calcien stains the cytoplasm of live cells. The location of Calcien staining was denoted by green pixels. Ethidium homodimer stains the nuclei of dead cells. The location of
Ethidium homodimer staining was denoted by red pixels. Each experiment was performed in triplicate (three different wells) for each G-gel, with each image of a row corresponding to a different well. Guo to GMP molar ratios and KCl concentration of the G-GeIs are indicated. [0048] Figure 17B shows the photomicrographic results of survival assays on cells in G-gels after 1 week. As in Figure 17A, assays were performed in triplicate.
Guo to GMP molar ratios and KCl concentration of the G-GeIs are indicated.
[0049] Figure 18A shows PLGA in G-gel (left) and buffer (right) 5 min after 10 seconds of vortexing. The transparent crystals in bottom of the vials are polymer crystals.
[0050] Figure 18B shows PLGA in G-gel (left) and buffer (right) after sonication for >2 h.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION [0051] Guanosine/GMP gels (G-gels) have been discovered to disperse and increase the stability of certain agents, that typically aggregate and/or are less stable in other aqueous solutions. G-gels have been found to be particularly useful in preparing high concentration formulations of such agents. In certain embodiments, the agent is an antibody. In some embodiments, the agent is a ligand. In some embodiments, the agent is a cell. In other embodiments, the agent is a particle, e.g., a microparticle or nanoparticle. In still other embodiments, the agent is a nucleic acid. In some embodiments, the agent is a targeting agent, e.g., an antibody or ligand, conjugated to a therapeutic agent, e.g., a cytotoxic compound, or a diagnostic e.g., an imaging agent. [0052] Some embodiments herein relate to the ability of the inventive guanosine/GMP compositions to produce gels having certain agents, e.g. , antibodies, nucleic acids, ligands, particles, etc., dispersed randomly throughout the gel. It will be appreciated that in certain embodiments, agents which are randomly dispersed in these guanosine/GMP gels exhibit less aggregation, agglomeration and instability, and maintain activity longer, than equivalent agents in preparations of the prior art, e.g., in certain preparations where such agents are non-randomly dispersed. [0053] In some embodiments, guanosine/GMP gels are provided that exhibit distinctive temperature sensitive gelation properties and that are effective for a variety of storage and delivery applications. In certain embodiments, guanosine/GMP gels are provided that are useful for high concentration antibody preparations. In other embodiments, guanosine/GMP gels are provided that are useful for nucleic acid preparations. In other embodiments, guanosine/GMP gels are provided that are useful for microparticle or nanoparticle preparations. In some embodiments, drug delivery compositions are provided that are capable of delivering a drug, e.g., an antibody, over an extended period of time at a predetermined rate. In some embodiments, guanosine/GMP gels are provided that are biocompatible and that are capable of releasing an agent over an extended period of time at a predetermined rate. In other embodiments, methods are provided for controlled release of an agent in a subject from biocompatible guanosine/GMP gels.
[0054] G-gels
[0055] Gels formed by individual guanosine derived compounds (e.g., guanosine, L-guanosine, ara-guanosine, guanosine 5 '-monophosphate, etc.) have been extensively studied (Gellert, M., et al., Proc. Natl. Acad. Sci. 1962, 48, 2013; Sasisekharan, V., and Zimmerman, S.; Davies, D.R. J. MoI. Biol. 1975, 92, 171; Proni, G., et al., Chem. Eur. J. 2000, 6, 3249; Walmsley, J.A. and Burnett, J.F. Biochemistry 1999, 38, 14063, Guschlbauer W, Journal of Biomolecular Structure and Dynamics. Vol. 8 (3); 1990, and Davis, J.T. Angew. Chem. Int. Ed. 2004, 43, 668). Accordingly, it is understood that some guanosine compounds self-associate to form planar tetrameric structures called G-quartets at high concentration, low temperature, or low pH. The basic building block is the G-quartet formed by Hoogsteen hydrogen bonding between each of four guanines and its two nearest neighbors. An exemplary G-quartet is shown below. As the monomer concentration increases, the G-quartets can aggregate into columnar stacks through π-π interactions or, in the case of 5 '-guanosine monophosphate (GMP), into continuous, hydrogen-bonded helices.
Figure imgf000012_0001
G-Ouartet
[0056] In some embodiments, the guanosine/GMP gels disclosed herein comprise guanosine and GMP compounds. In some embodiments, the guanosine is selected from the group consisting of: guanosine (Guo), L-guanosine, D-guanosine, ara-guanosine (guanine 9-β-D-arabinofuranoside), xylo-guanosine, guanosine-2 ',3'- isoproplidene, guanosine-5 '-sulfate, guanosine-5 '-chloride, N-methyl-guanosine, 8- bromo-guanosine, 8-iodo-guanosine, 8-bromo-D-guanosine, 8,2'-anhydro-guanosine, and isoguanosine. In some embodiments, the GMP is selected from the group consisting of: guanosine -2 '-monophosphate, guanosine-3' -monophosphate, guanosine- 5 '-monophosphate, guanosine-3 '(2')- monophosphate, guanosine-2', 3 '-cyclic phosphate, guanosine-2', 3'-cyclic phosphite, D-guanosine-5 '-monophosphate, 8- bromo-guanosine-5' -monophosphate, and 8-bromo-guanosine-3'-monophosphate. Other guanosine and GMPs, which may be used in the guanosine/GMP gels disclosed herein, will be apparent to the skilled artisan (See, e.g., Guschlbauer W, et al., Four- Stranded Nucleic Acid Structures 25 Years Later: From Guanosine Gels to Telomeric DNA, Journal of Biomolecular Structure and Dynamics, Vol. 8(3), 1990, the contents of which are incorporated herein by reference.)
[0057] In certain embodiments, the G-gel is made up of guanosine and guanosine-5 '-monophosphate as the only guanosine and GMP derivatives, respectively. In certain embodiments, the G-gel may contain another guanosine or GMP derivative. In some embodiments, the G-gel may contain only guanosines. In some embodiments, the G-gel may contain only GMPs. In some embodiments, the G-gel does not contain a guanosine-5'-hydrazide.
[0058] In some aspects, the present invention relates to the use of a reversible, biocompatible guanosine/GMP gels (G-gels) to stabilize certain agents in non- aggregate form at a high concentration. These guanosine/GMP gels are aqueous mixtures of guanosine compounds of different solubilities, such as the relatively insoluble guanosine (Guo) and the highly soluble guanosine 5 '-monophosphate (GMP). The properties of the inventive guanosine/GMP gels are highly tunable as a function of the total concentration of guanosine compounds, the ratio of the two different compounds, pH, ionic strength, and cation content. In some formulations, the gels are thermodissociative (gel with decreasing temperature) while in others the gels are thermoassociative (liquid at lower temperatures and a gel at room or body temperature). Since the G-gels comprise simple compounds that occur naturally in humans, the G- gels are biocompatible and well suited to administration to humans and other animals. [0059] Guanosine/GMP gels disclosed herein may be formed by mixing soluble guanosine 5 '-monophosphate (GMP) with insoluble guanosine (Guo). In such guanosine/GMP-gels, hydrophobic Guo facilitates the self-association of GMP while the hydrophilic GMP helps solubilize Guo in the aqueous solution. It is demonstrated herein that by combining the soluble GMP with the insoluble Guo in particular ratios in aqueous solution, the resulting solutions form reversible guanosine/GMP gels (Dowling, V. A., et al., Anal. Chem. 2004, 15, 4558; McGown, US Patent Application Publication 2004/0241718, published December 2, 2004, McGowan, International Patent Application Publication WO 2008/048352).
[0060] The ratio of guanosine to GMP may be at a variety of different ranges.
For example, the ratio of guanosine to GMP may be from about 0.01 to about 4, from about 0.05 to about 0.25. In some embodiments, the ratio of guanosine to GMP may be from about 0.01 to about 0.1, from about 0.02 to about 0.2, from about 0.03 to about 0.3, from about 0.04 to about 0.4, from about 0.05 to about 0.5, from about 0.06 to about 0.6, from about 0.07 to about 0.7, from about 0.08 to about 0.8, from about 0.09 to about 0.9, from about 0.1 to about 1, from about 0.2 to about 2, from about 0.3 to about 3, or from about 0.4 to about 4. In particular embodiments, the ratio of guanosine to GMP is about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.9, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.2, about 1.4, about 1.6, about 1.8, about 2, about 4, or more.
[0061] The mole fraction of GMP (XGMP) in a gel composition may be formulated at a variety of levels. For example, the mole fraction may be about 0.1 , about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or more. In particular embodiments, the mole fraction of GMP ranges from about 0.4 to 0.5, from about 0.5 to about 0.6, from about 0.6 to about 0.7, from about 0.7 to about 0.8, or from about 0.8 to about 0.9.
[0062] The GMP in a gel composition may be formulated at a variety of concentrations. For example, the GMP concentration may range from up to about 1 mM to about 500 mM or from about 50 mM to about 300 mM or more. In some embodiments, the GMP concentration is up to about 10 mM, from about 10 mM to about 20 mM, from about 20 mM to about 30 mM, from about 30 mM to about 40 mM, from about 40 mM to about 50 mM, from about 50 mM to about 60 mM, from about 6OmM to about 70 mM, from about 70 mM to about 80 mM, from about 80 mM to about 90 mM, from about 90 mM to about 100 mM, from about 100 mM to about 110 mM, from about 110 mM to about 120 mM, from about 120 mM to about 130 mM, from about 130 mM to about 140 mM, from about 140 mM to about 150 mM, from about 150 mM to about 160 mM, from about 160 mM to about 170 mM, from about 170 mM to about 180 mM, from about 180 mM to about 190 mM, from about 190 mM to about 200 mM, from about 200 mM to about 210 mM, from about 210 mM to about 220 mM, from about 220 mM to about 230 mM, from about 230 mM to about 240 mM, from about 240 mM to about 250 mM, from about 250 mM to about 260 mM, from about 260 mM to about 270 mM, from about 270 mM to about 280 mM, from about 280 mM to about 290 mM, from about 290 mM to about 300 mM, from about 300 mM to about 350 mM, from about 350 mM to about 400 mM, from about 400 mM to about 450 mM, or from about 450 mM to about 500 mM. In particular embodiments, the GMP concentration is about 10 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500 mM, or more.
[0063] Similarly, guanosine in a gel composition may be formulated at a variety of concentrations. For example, the guanosine concentration may range from up to about 0.1 mM to about 200 mM, from about 1 mM to about 100 mM, or from about 10 mM to about 70 mM. In some embodiments, the guanosine concentration is up to about 10 mM, from about 10 mM to about 20 mM, from about 20 mM to about 30 mM, from about 30 mM to about 40 mM, from about 40 mM to about 50 mM, from about 50 mM to about 60 mM, from about 60 mM to about 70 mM, from about 70 mM to about 80 mM, from about 80 mM to about 90 mM, from about 90 mM to about 100 mM, from about 100 mM to about 110 mM, from about 110 mM to about 120 mM, from about 120 mM to about 130 mM, from about 130 mM to about 140 mM, from about 140 mM to about 150 mM, from about 150 mM to about 160 mM, from about 160 mM to about 170 mM, from about 170 mM to about 180 mM, from about 180 mM to about 190 mM, or from about 190 mM to about 200 mM. In particular embodiments, the guanosine concentration is about 0.01 mM, about 0.1 mM, about 0.5 mM, about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 200 mM, or more.
[0064] In some cases the gels of the present invention are formed by self- association of guanosine and guanosine 5 '-monophosphate that are solution at low temperatures and then become a firm gel at higher temperatures before melting at even higher temperatures. As used herein, low temperatures are below room temperature and are between 2-20 °C while room temperature is defined as between 20-27 °C and high temperatures are defined as those above room temperature, particularly above 35 °C, above 45 °C, above 55 °C, above 65 °C or above 100 0C. It is noted that the actual transition temperatures will vary depending upon the specific gel composition, but typically the solution phase exists below room temperature, the gel exits at and above room temperature, and the higher temperature melting occurs above 40-50 0C. [0065] Thus, depending on the specific formulation of the guanosine composition, gelation may exhibit unique temperature dependencies. For some formulations, guanosine/GMP gels may form at up to about 0 °°C. For other formulations, guanosine/GMP gels may form within a range of from 0 0C to about 5 0C. For other formulations, guanosine/GMP gels may form within a range of from 15 °C to about 25 °C. For still other formulations, guanosine/GMP gels may form within a range of from 35 0C to about 40 °C or more.
[0066] Gelation at any particular temperature, constituent concentration, ratio, etc., may also depend on pH. For example, acidic pH may favor gelation for some formulations, whereas a basic pH may favor gelation for other formulations. Accordingly, in some formulations, the pH of a guanosine/GMP gel composition is up to about 6.5. In other formulations, the pH of a guanosine/GMP gel composition ranges from about 6.5 and about 7.5. In still other formulations, the pH of a guanosine/GMP gel composition is above 7.5. These pH ranges are not intended to be limiting and other ranges for guanosine/GMP gel compositions are envisioned. For example, the pH of a guanosine/GMP gel composition may range from about 2.0 to about 2.5, from about 2.5 to about 3.0, from about 3.0 to about 3.5, from about 3.5 to about 4.0, from about 4.0 to about 4.5, from about 4.5 to about 5.0, from about 5.0 to about 5.5, from about 5.5 to about 6.0, from about 6.0 to about 6.5, from about 6.5 to about 7.0, from about 7.0 to about 7.5, from about 7.5 to about 8.0, from about 8.0 to about 8.5, from about 8.5 to about 9.0, from about 9.0 to about 9.5, from about 9.5 to about 10.0, from about 10.0 to about 10.5, from about 10.5 to about 1 1.0, from about 11.0 to about 11.5, or from about 11.5 to about 12. In particular embodiments, the pH of a guanosine/GMP gel composition ranges from about 6.0 to about 6.2, from about 6.2 to about 6.4, from about 6.4 to about 6.6, from about 6.6 to about 6.8, from about 6.8 to about 7.0, from about 7.0 to about 7.2, from about 7.2 to about 7.4, from about 7.4 to about 7.6, from about 7.6 to about 7.8, or from about 7.8 to about 8.0. [0067] Guanosine/GMP gel compositions, in particular, comprise one or more cations. Exemplary cations include, but are not limited to, Li+, Na+, K+, Rb+, Cs+, NH4 +, Ag+, Ti+, Mg+*, Ca+^ Sr+*. N(CH3)4+ and Ba+^ Cations may be provided at a variety of concentrations including for example up to about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, or more. In some embodiments, the concentration of the cation ranges from about 10 mM to about 30 mM, from about 30 mM to about 60 mM, from about 60 mM to about 90 mM, from about 90 mM to about 120 mM, from about 120 mM to about 150 mM, from about 150 mM to about 180 mM, from about 180 mM to about 210 mM, from about 210 mM to about 240 mM, from about 240 mM to about 270 mM, from about 270 mM to about 300 mM, from about 350 mM to about 400 mM, or from about 400 to about 500 mM. [0068] In addition, Guanosine/GMP gel compositions, comprise one or more anions. Exemplary anions include, but are not limited to, halide ions including F, Cl, Br and I and non-halide anions including for example NO2, NO3, OCH3, CH3 COO, OH, O, CO3, SO4, HSO4, PO4, H2 PO4 and HPO4. Anions may be provided at a variety of concentrations including for example up to about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 1 10 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, or more. In some embodiments, the concentration of the anion ranges from about 10 mM to about 30 mM, from about 30 mM to about 60 mM, from about 60 mM to about 90 mM, from about 90 mM to about 120 mM, from about 120 mM to about 150 mM, from about 150 mM to about 180 mM, from about 180 mM to about 210 mM, from about 210 mM to about 240 mM, from about 240 mM to about 270 mM, from about 270 mM to about 300 mM, from about 350 mM to about 400 mM, or from about 400 to about 500 mM. [0069] It is to be understood that anions and cations of the gel compositions disclosed herein are typically provided as salts or combinations of salts. For example, a guanosine/GMP gel may comprise ammonium salt(s), e.g., (NH4)2SO4, (NH4)3PC>4, sodium salt(s), e.g., Na3PO4, NaCl, and/or a potassium salt(s), e.g., KCl. In particular embodiments, the guanosine/GMP gel comprises about 50 mM to about 200 mM of Na3PO4 and about 50 mM to about 200 mM of KCl. Following the methods disclosed herein, and methods available in the art, the skill artisan will be capable of selecting appropriate salts or combinations of salts to achieve desired cation and anion compositions, and gel properties. The guanosine/GMP gels may further comprise one or more buffering agents.
[0070] Formulations of guanosine/GMP gels (e.g. , pH, concentration of GMP, concentration of guanosine, salt types and concentrations, etc.) may be selected based on the desired properties of the gel, e.g., the predetermined rate of release of an agent, the temperature of gelation, etc. Following the methods disclosed herein, and methods available in the art, the skilled artisan will be capable of selecting appropriate formulations to achieve a gel composition with a desired functional characteristic. For example, Figure 15 (reproduced from Yu Y, et al., Tunable Thermoassociation of Binary Guanosine Gels, J. Phys. B 2008, 112, 1130-1134) provides an analysis of gelation properties of various GMP and guanosine combinations for a particular salt (0.05 M KCl) and pH (pH 7.2) conditions at a multiple temperature conditions. Following methods known in the art, such as those depicted in figure 15, in combination with those disclosed elsewhere herein, the skilled artisan will be capable of preparing gel compositions useful in the inventive storage and delivery (e.g., controlled-release) applications.
Guanosine/GMP gels comprising Therapeutic and Diagnostic Agents
[0071] Aspects of the invention provide guanosine/GMP gels that exhibit distinctive temperature sensitive gelation properties and that are effective for a variety of molecular storage and therapeutic and diagnostic delivery applications. For example, the guanosine/GMP gels disclosed herein are useful for high concentration antibody preparations. The guanosine/GMP gels disclosed herein are also useful for nucleic acids, e.g., siRNA, microRNA, DNA. Guanosine/GMP gels may also be used for microparticle, nanoparticle, or other particles, e.g., particles conjugated with, or encapsulating, therapeutic or diagnostic agents. Such preparations provide long-term storage solutions in which the conventional storage problems such as aggregation, agglomeration, decreased activity, and instability are minimized. [0072] Accordingly, the guanosine/GMP gels disclosed herein, e.g., gels comprising guanosine and guanosine 5 '-monophosphate (GMP), can maintain high concentrations of certain molecules or agents, e.g., antibodies, nucleic acids, and particles, e.g., microparticles and nanoparticles. Typically the agent is randomly dispersed in the guanosine/GMP gels. Thus, it is possible to achieve a concentration of an agent in the gel, that is at or above a concentration that results in aggregation, agglomeration, degradation, or decreased activity of the agent in a conventional formulation. Any one of a number of techniques known in the art may be used to assess aggregation, agglomeration, degradation, or activity of an agent stored in a gel preparation of the invention. Often, this assessment involves an evaluation of aggregation, agglomeration, degradation, or activity of an equivalent agent stored in a control solvent. Any appropriate control solvent made be used, but typically the control solvent is an aqueous solution, such as, for example, water or a mixture of water and an organic solvent, such as, for example, methanol, ethanol, isopropanol, n- propanol, tert-butyl alcohol, n-butyl alcohol, ethyl acetate, and acetonitrile. This solvent list in not limiting and other appropriate solvents may be used. [0073] With antibodies (including fragments thereof), for example, a broad range of concentrations of the antibody may be achieved in the guanosine/GMP gel. For example, antibodies may be maintained in guanosine/GMP gels at a concentration ranging from about 0.1 mg/ml to about 100 mg/ml or more, from about 1 mg/ml to about 25 mg/ml, or from about 5 mg/ml to about 15 mg/ml. In some embodiments, the concentration of the antibody in guanosine/GMP gels is from about 0.1 mg/ml to about 10 mg/ml, from about 10 mg/ml to about 20 mg/ml, from about 20 mg/ml to about 30 mg/ml, from about 30 mg/ml to about 40 mg/ml, from about 40 mg/ml to about 50 mg/ml, from about 50 mg/ml to about 60 mg/ml, from about 60 mg/ml to about 70 mg/ml, from about 70 mg/ml to aboutδO mg/ml, from about 80 mg/ml to about 90 mg/ml, from about 90 mg/ml to about 100 mg/ml, from about 100 mg/ml to about 110 mg/ml, from about 110 mg/ml to about 120 mg/ml, from about 120 mg/ml to about 130 mg/ml, from about 130 mg/ml to about 140 mg/ml, from about 140 mg/ml to about 150 mg/ml, from about 150 mg/ml to about 200 mg/ml, or from about 200 mg/ml to about 250 mg/ml. In particular embodiments, the concentration of the antibody in guanosine/GMP gels is about 0.1 mg/ml, about 1 mg/ml, about 10 mg/ml, about 20 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 110 mg/ml, about 120 mg/ml, about 130 mg/ml, about 140 mg/ml, about 150 mg/ml, about 160 mg/ml, about 170 mg/ml, about 180 mg/ml, about 190 mg/ml, about 200 mg/ml, about 250 mg/ml, or more. These concentrations are not intended to be limiting and other appropriate ranges are envisioned.
[0074] Other aspects of the invention provide guanosine/GMP gels that are capable of releasing certain molecules and therapeutic or diagnostic agents over an extended period of time at a predetermined rate. Accordingly, pharmaceutical compositions are provided that are capable of delivering an agent, e.g., an antibody, over an extended period of time at a predetermined rate to a subject from a biocompatible guanosine/GMP gel.
Antibodies
[0075] In the methods and composition disclosed herein, an antibody may serve as an agent, e.g., therapeutic or diagnostic agent, e.g., an antibody that binds specifically to and inhibits activity of a disease associated molecule. As used herein, unless otherwise noted, the term "antibody" refers to antibodies, including for example polyclonal, monoclonal, chimeric, and humanized antibodies, and fragments of antibodies that specifically bind to antigens. In certain embodiments, the antibody is a monoclonal antibody. In certain embodiments, the antibody is a polyclonal antibody. In certain embodiments, the antibody is a humanized antibody. Antibodies may comprise an immunoglobulin of any class, e.g., IgG, IgM, IgA, IgD, IgE. [0076] As is well known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W.R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The pFc1 and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
[0077] Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FRl through FR4) separated respectively by three complementarity determining regions (CDRl through CDR3). The CDRs, and in particular the CDR3 regions, and more particularly the heavy chain CDR3, are largely responsible for antibody specificity.
[0078] It is now well established in the art that the non-CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecifϊc antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody. See, e.g., U.S. Patents 4,816,567, 5,225,539, 5,585,089, 5,693,762 and 5,859,205; each of which is incorporated herein by reference.
[0079] Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti- mouse antibody (HAMA) responses when administered to humans. [0080] Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab')2, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDRl and/or CDR2 regions have been replaced by homologous human or non-human sequences. The present invention also includes so-called single chain antibodies. [0081] Therapeutic antibodies may bind to, and inhibit the activity of, a protein of a variety of disease relevant signaling pathways, including for example Integrin, Androgen Receptor, B Cell Receptor, EGFRl, IL-2, IL-4, IL-6, Kit, Notch, T Cell Receptor, TGFβ, TNF-α, or WNT. Abciximab, for example, is a platelet aggregation inhibitor that targets the glycoprotein Ilb/IIIa receptor on the platelet membrane and is mainly used during and after coronary artery procedures like angioplasty to prevent platelets from sticking together and causing thrombus formation within the coronary artery. Adalimumab is another example that binds to TNFα, preventing it from activating TNF receptors and is useful as an immunosuppressant for treating a variety of inflammatory conditions, including for example Crohn's disease. [0082] In some embodiments, the antibody is selected from the group consisting of: Abciximab, Adalimumab, Alemtuzumab, Basiliximab, Bevacizumab, Cetuximab, Daclizumab, Eculizumab, Efalizumab, Gemtuzumab, Ibritumomab tiuxetan, Infliximab, Muromonab-CD3, Natalizumab, Omalizumab, Palivizumab, Panitumumab, Ranibizumab, Rituximab, Tositumomab, and Trastuzumab. In other embodiments, the antibody is an antibody that binds specifically to an antigen of the foregoing list of antibodies. [0083] In some embodiments, the antibody binds to a cell surface receptor, including for example a cell surface receptor selected from the group consisting of insulin receptor (insulin), insulin-like growth factor receptor (including both IGF-I and IGF-2), growth hormone receptor, glucose transporters (particularly GLUT 4 receptor), transferring receptor (transferring), epidermal growth factor receptor (EGF), low density lipoprotein receptor, high density lipoprotein receptor, leptin receptor, estrogen receptor (estrogen); interleukin receptors including IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, 1L-9, IL-11, IL-12, IL-13, IL-15, and IL-17 receptors, human growth hormone receptor, vascular endothelial growth factor ("VEGF") receptor, platelet- derived growth factor ("PDGF") receptor, transforming growth factor receptor (including TGF-α and TGF-β), erythropoietin ("EPO") receptor, thrombopoietin ("TPO") receptor, ciliary neurotrophic factor receptor, prolactin receptor, and T-cell receptors.
[0084] In some embodiments, the antibody binds to a G-protein coupled receptor, including for example a G-protein coupled receptor selected from the group consisting of: αl A-adrenergic receptor, αlB-adrenergic receptor, α2-adrenergic receptor, α2B-adrenergic receptor, βl -adrenergic receptor, β2-adrenergic receptor, β3- adrenergic receptor, ml acetylcholine receptor (AChR), m2 AChR, m3 AChR, m4 AChR, m5 AChR, Dl dopamine receptor, D2 dopamine receptor, D3 dopamine receptor, D4 dopamine receptor, D5 dopamine receptor, Al adenosine receptor, A2b adenosine receptor, 5-HTla, 5-HTlb, 5HTl-like, 5-HTld, 5HTld-like, 5HTId beta, substance K (neurokinin A), fMLP receptor, fMLP-like receptor, angiotensin II type 1 , endothelin ETA, endothelin ETB, thrombin, growth hormone-releasing hormone (GHRH), vasoactive intestinal peptide, oxytocin, somatostatin SSTRl and SSTR2, SSTR3, cannabinoid, follicle stimulating hormone (FSH), leutropin (LH/HCG), thyroid stimulating hormone (TSH), thromboxane A2, platelet-activating factor (PAF), C5a anaphylatoxin, Interleukin 8 (IL-8) IL-8RA, IL-8RB, Delta Opioid, Kappa Opioid, mip-1 /RANTES, Rhodopsin, Red opsin, Green opsin, Blue opsin, metabotropic glutamate mGluRl-6, histamine H2, ATP, neuropeptide Y, amyloid protein precursor, insulin-like growth factor II, bradykinin, gonadotropin-releasing hormone, cholecystokinin, melanocyte stimulating hormone receptor, antidiuretic hormone receptor, glucagon receptor, and adrenocorticotropic hormone II. [0085] Antibodies may also be conjugated to other therapeutic or diagnostic agents, e.g., cytotoxic compound, toxins, small molecules, nucleic acids, or radioisotopes. Such antibodies may be useful for targeting certain therapeutic agents to specific cell types, e.g., cancer cells. Exemplary cytotoxic compounds which may be conjugated to a therapeutic antibody include for example, calicheamicin, esperamicin, methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin C, cisplatinum, etopside, bleomycin and 5-fluorouracil. Still other cytotoxic compounds are disclosed herein and will be apparent to the skilled artisan.
Ligands
[0086] In the methods and composition disclosed herein, a ligand, e.g., a natural or synthetic ligand, may serve as an agent, e.g., a therapeutic or diagnostic agent. [0087] In some embodiments, the ligand is all or a portion (e.g. a binding portion) of a ligand for a cell surface receptor. Suitable ligands include, but are not limited to, all or a functional portion of the ligands that bind to a cell surface receptor selected from the group consisting of a G-protein-coupled receptor, insulin receptor (insulin), insulin-like growth factor receptor (including both IGF-I and IGF -2), growth hormone receptor, glucose transporters (particularly GLUT 4 receptor), transferring receptor (transferring), epidermal growth factor receptor (EGF), low density lipoprotein receptor, high density lipoprotein receptor, leptin receptor, estrogen receptor (estrogen); interleukin receptors including IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 11, IL-12, IL-13, IL-15, and IL-17 receptors, human growth hormone receptor, vascular endothelial growth factor ("VEGF") receptor, platelet-derived growth factor ("PDGF") receptor, transforming growth factor receptor (including TGF-α and TGF-β), erythropoietin ("EPO") receptor, thrombopoietin ("TPO") receptor, ciliary neurotrophic factor receptor, prolactin receptor, and T-cell receptors. Hormone ligands are also envisioned. Hormones include both steroid hormones and proteinaceous hormones, including, but not limited to, epinephrine, thyroxine, oxytocin, insulin, thyroid- stimulating hormone, calcitonin, chorionic gonadotropin, cortictropin, follicle- stimulating hormone, glucagon, leuteinizing hormone, lipotropin, melanocyte- stimutating hormone, norepinephrine, parathyroid hormone, thyroid-stimulating hormone (TSH), vasopressin, enkephalins, seratonin, estradiol, progesterone, testosterone, cortisone, and glucocorticoids and the hormones listed above. Receptor ligands include ligands that bind to receptors such as cell surface receptors, which include hormones, lipids, proteins, glycoproteins, signal transducers, growth factors, cytokines, and others.
[0088] Ligands may also be conjugated to other therapeutic or diagnostic agents, e.g., cytotoxic compound, toxins, small molecules, nucleic acids, or radioisotopes. Such antibodies may be useful for targeting certain therapeutic agents to specific cell types, e.g., cancer cells.
Particles
[0089] In the methods and composition disclosed herein, a particle may serve as an agent, e.g., therapeutic or diagnostic agent. As used herein, the term "particle" refers to any particle, in terms of composition, shape, and size, that is suitable for delivery of an agent to a subject or cell. A "microparticle" is a particle having an average diameter on the order of micrometers (e.g., between about 1 micrometer and about 1 mm), while a "nanoparticle" is a particle having an average diameter on the order of nanometers (e.g., between about 1 nm and about 1 micrometer). [0090] Particles may have any shape or size. For instance, particles may have an average diameter of less than about 5 mm or 2 mm, or less than about 1 mm, or less than about 500 microns, less than about 200 microns, less than about 100 microns, less than about 60 microns, less than about 50 microns, less than about 40 microns, less than about 30 microns, less than about 25 microns, less than about 10 microns, less than about 3 microns, less than about 1 micron, less than about 300 nm, less than about 100 nm, less than about 30 nm, or less than about 10 nm. The particles may be spherical or non-spherical. The average diameter of a non-spherical particle is the diameter of a perfect sphere having the same volume as the non-spherical particle. [0091] The particles may be formed of any suitable material, depending on the application. For example, the particles may comprise a glass, and/or a polymer such as polyethylene, polystyrene, silicone, polyfluoroethylene, polyacrylic acid, a polyamide (e.g., nylon), polycarbonate, polysulfone, polyurethane, polybutadiene, polybutylene, polyethersulfone, polyetherimide, polyphenylene oxide, polymethylpentene, polyvinylchloride, polyvinylidene chloride, polyphthalamide, polyphenylene sulfide, polyester, polyetheretherketone, polyimide, polymethylmethacylate and/or polypropylene. In some cases, the particles may comprise a ceramic such as tricalcium phosphate, hydroxyapatite, fluorapatite, aluminum oxide, or zirconium oxide. In some cases (for example, in certain biological applications), the particles may be formed from biocompatible and/or biodegradable polymers such as polylactic and/or polyglycolic acids, polyanhydride, polycaprolactone, polyethylene oxide, polybutylene terephthalate, starch, cellulose, chitosan, proteins, carbohydrates, and/or combinations of these. In some embodiments, the particles may comprise a hydrogel, such as agarose, collagen, fibrin.
[0092] The particles may include a magnetically susceptible material in some cases, e.g. , a material displaying paramagnetism or ferromagnetism. For instance, the particles may include iron, iron oxide, magnetite, hematite, or some other compound containing iron. In another embodiment, the particles can include a conductive material (e.g., a metal such as titanium, copper, platinum, silver, gold, tantalum, palladium, rhodium, etc.), or a semiconductive material (e.g., silicon, germanium, CdSe, CdS, etc.). Other particles include ZnS, ZnO, ITO, TiO2, AgI, AgBr, HgI2, PbS, PbSe, ZnTe, CdTe, In2S3, In2Se3, Cd3P2, Cd3As2, InAs, or GaAs.
[0093] In particular embodiments, the particles are conjugated to a therapeutic agent, including for example peptide, polypeptide, protein drugs, other large hydrophilic molecules, small molecule compounds, and nucleic acids. In other embodiments, microparticles or nanoparticles encapsulate a therapeutic agent, including for example peptide, polypeptide, protein drugs, other large hydrophilic molecules, small molecule compounds, and nucleic acids. In certain embodiments the particle is conjugated to a targeting agent, e.g., an antibody or ligand.
Nucleic Acids
[0094] Agents may also be nucleic acids. Nucleic acids include, but are not limited to, RNA, DNA, peptide nucleic acids (PNA), and combinations thereof. Nucleic acids include expression vectors for delivering exogenous gene products, e.g., cDNA, non-protein coding RNAs, and short interfering nucleic acids (siNA) that include, for example: microRNA (miRNA), short interfering RNA (siRNA), double- stranded RNA (dsRNA), and short hairpin RNA (shRNA) molecules, for example. [0095] An siNA of the invention can be unmodified or chemically-modified.
An siNA of the instant invention can be chemically synthesized, expressed from a vector or enzymatically synthesized. The disclosure also features various chemically- modified synthetic short interfering nucleic acid molecules capable of modulating gene expression or activity in cells by RNA interference (RNAi). The use of chemically- modified siNA improves various properties of native siNA molecules through, for example, increased resistance to nuclease degradation in vivo and/or through improved cellular uptake. Furthermore, siNA having multiple chemical modifications may retain RNAi activity. The siNA molecules of the instant invention provide useful reagents and methods for a variety of therapeutic applications.
[0096] Chemically synthesizing nucleic acid molecules with modifications
(base, sugar and/or phosphate) that prevent their degradation by serum ribonucleases can increase their potency (see, e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; and Sproat, U.S. Pat. No. 5,334,711, all of these describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules herein). Modifications which enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired. [0097] There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'amino, 2'-C-allyl, 2'-flouro, 2'-O-methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry , 35, 14090). Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344, 565 568; Pieken et al. Science, 1991, 253, 314317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334 339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,71 1 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., molecule comprises one or more chemical modifications.
[0098] Other nucleic acids known in the art may also serve as therapeutic agents in the instant invention. For example, triple helix approaches may be used to mediate sequence-specific gene suppression. Triple helix forming oligonucleotides have been found in some cases to bind in a sequence-specific manner (Postel et al., Proc. Natl. Acad. Sci. U.S.A. 88(18):8227-31, 1991; Duval-Valentin et al., Proc. Natl. Acad. Sci. U.S.A. 89(2):504-8, 1992; Hardenbol and Van Dyke Proc. Natl. Acad. Sci. U.S.A. 93(7):2811-6, 1996; Porumb et al., Cancer Res. 56(3):515-22, 1996). Similarly, peptide nucleic acids have been shown to inhibit gene expression (Hanvey et al., Antisense Res. Dev. l(4):307-17, 1991; Knudsen and Nielson Nucleic Acids Res. 24(3):494-500, 1996; Taylor et al., Arch. Surg. 132(11):1177-83, 1997). DNA and/or RNA aptamers may also be used to, e.g., inhibit the activity of target a protein of interest.
[0099] Nucleic acid molecules of the invention may be provided in any one of a number of different vectors. As used herein, a "vector" may be any of a number of nucleic acid molecules into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA although RNA vectors are also available. Vectors include, but are not limited to, plasmids, phagemids and virus genomes or portions thereof.
[00100] An expression vector is one into which a desired sequence may be inserted, e.g., by restriction and ligation such that it is operably joined to regulatory sequences {e.g., a promoter sequence, enhancer sequence) and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells that have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins that increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes that encode enzymes whose activities are detectable by standard assays known in the art {e.g., β-galactosidase or alkaline phosphatase), and genes that visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques {e.g., green fluorescent protein). [00101] As used herein, a coding sequence and regulatory sequences are said to be "operably" joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide. It will be appreciated that a coding sequence need not encode a protein but may instead, for example, encode a functional RNA such as an shRNA. [00102] The precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5 '-non-transcribed and 5 '-non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Such 5 '-non-transcribed regulatory sequences will include a promoter region that includes a promoter sequence for transcriptional control of the operably joined gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired. The vectors of the invention may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art. One of skill in the art will be aware of appropriate regulatory sequences for expression of interfering RNA, e.g., shRNA, miRNA, etc.
[00103] In some embodiments an siNA is an shRNA, shRNA-mir, or microRNA molecule encoded by and expressed from a plasmid-based expression vector. Thus, in some embodiments a nucleic acid is a transgene incorporated in a plasmid-based expression vector that encodes a small-interfering nucleic acid. Such transgenes and expression vectors can employ either polymerase II or polymerase III promoters to drive expression of these shRNAs and result in functional siRNAs in cells. The former polymerase permits the use of classic protein expression strategies, including inducible and tissue-specific expression systems. In some embodiments, transgenes and expression vectors are controlled by tissue specific promoters. In other embodiments transgenes and expression vectors are controlled by inducible promoters, such as tetracycline inducible expression systems.
[00104] In another embodiment, a small interfering nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. The recombinant mammalian expression vector may be capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the myosin heavy chain promoter, albumin promoter, lymphoid-specific promoters, neuron specific promoters, pancreas specific promoters, and mammary gland specific promoters. Developmentally-regulated promoters are also encompassed, for example the murine hox promoters and the a-fetoprotein promoter. [00105] In some embodiments, a virus vector for delivering a nucleic acid molecule is selected from the group consisting of adenoviruses, adeno-associated viruses, poxviruses including vaccinia viruses and attenuated poxviruses, Semliki Forest virus, Venezuelan equine encephalitis virus, retroviruses, Sindbis virus, and Ty virus-like particle. Examples of viruses and virus-like particles which have been used to deliver exogenous nucleic acids include: replication-defective adenoviruses (e.g., Xiang et al., Virology 219:220-227, 1996; Eloit et al., J. Virol. 7:5375-5381, 1997; Chengalvala et al., Vaccine 15:335-339, 1997), a modified retrovirus (Townsend et al., J. Virol. 71 :3365-3374, 1997), a nonreplicating retrovirus (Irwin et al., J. Virol. 68:5036-5044, 1994), a replication defective Semliki Forest virus (Zhao et al., Proc. Natl. Acad. Sci. USA 92:3009-3013, 1995), canarypox virus and highly attenuated vaccinia virus derivative (Paoletti, Proc. Natl. Acad. Sci. USA 93:11349-11353, 1996), non-replicative vaccinia virus (Moss, Proc. Natl. Acad. Sci. USA 93:11341-11348, 1996), replicative vaccinia virus (Moss, Dev. Biol. Stand. 82:55-63, 1994), Venzuelan equine encephalitis virus (Davis et al., J. Virol. 70:3781-3787, 1996), Sindbis virus (Pugachev et al., Virology 212:587-594, 1995), lentiviral vectors (Naldini L, et al., Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11382-8) and Ty virus-like particle (Allsopp et al., Eur. J. Immunol 26: 1951-1959, 1996). [00106] Another virus useful for certain applications is the adeno-associated virus, a double-stranded DNA virus. The adeno-associated virus is capable of infecting a wide range of cell types and species and can be engineered to be replication-deficient. It further has advantages, such as heat and lipid solvent stability, high transduction frequencies in cells of diverse lineages, including hematopoietic cells, and lack of superinfection inhibition thus allowing multiple series of transductions. The adeno- associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.
[00107] In general, other useful viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non-cytopathic viruses include certain retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. In general, the retroviruses are replication- deficient (i.e., capable of directing synthesis of the desired transcripts, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, M., "Gene Transfer and Expression, A Laboratory Manual," W.H. Freeman Co., New York (1990) and Murry, EJ. Ed. "Methods in Molecular Biology," vol. 7, Humana Press, Inc., Clifton, New Jersey (1991).
Cells
[00108] In the methods and composition disclosed herein, a cell, e.g., a mammalian cell, e.g., a human cell, may serve as an agent, e.g., a therapeutic agent. [00109] The cells may be any mammalian cells. The cells may be any human cells. The cells may be selected from the group consisting of Lymphocytes, B cells, T cells, cytotoxic T cells, natural killer T cells, regulatory T cells, T helper cells, myeloid cells, granulocytes, basophil granulocytes, eosinophil granulocytes, neutrophil granulocytes, hypersegmented neutrophils, monocytes, macrophages, reticulocytes, platelets, mast cells, thrombocytes, megakaryocytes, dendritic cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, oxyphil cells, adrenal cells, chromaffin cells, pineal cells, pinealocytes, glial cells, glioblasts, astrocytes, oligodendrocytes, microglial cells, magnocellular neurosecretory cells, stellate cells, boettcher cells; pituitary cells, gonadotropes, corticotropes, thyrotropes, somatotrope, lactotrophs, pneumocyte, type I pneumocytes, type II pneumocytes, Clara cells; goblet cells, alveolar macrophages, myocardiocytes, pericytes, gastric cells, gastric chief cells, parietal cells, goblet cells, paneth cells, G cells, D cells, ECL cells, I cells, K cells, S cells, enteroendocrine cells, enterochromaffin cells, APUD cell, liver cells, hepatocytes, Kupffer cells, bone cells, osteoblasts, osteocytes, osteoclast, odontoblasts, cementoblasts, ameloblasts, cartilage cells, chondroblasts, chondrocytes, skin cells, hair cells, trichocytes, keratinocytes, melanocytes, nevus cells, muscle cells, myocytes, myoblasts, myotubes, adipocyte, fibroblasts, tendon cells, podocytes, juxtaglomerular cells, intraglomerular mesangial cells, extraglomerular mesangial cells, kidney cells, kidney cells, macula densa cells, spermatozoa, Sertoli cells, leydig cells, oocytes, and mixtures thereof. Accordingly, the cells may be of mesenchymal, ectodermal, and endodermal origin. [00110] The cells may be selected from the group consisting of cord-blood cells, stem cells, embryonic stem cells, adult stem cells, progenitor cells, induced progenitor cells, autologous cells, isograft cells, allograft cells, xenograft cells, and genetically engineered cells.
Other Therapeutic/Diagnostic Agents
[00111] It will be appreciated that certain agents disclosed herein may be dispersed in the guanosine/GMP gel compositions alone or, preferably, conjugated to another agent, e.g., an antibody, a ligand, a nucleic acid, or a particle, e.g. microparticle or nanoparticle. Therapeutic agents having molecular weights less than 100,000 Da, more preferably less than 20,000 Da, and most preferably less than 2,000 Da, advantageously may be used in the compositions and methods of the present invention, e.g., alone or conjugated to particles or antibodies. These therapeutic agents include, for example, physiologically active materials or medicinal drugs, such as agents affecting the central nervous system, anti-allergic agents, cardiovascular agents, agents affecting respiratory organs, agents affecting digestive organs, hormone preparations, agents affecting metabolism, cytotoxic agents, antibiotic preparations, chemotherapeutics, antimicrobials, local anesthetics, antihistaminics, antiphlogistics, astringents, vitamins, antifungal agents, peripheral nervous anesthetics, vasodilators, crude drug essences, tinctures, crude drug powders, hypotensive agents, nucleic acids or immunosuppressants. Antibodies, e.g., antibodies conjugated with other therapeutic agents, e.g., a cytotoxic compound, a radioisotope, or a nucleic acid, may also be considered therapeutic agents.
[00112] Several polypeptides, including cytokines and growth factors, also may be used as therapeutic agents. Cytokines suitable for use in the compositions and methods of the present invention include, for example, interferons (IFNs), tumor necrosis factors (TNFs), interleukins, colony stimulating factors (CSFs), growth factors such as osteogenic factor extract (OFE), epidermal growth factor (EGF), transforming growth factor (TGF) alpha, TGF-β (including any combination of TGF-βs), TGF-βl, TGF-β2, platelet derived growth factor (PDGF-AA, PDGF-AB, PDGF-BB), acidic fibroblast growth factor (FGF), basic FGF, connective tissue activating peptides (CTAP), β-thromboglobulin, insulin-like growth factors, erythropoietin (EPO), nerve growth factor (NGF), bone morphogenic protein (BMP), osteogenic factors, and others. Other therapeutic agents which may be used in the methods and compositions disclosed herein include, for example, oxytocin, vasopressin, adrenocorticotrophic hormone (ACTH), transforming growth factor antagonists, prolactin, luliberin or luteinizing hormone releasing hormone (LH-RH), LH-RH agonists or antagonists, growth hormone, growth hormone releasing factor, insulin, somatostatin, bombesin antagonists, glucagon, interferon, gastrin, tetragastrin, pentagastrin, urogastrone, secretin, calcitonin, enkephalins, endomorphins, angiotensins, renin, bradykinin, bacitracins, polymyzins, colistins, tyrocidin, gramicidines, monoclonal antibodies, soluble vaccines, and synthetic analogues, modifications and pharmaceutically-active fragments thereof. Polypeptides can be natural or synthetic. [00113] Other suitable therapeutic agents for use in the invention include antiinflammatory agents such as hydrocortisone and prednisone; antibacterial agents such as penicillin, cephalosporins, and bacitracin; antiparasitic agents such as quinacrine, and chloroquine; antifungal agents such as nystatin, and gentamicin; antiviral agents such as acyclovir, ribavirin, and interferons; antineoplastic agents such as methotrexate, 5-fluorouracil, and adriamycin, and tumor necrosis factor; analgesic agents such as salicylic acid, acetaminophen, ibuprofen, flurbiprofen, and morphine; local anesthetics such as lidocaine, bupivacaine, and benzocaine; vaccines such as hepatitis, influenza, measles, rubella, tetanus, polio, and rabies; central nervous system agents such as a tranquilizer, β-adrenergic blocking agent, and dopamine; growth hormones such as human growth hormone and insulin-like growth factor; other hormones such as progesterone, follicle stimulating hormone, insulin, and somatotropins; antihistamines such as diphenhydramine, and chlorphencramine; cardiovascular agents such as digitalis, nitroglycerine, papaverine, and streptokinase; vasodilators such as theophylline, niacin, and minoxidil; and other similar substances. [00114] Examples of cytotoxic compounds that may be suitable as therapeutic agents in the methods and compositions disclosed herein include, but are not limited to, the following: Antineoplastic agents such as: Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Adriamycin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Buniodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; DACA (N [2- (Dimethyl amino)ethyl]acridine 4 carboxamide); Dactinomycin; Daunorubicin Hydrochloride; Daunomycin; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Ifesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflornithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Ethiodized Oil I 131 ; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; 5 FdUMP; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa 2a; Interferon Alfa 2b; Interferon Alfa nl ; Interferon Alfa n3; Interferon Beta- Ia; Interferon Gamma- Ib; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin,. Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safϊngol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Thymitaq; Tiazofurin; Tirapazamine; Tomudex; TOP 53; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine; Vinblastine Sulfate; Vincristine; Vincristine Sulfate, Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride; 2 Chlorodeoxyadenosine; 2' Deoxyformycin; 9 aminocamptothecin; raltitrexed; N propargyl 5, 8 dideazafolic acid, 2-chloro 2' arabino fluoro 2' deoxyadenosine; 2 chloro 2' deoxyadenosine; anisomycin; trichostatin A; hPRL Gl 29R; CEP 751 ; linomide; Piritrexim Isethionate; Sitogluside; Tamsulosin Hydrochloride and Pentomone. [00115] Examples of antibiotics that may be suitable as therapeutic agents in the methods and compositions disclosed herein include, but are not limited to, the following: Aminoglycosides, such as Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Streptomycin, Tobramycin, Paromomycin; Ansamycins such as Geldanamycin and Herbimycin; the Carbacephem, Loracarbef; Carbapenems such as Ertapenem, Doripenem, Imipenem, and Meropenem; Cephalosporins, such as Cefadroxil, Cefazolin, Cefalotin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefdinir, Cefotetan, and Cefepime; Glycopeptides such as Teicoplanin and Vancomycin; Macrolides such as Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, and Spectinomycin; Monobactams such as Aztreonam; Penicillins such as Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Meticillin, Nafcillin, Oxacillin, Penicillin, Penicillin G, Piperacillin, and Ticarcillin; Polypeptides such as Bacitracin, Colistin and Polymyxin B; Quinolones and Fluoroquinolones such as Ciprofloxacin, Enoxacin, Gatifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Norfloxacin, Ofloxacin, and Trovafloxacin; Sulfonamides such as, Mafenide, Prontosil, Sulfacetamide, Sulfamethizole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Sulfadiazine, Trimethoprim, and Trimethoprim-Sulfamethoxazole; Tetracyclines such as Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, and Tetracycline; and others including Arsphenamine, Chloramphenicol, Clindamycin, Lincomycin, Ethambutol, Fosfomycin, Fusidic acid, Furazolidone, Isoniazid, Linezolid, Metronidazole, Mupirocin, Nitrofurantoin, Platensimycin, Pyrazinamide, Quinupristin/Dalfopristin, Rifampicin (Rifampin), and Tinidazole. [00116] In some embodiments, radioactive molecules may be used as therapeutic and/or diagnostic agents in the compositions and methods disclosed herein. Exemplary radioactive agents for use in the gels include but are not limited to Fibrinogen I 125; Fludeoxyglucose F18; Fluorodopa F 18; Insulin I 125; Insulin 1 131; Iobenguane I 123; Iodipamide Sodium I 131; Iodoantipyrine 1 131; Iodocholesterol 1 131; Iodohippurate Sodium I 123; Iodohippurate Sodium I 125; Iodohippurate Sodium I 131 ; Iodopyracet I 125; Iodopyracet 1 131 ; Iofetamine Hydrochloride I 123; Iomethin I 125; Iomethin I 131; Iothalamate Sodium I 125; Iothalamate Sodium I 131 ; Iotyrosine 1 131 ; Liothyronine I 125; Liothyronine 1 131; Merisoprol Acetate Hg 197; Merisoprol Acetate Hg 203; Merisoprol Hg 197; Selenomethionine Se 75; Technetium Tc 99m Atimony Trisulfide Colloid; Technetium Tc 99m Bicisate; Technetium Tc 99m Disofenin; Technetium Tc 99m Etidronate; Technetium Tc 99m Exametazime; Technetium Tc 99m Furifosmin; Technetium Tc 99m Gluceptate; Technetium 99m Lidofenin; Technetium Tc 99mm Mebrofenin; Technetium Tc 99m Medronate; Technetium Tc 99m Medronate Disodium; Technetium Tc 99m Mertiatide; Technetium Tc 99m Oxidronate; Technetium Tc 99m Pentetate; Technetium Ic 99m Pentetate Calcium Trisodium; Technetium Tc 99m Sestamibi; Technetium Tc 99m Siboroxime; Technetium Tc 99m Succimer; Technetium Tc 99m Sulfur Colloid; Technetium Tc 99m Teboroxime; Technetium Tc 99m Tetrofosmin; Technetium Tc 99m Tiatide; Thyroxine I 125: Thyroxine 1 131 ; Tolpovidone 1 131; Triolein I 125; and Triolein 1 131.
[00117] Radioisotopes may also be conjugated to agents, e.g., antibodies, to create therapeutic and diagnostic agents, e.g., radiolabel antibody cancer therapies. Radioisotopes may be selected from the group consisting of: 225Ac, 11At, 21 Bi, ' Bi,
186D Rh. , 188D RIh, 177 LT u, 90V Y, '3 IT 1, 67^ C,u,, 125τ 1, 123 IT, 77D B,r, 153ς S,m, 166Q BΛo, 64 C,-.u, 212™ Pb, 224D Rao a _n_d, 223Ra. Other appropriate isotopes will be apparent to the skilled artisan. [00118] Other examples of suitable materials for use as diagnostic agents, e.g. , contrast agents, in MRI include but are not limited to the gadolinium chelates currently available, such as diethylene triamine pentacetic acid (DTPA) and gadopentotate dimeglumine, as well as iron, magnesium, manganese, copper and chromium. [00119] Examples of materials useful for CAT and x-rays include iodine based materials for intravenous administration, such as ionic monomers typified by diatrizoate and iothalamate, non-ionic monomers such as iopamidol, isohexyl, and ioversol, non-ionic dimers, such as iotrol and iodixanol, and ionic dimers, for example, ioxagalte.
Agent Dispersion
[00120] An agent may be dispersed in a guanosine/GMP gel in a variety of ways.
Typically, an agent is first suspended or diluted in an aqueous solution comprising guanosine and GMP, which may also comprise appropriate salts and buffering agents. Alternatively, guanosine and/or GMP may be suspended or diluted in an aqueous solution comprising an agent. When an agent is diluted or suspended in an aqueous solution comprising guanosine and GMP, the suspension or dilution is typically performed under conditions, e.g., at a temperature, ionic strength, and pH, in which gelation of the solution is minimized, e.g., in which the solution remains a liquid. Prior to addition to the aqueous solution, the agent may be in a solid form, e.g., as a lyophilized solid, or in a liquid form, e.g., in an aqueous solution. Typically, after the agent has been combined in an aqueous solution with the guanosine and GMP, the solution is maintained under conditions, e.g., at a temperature, ionic strength, and pH, in which gelation of the solution is minimized, and is mechanically agitated to disperse the agent throughout the solution. The mechanical agitation may be accomplished by any one of a variety of methods disclosed herein and known in the art. For example, mechanical agitation may be accomplished by shaking, vortexing, sonicating or passing through a small orifice the composition comprising the agent, guanosine, and GMP. [00121] Following agitation, e.g., immediately following, the conditions of the composition comprising the agent, guanosine, and GMP are altered to facilitate gelation of the guanosine and GMP. For example, gelation may be induced by adjusting the pH of the composition, e.g., by decreasing the pH. Gelation may also be induced by adjusting the temperature of the composition, e.g., by decreasing the temperature. In some embodiments, the agitation is performed at a temperature in a range of about 15 °C to about 25 °C and gelation is brought about following agitation by cooling the composition to a temperature below about 15 °C, e.g., to a range of about 0 °C to about 10 0C. Gelation may also be induced by adjusting the ionic strength of the composition, e.g., by addition of salts. For example, the addition of potassium cations is known to promote G-quartet formation.
Pharmaceutical Compositions
[00122] It is often desirable to control the release rate of certain molecules (e.g., diagnostic agents or therapeutic agents) that are dispersed within the inventive guanosine/GMP gels disclosed herein, e.g., for delivery of the molecules or therapeutic agents to a subject. It may be desirable for this release rate to occur over several days, weeks, or even months. Also, it may be desirable to have a release rate that is controlled and gradual over this time period, with zero order kinetics or time independent release being most preferred and desirable. [00123] Accordingly, methods are provide for tuning the guanosine/GMP gels to achieve a predetermined release rate, which is typically a rate desirable for a particular application, e.g., for bringing about desired therapeutic or diagnostic result. Tuning of guanosine/GMP gels typically comprises setting a parameter of the gel to a level that results in a gel that releases the agent at the predetermined rate. Such parameters include, for example, pH, viscosity, ionic strength, cation concentration, nucleoside concentration, ratio of guanosine to GMP, and agent concentration. Thus, the methods typically include an evaluation or determination of the level of particular parameter that results in a gel that releases the agent at the predetermined rate. The examples section provides exemplary tuning methods, e.g., based on pH levels. Other tuning methods can be achieved using methods well known in the art.
[00124] For example, the level of particular parameter that results in a gel that releases an agent, e.g., an antibody, a ligand, a nucleic acid, a particle, at the predetermined rate may be determined by preparing a gel at a first test level of the parameter; determining a rate of release of an agent from the gel at the first test level; preparing a gel at a second test level of the parameter; determining a rate of release of an agent from the gel at the second test level; and evaluating the rates of release of the agent at the first and second test levels to establish a level of the parameter that results in a gel that releases the agent at the predetermined rate. It is to be understood that the methods are not limited to evaluating only two test levels and that any number of test levels may be evaluated to establish appropriate parameter levels for tuning gels. [00125] The rate of release of the agent from the gel may be determined by maintaining the gel in a medium, e.g., a aqueous solution, a buffer solution, a tissue, in vivo or ex vivo, for a predetermined time, and measuring the concentration of the agent in the medium on one or more occasions during the predetermined time. For therapeutic applications, the predetermined rate is typically a rate of release of an agent from the gel into a medium that maintains, for a predetermined time, a concentration of the agent, in the medium, that is at or above a concentration of the agent in a subject which effects a desired therapeutic response in the subject. For diagnostic applications, the predetermined rate is typically a rate of release of an agent from the gel into a medium that maintains, for a predetermined time, a concentration of the agent, in the medium, that is at or above a concentration of the agent in a subject which facilitates a desired diagnostic read-out, e.g., an imaging read-out, in the subject. [00126] In some applications, the predetermined rate may be 50% of the agent in the gel being released from the gel within up to about 1 day. In other applications, the predetermined rate may be 50% of the agent in the gel being released from the gel within a range of about 1 day to about 30 days. In other applications, the predetermined rate may be 50% of the agent in the gel being released from the gel within a range of about 1 week to about 26 weeks. In still other applications, the predetermined rate may be 50% of the agent in the gel being released from the gel within a range of up to about 1 month to about 12 months.
[00127] As used herein, a "therapeutically effective amount" is an amount of a compound or composition that produces a desired therapeutically beneficial result in a subject. A therapeutically effective amount can refer to any compounds, e.g., therapeutic agents, or compositions described herein. The therapeutically effective amount of the active agent to be included in pharmaceutical compositions depends, in each case, upon several factors, e.g., the type, size and condition of the patient to be treated, the intended mode of administration, the capacity of the patient to incorporate the intended dosage form, etc. Generally, an amount of active agent is included in each dosage form to provide from about 0.1 to about 250 mg/kg, and preferably from about 0.1 to about 100 mg/kg. One of ordinary skill in the art would be able to determine empirically an appropriate therapeutically effective amount. Methods for establishing a therapeutically effective amount for any compounds or compositions described herein will be known to one of ordinary skill in the art. As used herein, pharmacological compositions comprise compounds or compositions that have therapeutic utility, and a pharmaceutically acceptable carrier, i.e., that facilitate delivery of compounds or compositions, in a therapeutically effective amount.
[00128] The disclosure in other embodiments provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such container(s) can be various written materials (written information) such as instructions (indicia) for use, or a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. [00129] As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the compositions of this invention, its use in the therapeutic formulation is contemplated. Supplementary active ingredients can also be incorporated into the pharmaceutical formulations. A composition is said to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by a recipient patient. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers are well-known in the art. See, for example, Remingon's Pharmaceutical Sciences, 18th Ed. (1990). [00130] It will be understood by those skilled in the art that any mode of administration, vehicle or carrier conventionally employed and which is inert with respect to the active agent may be utilized for preparing and administering the pharmaceutical compositions of the present invention. Illustrative of such methods, vehicles and carriers are those described, for example, in Remington's Pharmaceutical Sciences, 4th ed. (1970), the disclosure of which is incorporated herein by reference. Those skilled in the art, having been exposed to the principles of the invention, will experience no difficulty in determining suitable and appropriate vehicles, excipients and carriers or in compounding the active ingredients therewith to form the pharmaceutical compositions of the invention.
[00131] The pharmaceutical compositions of the present invention preferably contain a pharmaceutically acceptable carrier or excipient suitable for rendering the compound or mixture administrable orally as a tablet, capsule or pill, or parenterally, intravenously, intradermally, intramuscularly or subcutaneously, or transdermally. The active ingredients may be admixed or compounded with any conventional, pharmaceutically acceptable carrier or excipient. The pharmaceutical compositions may also comprise suitable buffering agents. The pharmaceutical composition of the invention are also produced under sterile conditions using Good Manufacturing Practices (GMP).
[00132] The compositions disclosed herein may also be formulated with pharmaceutically acceptable salts. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.
[00133] The pharmaceutical compositions disclosed herein may be administered by any suitable means such as orally, intranasally, subcutaneously, intramuscularly, intravenously, intra-arterially, parenterally, intraperitoneally, intrathecally, intratracheally, ocularly, sublingually, vaginally, rectally, dermally, or as an aerosol. Depending upon the type of condition (e.g., cancer) to be treated, compounds of the invention may, for example, be inhaled, ingested or administered by systemic routes. Thus, a variety of administration modes, or routes, are available. The term "parenteral" includes subcutaneous, intravenous, intramuscular, intraperitoneal, and intrasternal injection, or infusion techniques. The particular mode selected will depend, of course, upon the particular composition selected, the particular condition being treated and the dosage required for therapeutic efficacy. The methods, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces acceptable levels of efficacy without causing clinically unacceptable adverse effects. The guanosine/GMP gel compositions may also be implanted, e.g., within, partially or completely, or adjacent to a target tissue, e.g., a diseased tissue, to effect local delivery of a therapeutic or diagnostic agent to the tissue.
EXAMPLES
Example 1: Stabilized Antibody Compositions
[00134] Guanosine (Guo) and guanosine 5 '-monophosphate (GMP) were purchased in solid form from Sigma Aldrich.
[00135] To measure the antibody aggregation at high concentration in various media, lyophilized anti-goat IgG antibody powder produced from rabbit containing sodium chloride and sodium phosphate buffer crystals at pH 7.2 was applied. The sample was 1.7 mg lyophilized antibody powder (containing 0.6 mg protein and 1.1 mg Na3PO4ZNaCl buffer crystal) diluted into 50 μL guanosine/GMP gel composed of 0.25 M GMP and 0.020 M Guo, which gave a final antibody concentration of 12 mg/mL and buffer concentration of 0.10 M Na3PO4, 0.15 M KCl. The control was the same amount of lyophilized antibody powder diluted into 50 μL HPLC-grade water, which gave the same final antibody and buffer concentrations. The same media can be used to stabilize and deliver other proteins such as enzymes, ligands, nucleic acids and receptors, and for peptide release as well.
[00136] To visualize antibody aggregation, 4 μL of 1 mg/mL anti-rabbit whole
IgG antibody labeled with atto-540 fluorescence dye was added into each vial and vortexed for 15 seconds to ensure even distribution. This served as a secondary antibody to the anti-goat IgG antibody powder produced from rabbit and provided a fluorescent signal.
[00137] Immediately after vortexing, 10 μL of the sample or control solution was dropped on a pre-cleaned glass slide and covered with a glass cover slide for imaging. The remainders of the sample and control solutions were stored in ice for later measurements.
[00138] Antibody aggregation was tested using a Zeiss LSM 510 Meta laser scanning confocal microscope equipped with a Meta detector that can capture emissions ranging from 411-754 run, in 10.7 nm bins across 32 channels. In all tests, the XlOO oil objective was used and the 518 F oil was applied between the sample slice and objective to achieve the best light transmission. The 1 mW 543 nm green line from a HeNe laser was applied as excitation with an output at around 90% and the detector was set with gain at around 700.
Results
[00139] Figures 1 shows two randomly selected, representative spots of freshly prepared antibody in water. Figure 2 shows two randomly selected, representative spots of fresh prepared antibody in the G-gel. Figure 3 shows the antibody in water after 25 min storage in ice and Figure 3 shows antibody in G-gel after 30 min storage in ice. Figure 4 shows images of a different sample of antibody dispersed in H2O (left) and G-gel (right) after 3 h storage in the refrigerator. The preparation and microscopy were performed identically to the samples used in Figures 1-3.
Example 2: Extended Release Delivery [00140] The guanosine/GMP gels are able to be tuned for timed release of pharmaceutical compounds. To demonstrate this, we made solutions of the fluorescent dye fluorescein in different G-gel formulations, added a layer of water on top of the gel, and observed the diffusion of the fluorescein dye from the gel into the water. As shown in Figure 5, fluorescein is released much faster from the low viscosity G-gel than high viscosity G-gel, demonstrating the tunability of the G-gel to specify the rate of release. [00141] In a second set of experiments, we tested the effects of different G-gel conditions including pH and G-gel composition on diffusion of a different fluorescent dye, Rhodamine, from the G-gel into the water layer. As shown in Figure 6, both pH and composition are important determinants of diffusion rate and they can be combined to tailor delivery for specific conditions for drug delivery. For example, diffusion tends to be slow at acidic pH and rapid at basic pH, especially at the lower Guo content. If a drug in this gel were ingested orally, it would not diffuse in the acidic environment of the stomach but would be released once it reached the higher pH environment of the intestines. G-gel composition could be optimized for different administration routes as well, for example, to control the rate of release of a drug that is delivered intravenously into the near-neutral pH of blood.
Example 3: Nanoparticle Dispersion in G-gelsfor " Nanopharmaceuticals" [00142] Inorganic nanoparticles have low solubility in aqueous solution and high tendency to agglomerate. Existing methods generally involve surface treatment to stabilize the particles in aqueous solution. Drawbacks are that these approaches are time consuming, they add to the production cost, the processes may not be scalable, and the modifications may limit the product applications. The G-gels discussed in this paper are capable of dispersing individual, unmodified, inorganic nanoparticles such as TiO2, ITO, and ZnO in suspensions that are stable for weeks to years. The mechanism is not yet understood but appears to be a random encapsulation of the particles by the gel.
[00143] Inorganic nanoparticles could be used to fine-tune the G-gel media for protein dispersion and drug delivery. Further, the nanoparticles could be used to form nanostructured proteins and drugs within the G-gel media to enable delivery on a nano- scale. The facile, individual dispersion of nanoparticles in G-gels will provide a simple and scalable means for development and implementation of "nanopharmaceuticals". [00144] Without being bound by a particular theory, the mechanism of solubilization appears to be encapsulation of the particles by the gel through nonspecific interactions. This is in contrast to previously discovered media formed by selective dispersion of carbon nanotubes in guanosine/GMP gels in which the rod-like tubes are preferentially solubilized based on chirality and diameter through highly specific interactions of the tubes with the linear, rod-like guanosine assemblies that form the organized gel, and result in a medium in which the carbon nanotubes are selectively solubilized and aligned in the G-gel; the nanotubes actually become a component of the G-gel architecture through close and selective interactions with the nanostructures that comprise the G-gel scaffold. This is illustrated in Figure 7. In some embodiments of the present invention, the nanoparticles show no evidence of alignment or other non-random organization; this indicates a mechanism of solubilization and dispersion that is different than that observed in the carbon nanotube containing gels noted above. In some embodiments of the present invention, the nanoparticles are randomly dispersed in the G-gel without actually becoming a part of the G-gel architecture itself. The nanoparticles are merely encapsulated in random sites in the bulk G-gel medium and are therefore more readily released from the gel simply by changing the properties of the bulk solution such as pH.
Example 4: Medium for Nanoparticles Dispersion
[00145] Suspensions of spherical, inorganic nanoparticles such as TiO2, ZnO,
Indium tin Oxide (ITO), etc., in a reversible gel medium formed by binary mixtures of guanosine compounds are disclosed herein. TiO2 is the mostly used white pigment and has applications in wide-ranging areas including paints and coatings, sunscreens and solar cells. ITO nanoparticles can be utilized to prepare transparent conductive film and are widely used in touch screens. Quantum dots have numerous applications in chemical and biological imaging and analysis as well as nanostructured devices. Inorganic nanoparticles are difficult to handle due to their low solubility in aqueous solution and high tendency to agglomerate. Existing methods generally involve surface treatment to stabilize the particles in aqueous solution. Drawbacks are that these approaches are time consuming, they add to the production cost, the processes may not be scalable, and the modifications may limit the product applications. [00146] As disclosed herein, unmodified particles are readily and rapidly solubilized to form suspensions that are stable for weeks or longer. Without being bound by a particular theory, the mechanism appears to be encapsulation of the particles by the gel through nonspecific interactions. This is in contrast to previously discovered media formed by selective dispersion of carbon nanotubes in guanosine/GMP gels, in which case the rod-like tubes are preferentially solubilized based on chirality and diameter, presumably through specific interactions with the linear, rod-like guanosine assemblies that form the organized gel. The result is a medium in which the carbon nanotubes are selectively aligned in the gel. In certain embodiments of the present invention, the nanoparticles are spherical and show no evidence of alignment or other organization, indicating a different mechanism of solubilization and dispersion. The gel medium is nontoxic and biocompatible. It is formed by simple, commercial compounds in aqueous solution. The properties such as viscosity and thermal stability of the nanoparticle suspensions can be tuned by varying the composition of the gel medium. For example, the temperature response of the suspensions can be tuned so that they transition from liquid to gel with increasing or decreasing temperature to facilitate handline, formation of homogeneous dispersions, injectability, etc. The rate of gelation can be controlled as well using thermal, pH or cation gradients.
The following are specific examples of nanoparticle dispersion:
TiO 2 Nanoparticles
[00147] Figure 8 shows dispersion of 6 different samples of TiO2 (mean diameter approximately 6-8 run) in the aqueous gel medium immediately following 30 s sonication at 0°C and the same coated with ZnO2 and/or Al2O3. The results demonstrate the long-term stability of the aqueous suspensions. Figure 9 shows suspensions of TiO2 samples 1 and 5 in water prepared using the same protocol as for the gel suspensions. The photograph was taken 3 days after sample preparation. The TiO2 begins to precipitate within 12 hours following sonication. Figure 9 also shows a side-by-side comparison OfTiO2 sample 1 in water and in the gel 3 days following preparation. The TiO2 has precipitated out of the water but remains dispersed in the gel. Figure 10 shows AFM images Of TiO2 that was sonicated for 10 min in water and in the gel, dropped on a silicon substrate and air dried overnight. The TiO2 exists as large aggregates in water but is highly dispersed as small (individual) particles in the gel.
ZnO Nanoparticles
[00148] Figure 11 shows untreated ZnO nanoparticles prepared in water and in gel after 2 days. The vials were inverted for the photograph in order to show phase separation in both vials. In water, the solution at the bottom of the inverted vial contains essentially no nanoparticles, which remained instead as a precipitate at the top of the inverted vial and coating the surrounding vial wall. In gel, there is a highly viscous phase at the top of the inverted vial that contains nanoparticles as well. Figure 12 shows AFM images of ZnO in water and in gel, prepared as described above for AFM images of TiO2. Again, the particles show heavy aggregation and clumping in water but are much more individually dispersed in the gel.
ITO Nanoparticles
[00149] Figure 13 shows ITO (approximately 50 nm mean diameter) prepared in water and in gel after 1 day. The nanoparticles have begun to precipitate from the water solution but remain dispersed in the gel. Figure 14 shows an AFM image of the ITO in gel. The particles are well-dispersed and discrete. The ITO in water was too clumped and rough to be imaged by AFM.
[00150] References
1. On the web at blog.leinco.com/tag/therapeutic-antibodies/, Posts tagged "Therapeutic Antibodies: In vivo functional formulation monoclonal antibodies and recombinant proteins," 2008.
2. On the web at genengnews.com/articles/chitem.aspx?aid=949&chid=3, Addressing stability of biological drugs: Forced degradation studies predict effects on bioproducts in drug development and manufacture, Genetic Engineering and Biotechnology News 2005, Vol. 25.
3. Cromwell, MEM; Hilario, E.; Jacobson, F., Protein aggregation and bioprocessing, The AAPS Journal 2006, E572-E579. Example 5: Binary Guanosine Gels For Encapsulation Of Living Cells [00151] Certain in vivo applications involve cells being encapsulated in a G-GeI in a form that establishes a boundary between the cells and a host organism. Encapsulation may sequester the living cells and protect them from degradation and immunological attack by the host organism while sustaining them during the course of their desired function. As will be appreciated by the skilled artisan, specific requirements for a given application will depend upon various factors including the type of cell (e.g., stem, islet, hepatocyte, non-human, microorganism, either natural or genetically engineered), its therapeutic role (e.g., gene delivery, tissue regeneration, hormone production, tumor destruction, vaccine production) and where that functional role will be targeted (e.g., external use or in vivo in skin, eye, bone, brain, internal organ, gastrointestinal tract, etc.). For particular in vivo applications, a cell encapsulating G-gel will be prepared with consideration of the following attributes:
• Mechanical strength and structural flexibility
• Biocompatibility and non-immunogenicity
• Selective permeability, e.g., allowing exchange of small ions and molecules (oxygen, nutrients, electrolytes, hormones, wastes) with the external surroundings while preventing transfer of antigenic macromolecules to the host and immunogenic responders such as antibodies and T-cells from the host
• Homogeneous cell dispersion, high purity and batch-to-batch reproducibility
• Ability to encapsulate cells under conditions that will not harm the cells
• Ability to regulate cell proliferation to avoid overgrowth and overcrowding
• Inexpensive and simple preparation
[00152] Additionally, some applications may require that the capsules be scaled up in size without losing mechanical stability and selective permeability.
Binary G-gelsfor Encapsulation of Living Cells
[00153] Reversible guanosine gels (G-gels) as biomaterials for encapsulation of living cells are disclosed. G-gels are self-assembled networks of hydrogen-bonded guanine tetrads formed by guanosine nucleosides and nucleotides. Their organization and viscosity are dependent upon monomer concentration, temperature, pH and cation content, providing multiple variables for controlling and modulating the properties of the gels.
[00154] Advantages offered by thermoassociative G-gels formed by mixtures of guanosine and guanosine monophosphate in certain proportions that have the potential to overcome certain challenges that have limited the success of other materials for cell encapsulation:
• Cells can be readily and homogeneously incorporated into the G-gels simply by adding them to refrigerated solutions and bringing the resulting mixtures to room or physiological temperature.
• The negatively charged surface of the G-gels will inhibit adhesion and adsorption of host cells and proteins.
• Unlike the polymeric gels, G-gels are formed by small molecules that occur naturally in the body and may therefore be more easily eliminated from the body and less likely to trigger immune responses.
• The G-gel scaffold offers higher regularity that may provide better control of "pore" size and cell distribution compared to randomly cross-linked polymers.
• The G-gels are formed from simple compounds that are readily available at high purity and relatively low cost, unlike gels formed from alginates and other biomaterials derived from natural sources that may require purification, adding to their cost, and are difficult to standardize, to standardize.
• G-gels are stable at neutral pH and their stability increases with decreasing pH, which makes them excellent candidates for certain uses in the gastrointestinal tract.
• Like hydrogels, G-gels are highly responsive to temperature and pH, as well as specific cations such as K+, providing several independent variables for manipulation of gelation.
Results
[00155] We evaluated the extent to which thermoassociative G-gels incorporate and sustain living cells. We investigated the viability of human neonatal dermal fibroblasts in thermoassociative G-gels of 6 different Guo/5'GMP ratios. The fibroblast cells first were cultured on standard tissue culture plastic in 50/50 Dulbecco's Modified Eagle Medium (DMEMf) with 10% fetal bovine serum, 10 U/ml Penicillin and 100 μg/ml Streptomycin, and 2 mM L-glutamine at pH 7.2., and were passaged at confluence. For embedding in gels, cells at passage 9 were collected by trypsinization and then washed to remove any remaining trypsin. Cells were centrifuged into pellet at 1000 rpm for 5 min. in preparation for being encapsulated. The G-gel was prepared in 50/50 DMEMf and kept in the solution phase until cells were ready to be inserted. The G-gel and cells were then mixed with a pipette and transferred into a 6- well plate. Following gelation the plate was covered and the constructs were incubated at 37 0C and 5.0 % CO2 for 3 hours. Digital images were taken of the entire plate and of each construct at 1OX magnification using a light microscope. 5 ml of medium was added to each well and gels were separated from the sides and bottoms of the wells. After three days a live/dead assay was performed on the constructs. Control gels that were prepared and incubated under identical conditions but without cells were also analyzed. [00156] As shown in Figure 16, there is evidence of cell growth in the gels seeded with fibroblasts. Live cells were indicated in the live/dead assay on Day 3 (data not shown). There was no evidence of significant cell growth in the cell-free control gels. Results for the other four gels were consistent with these, although with differing extents of cell growth.
[00157] We performed further experiments to evaluate cell encapsulation ability as a function of binary G-gel composition. Rat aortic smooth muscle cells were used in these studies. The results are shown in images of live/dead assays obtained using confocal microscopy. (Figure 17A.) Calcien and Ethidium Homodimer were used to indicate the live and dead cells, respectively. Green indicates stained cytoplasm of live cells, red indicates stained nuclei of dead cells. Each experiment was performed in triplicate, and so there are three images (corresponding to three different wells) for each G-gel. The results indicate that some gel compositions are much better than others for cell encapsulation.
[00158] Next we performed live/dead assays on cell in G-gels after 1 week. The results Figure 17B show that cells remain viable after 1 week. [00159] The results disclosed herein show that living cells can be incorporated and dispersed within binary G-gels without destroying them and that they remain viable even after a week of incubation.
Example 6: Evaluation of PLGA in G-gels [00160] Poly(DL-lactide-co-glycolide) (PLGA) (average Mw 5,000-15,000, lactide:glycolide (50:50), Sigma- Aldrich) was dissolved at a concentration of 1 mg/mL in G-gel (0.25 M GMP, 0.02 M Guo), and 0.04 M phosphate buffer (pH 7.4) as a control. The solutions were gently vortexed for ~10 s and monitored for precipitation for 1 h post solubilization. Initially, no precipitation of polymer was observed in G-gel or control buffer, however, precipitation was apparent within 5 min post solubilization. Precipitants could not be brought back into solution in spite of subjecting it to extended periods of vortexing. Figure 18A shows precipitation in both G-gel (left) and buffer (right) 5 min after solubilization. The precipitation of polymer crystals indicated poor dispersability of PLGA in G-gels.
[00161] Efforts to facilitate PLGA dispersion by sonication in a separate set of experiments also were unsuccessful. Figure 18B shows results for PLGA in G-gel (left) and buffer (right) after more than 2 h sonication. These results confirm our conclusion that PLGA cannot be stably dispersed in G-gels. The observed change of PLGA color in G-gels (left) in comparison to buffer (right) may be attributed to surface modification of PLGA by G-gel components.

Claims

CLAIMS What is claimed is:
1. A composition comprising an antibody dispersed in a guanosine/GMP gel.
2. The composition of claim 1, wherein the guanosine/GMP gel comprises guanosine and guanosine 5 '-monophosphate (GMP).
3. The composition of claim 1 or 2, wherein the antibody is randomly dispersed in the gel.
4. The composition of any one of claims 1 to 3, wherein the concentration of the antibody in the gel, is at or above a concentration that results in aggregation, degradation, or decreased activity of the antibody in a control solvent.
5. The composition of any one of claims 1 to 4, wherein the control solvent is an aqueous solution.
6. The composition of any one of claims 1 to 5, wherein the aqueous solution is water or a mixture of water and an organic solvent, optionally wherein the organic solvent is selected from the group consisting of: methanol, ethanol, isopropanol, n-propanol, tert-butyl alcohol, n-butyl alcohol, ethyl acetate, and acetonitrile.
7. The composition of any one of claims 1 to 6, wherein the concentration of the antibody ranges from about 0.1 mg/ml to about 100 mg/ml.
8. The composition of any one of claims 1 to 7, wherein the concentration of the antibody ranges from about 1 mg/ml to about 25 mg/ml.
9. The composition of any one of claims 1 to 8, wherein the concentration of the antibody ranges from about 5 mg/ml to about 15 mg/ml.
10. The composition of any one of claims 1 to 9, wherein the antibody is a human antibody, a humanized antibody, a monoclonal antibody, a polyclonal antibody, a single-chain antibody or an antibody fragment.
11. The composition of claim 10, wherein the antibody fragment is a F(ab')2, Fab, Fd, or Fv fragment.
12. The composition of any one of claims 1 to 1 1, wherein the antibody binds to a protein of a signaling pathway selected from: VEGF, Integrin, Androgen Receptor, B-CeIl Receptor, EGFRl, IL-2, IL-4, IL-6, Kit, Notch, T Cell Receptor, TGFβ, TNF-α, and WNT.
13. The composition of any one of claims 1 to 12, wherein the antibody is conjugated to a therapeutic or diagnostic agent.
14. The composition of claim 13, wherein the therapeutic agent is a cytotoxic compound, a nucleic acid, a toxin, or a radioisotope.
15. The composition of claim 14, wherein the cytotoxic compound is selected from the group consisting of: calicheamicin, esperamicin, methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin C, cisplatinum, etopside, bleomycin and 5-fluorouracil.
16. The composition of claim 14, wherein the radioisotope is selected from the group consisting of: 225Ac, 211At, 212Bi, 213Bi, 186Rh, 188Rh, 177Lu, 90Y, 131I, 67Cu, 1251, 1231, 77Br, 153Sm, 166Bo, 64Cu, 212Pb, 224Ra and 223Ra.
17. The composition of any one of claims 1 to 16, wherein the antibody is selected from the group consisting of: Abciximab, Adalimumab, Alemtuzumab, Basiliximab, Bevacizumab, Cetuximab, Daclizumab, Eculizumab, Efalizumab, Gemtuzumab, Ibritumomab tiuxetan, Infliximab, Muromonab-CD3, Natalizumab, Omalizumab, Palivizumab, Panitumumab, Ranibizumab, Rituximab, Tositumomab, and Trastuzumab.
18. The composition of any one of claims 1 to 16, wherein the antibody binds specifically to an antigen of an antibody selected from the group consisting of: Abciximab, Adalimumab, Alemtuzumab, Basiliximab, Bevacizumab, Cetuximab, Daclizumab, Eculizumab, Efalizumab, Gemtuzumab, Ibritumomab tiuxetan, Infliximab, Muromonab-CD3, Natalizumab, Omalizumab, Palivizumab, Panitumumab, Ranibizumab, Rituximab, Tositumomab, and Trastuzumab.
19. The composition of any one of claims 1 to 18, wherein the ratio of guanosine to GMP ranges from about 0.01 to about 1.
20. The composition of any one of claims 1 to 19, wherein the ratio of guanosine to GMP ranges from about 0.05 to about 0.25.
21. The composition of any one of claims 1 to 20, wherein the ratio of guanosine to GMP is about 0.05.
22. The composition of any one of claims 1 to 20, wherein the ratio of guanosine to GMP is about 0.15
23. The composition of any one of claims 1 to 20, wherein the ratio of guanosine to GMP is about 0.25.
24. The composition of any one of claims 1 to 23, wherein the GMP concentration ranges from about 1 mM to about 500 mM.
25. The composition of any one of claims 1 to 24, wherein the GMP concentration ranges from about 50 mM to about 300 mM.
26. The composition of any one of claims 1 to 25, wherein the GMP concentration is about 200 mM.
27. The composition of any one of claims 1 to 25, wherein the GMP concentration is about 250 mM.
28. The composition of any one of claims 1 to 23, wherein the guanosine concentration ranges from 0.1 mM to about 100 mM.
29. The composition of any one of claims 1 to 23 and 28, wherein the guanosine concentration ranges from about 1 mM and 50 mM.
30. The composition of any one of claims 1 to 23, 28 and 29, wherein the guanosine concentration is about 10 mM.
31. The composition of any one of claims 1 to 23, 28 and 29, wherein the guanosine concentration is about 30 mM.
32. The composition of any one of claims 1 to 23, 28 and 29, wherein the guanosine concentration is about 50 mM.
33. The composition of any one of claims 1 to 32, further comprising a cation selected from: Li+, Na+, K+, Rb+, Cs+, NH4 +, Ag+, Ti+, Mg"", Ca+^ Sr" N(CH3)4+ and Ba4+.
34. The composition of any one of claims 1 to 33, wherein the pH of the composition is up to about 6.5.
35. The composition of any one of claims 1 to 33, wherein the pH of the composition ranges from about 6.5 and about 7.5.
36. The composition of any one of claims 1 to 33, wherein the pH of the composition is above 7.5.
37. The composition of any one of claims 1 to 36, wherein the temperature of the gel is up to about 0 0C.
38. The composition of any one of claims 1 to 36, wherein the temperature of the gel ranges from 0 0C to about 5 0C.
39. The composition of any one of claims 1 to 36, wherein the temperature of the gel ranges from about 15 0C to about 25 0C.
40. The composition of any one of claims 1 to 36, wherein the temperature of the gel ranges from about 35 0C to about 40 0C.
41. The composition of any one of claims 1 to 40, further comprising a salt or a buffering agent.
42. A composition comprising an isolated antibody, guanosine, and GMP.
43. The composition of claim 42, wherein the guanosine and GMP are of a gel form.
44. A composition comprising a nucleic acid dispersed in a guanosine/GMP gel.
45. The composition of claim 44, wherein the guanosine/GMP gel comprises guanosine and guanosine 5 '-monophosphate (GMP).
46. The composition of claim 44 or 45, wherein the nucleic acid is randomly dispersed in the gel.
47. The composition of any one of claims 44 to 46, wherein the nucleic acid is a RNA, DNA, peptide nucleic acid (PNA), or a combination thereof.
48. The composition of any one of claims 44 to 47, wherein the nucleic acid is an small interfering nucleic acid.
49. The composition of claim 48, wherein the small interfering nucleic acid is a shRNA, microRNA, siRNA, antisense oligonucleotide, or a miRNA sponge.
50. The composition of any one of claims 44 to 47, wherein the nucleic acid is a plasmid vector.
51. The composition of claim 50, wherein the plasmid vector is a vector that expresses a mRNA or a small interfering nucleic acid.
52. The composition of any one of claims 44 to 47, wherein the nucleic acid is in a virus vector.
53. The composition of claim 52, wherein the virus vector is an adenovirus, lentivirus, retrovirus, herpesvirus, or adeno-associated virus.
54. The composition of any one of claims 44 to 53, wherein the nucleic acid is conjugated to an antibody, ligand or a particle.
55. The composition of any one of claims 44 to 53, wherein the nucleic acid is encapsulated in a particle.
56. A composition comprising a particle dispersed a guanosine/GMP gel.
57. The composition of claim 56, wherein the guanosine/GMP gel comprises guanosine and guanosine 5 '-monophosphate (GMP).
58. The composition of claim 56 or 57, wherein the particle is randomly dispersed in the gel.
59. The composition of any one of claims 56 to 58, wherein the particle is a nano-particle.
60. The composition of any one of claims 56 to 58, wherein the particle is a micro-particle.
61. The composition of any one of claims 56 to 58, wherein the particle is in a range of about 1 nM to less than 1 μM in diameter.
62. The composition of any one of claims 56 to 58, wherein the particle is in a range of about 1 μM to less than 1 mM in diameter.
63. The composition of any one of claims 56 to 58, wherein the particle is in a range of about 1 nM to about 10 nM in diameter.
64. The composition of any one of claims 56 to 58, wherein the particle is conjugated to a therapeutic or diagnostic agent.
65. The composition of any one of claims 56 to 64, wherein the particle encapsulates a therapeutic or diagnostic agent.
66. The composition of any one of claims 56 to 65, wherein the particle is at a concentration in a range of about 0.1 to about 100 mg/ml.
67. The composition of any one of claims 56 to 66, wherein the particle is at a concentration in a range of about 0.5 to about 50 mg/ml.
68. The composition of any one of claims 56 to 67, wherein the particle is at a concentration in a range of about 1 to about 10 mg/ml.
69. The composition of any one of claims 56 to 68, wherein the particle is a ZnS, ZnO, TiO2, AgI, AgBr, HgI2, PbS, PbSe, ZnTe, CdTe, In2S3, In2Se3, Cd3P2, Cd3As2, InAs, or GaAs particle.
70. The composition of any one of claims 56 to 69, wherein the particle comprises glass, polyethylene, polystyrene, silicone, polyfluoroethylene, polyacrylic acid, a polyamide, polycarbonate, polysulfone, polyurethane, polybutadiene, polybutylene, polyethersulfone, polyetherimide, polyphenylene oxide, polymethylpentene, polyvinylchloride, polyvinylidene chloride, polyphthalamide, polyphenylene sulfide, polyester, polyetheretherketone, polyimide, polymethylmethacylate or polypropylene.
71. A composition comprising a ligand dispersed a guanosine/GMP gel.
72. The composition of claim 71, wherein the ligand is conjugated to a therapeutic or diagnostic agent.
73. The composition of any one of claims 44 to 72, wherein the ratio of guanosine to GMP ranges from about 0.01 to about 1.
74. The composition of any one of claims 44 to 73, wherein the ratio of guanosine to GMP ranges from about 0.05 to about 0.25.
75. The composition of any one of claims 44 to 74, wherein the ratio of guanosine to GMP is about 0.05.
76. The composition of any one of claims 44 to 74, wherein the ratio of guanosine to GMP is about 0.15
77. The composition of any one of claims 44 to 74, wherein the ratio of guanosine to GMP is about 0.25.
78. The composition of any one of claims 44 to 77, wherein the GMP concentration ranges from about 1 mM to about 500 mM.
79. The composition of any one of claims 44 to 78, wherein the GMP concentration ranges from about 50 mM to about 300 mM.
80. The composition of any one of claims 44 to 79, wherein the GMP concentration is about 200 mM.
81. The composition of any one of claims 44 to 79, wherein the GMP concentration is about 250 mM.
82. The composition of any one of claims 44 to 77, wherein the guanosine concentration ranges from 0.1 mM to about 100 mM.
83. The composition of any one of claims 44 to 77 and 82, wherein the guanosine concentration ranges from about 1 mM and 50 mM.
84. The composition of any one of claims 44 to 77, 82 and 83, wherein the guanosine concentration is about 10 mM.
85. The composition of any one of claims 44 to 77, 82 and 83, wherein the guanosine concentration is about 30 mM.
86. The composition of any one of claims 44 to 77, 82 and 83, wherein the guanosine concentration is about 50 mM.
87. The composition of any one of claims 44 to 77, 82 and 83, further comprising a cation selected from: Li+, Na+, K+, Rb+, Cs+, NH4 +, Ag+, Ti+, Mg4+, Ca+^ Sr+^ N(CH3)4+ and Ba+^
88. The composition of any one of claims 44 to 87, wherein the pH of the composition is up to about 6.5.
89. The composition of any one of claims 44 to 87, wherein the pH of the composition ranges from about 6.5 and about 7.5.
90. The composition of any one of claims 44 to 87, wherein the pH of the composition is above 7.5.
91. The composition of any one of claims 44 to 90, wherein the temperature of the gel is up to about 0 0C.
92. The composition of any one of claims 44 to 90, wherein the temperature of the gel ranges from 0 °C to about 5 °C.
93. The composition of any one of claims 44 to 90, wherein the temperature of the gel ranges from about 15 °C to about 25 °C.
94. The composition of any one of claims 44 to 90, wherein the temperature of the gel ranges from about 35 °C to about 40 °C.
95. The composition of any one of claims 44 to 94, further comprising a salt or a buffering agent.
96. A method for producing a stable antibody composition, the method comprising: dispersing an antibody in a guanosine/GMP gel.
97. The method of claim 96, wherein the gel comprises guanosine and guanosine 5 '-monophosphate.
98. The method of claim 96 or 97, wherein the dispersing comprises shaking, vortexing, sonicating or passing through a small orifice a composition comprising the antibody and constituents of the guanosine/GMP gel.
99. The method of any one of claims 96 to 98, further comprising determining if the distribution of the antibody in the gel is random.
100. The method of any one of claims 96 to 99, wherein the dispersing is performed at a temperature in a range of about 15 0C to about 25 °C.
101. The method of any one of claims 96 to 100, further comprising cooling the composition to a temperature below about 15 °C.
102. The method of any one of claims 96 to 101, wherein the concentration of the antibody in the gel, is at or above a concentration that results in aggregation, degradation, or decreased activity of the antibody in a control solvent.
103. The method of any one of claims 96 to 102, wherein the control solvent is an aqueous solution.
104. The method of claim 103, wherein the aqueous solution is water or a mixture of water and an organic solvent, optionally wherein the organic solvent is selected from the group consisting of: methanol, ethanol, isopropanol, n-propanol, tert- butyl alcohol, n-butyl alcohol, ethyl acetate, and acetonitrile.
105. The method of any one of claims 96 to 104, wherein the antibody is dispersed in the gel at a concentration in a range of from about 0.1 mg/ml to about 100 mg/ml.
106. The method of any one of claims 96 to 105, wherein the antibody is dispersed in the gel at a concentration in a range of from about 1 mg/ml to about 25 mg/ml.
107. The method of any one of claims 96 to 106, wherein the antibody is dispersed in the gel at a concentration in a range of from about 5 mg/ml to about 15 mg/ml.
108. A method for producing a stable nucleic acid composition, the method comprising: dispersing a nucleic acid in a guanosine/GMP gel.
109. The method of claim 108, wherein the gel comprises guanosine and guanosine 5 '-monophosphate.
110. The method of claim 108 or 109, wherein the dispersing comprises shaking, vortexing, sonicating or passing through a small orifice a composition comprising the nucleic acid and constituents of the guanosine/GMP gel.
111. The method of any one of claims 108 to 110, further comprising determining if the distribution of the nucleic acid in the gel is random.
112. A method for producing a stable particle composition, the method comprising: dispersing a particle in a guanosine/GMP gel.
113. The method of claim 112, wherein the gel comprises guanosine and guanosine 5 '-monophosphate.
1 14. The method of claim 1 12 or 113, wherein the dispersing comprises shaking, vortexing, sonicating or passing through a small orifice a composition comprising the particle and constituents of the guanosine/GMP gel.
115. The method of any one of claims 112 to 1 14, further comprising determining if the distribution of the particle in the gel is random.
116. A method for producing a composition that effects controlled-release of an antibody in a subject, the method comprising: dispersing an antibody in a guanosine/GMP gel that is tuned to release the antibody from the gel into a surrounding medium at a predetermined rate.
117. A method for producing a composition that effects controlled-release of a nucleic acid in a subject, the method comprising: dispersing a nucleic acid in a guanosine/GMP gel that is tuned to release the nucleic acid from the gel into a surrounding medium at a predetermined rate.
118. A method for producing a composition that effects controlled-release of a particle in a subject, the method comprising: dispersing the particle in a guanosine/GMP gel that is tuned to release the particle from the gel into a surrounding medium at a predetermined rate.
119. A method for producing a composition that effects controlled-release of an agent in a subject, the method comprising: tuning a guanosine/GMP gel to release an agent from the gel into a surrounding medium at a predetermined rate, and dispersing the agent in a guanosine/GMP gel.
120. The method of claim 119, wherein tuning comprises setting a parameter of the gel to a level that results in a gel that releases the agent at the predetermined rate, wherein the parameter is selected from: pH, viscosity, ionic strength, cation concentration, nucleoside concentration, ratio of guanosine to GMP, and agent concentration.
121. The method of claim 119 or 120, further comprising determining the level that results in a gel that releases the agent at the predetermined rate,
122. The method of claim 120 or 121, wherein the level is determined by preparing a gel at a first test level of the parameter; determining a rate of release of an agent from the gel at the first test level; preparing a gel at a second test level of the parameter; determining a rate of release of an agent from the gel at the second test level; and evaluating the rates of release of the agent at the first and second test levels to establish a level of the parameter that results in a gel that releases the agent at the predetermined rate.
123. The method of any one of claims 119 to 122, wherein the rate of release of the agent from the gel is determined by maintaining the gel in a medium for a predetermined time, and measuring the concentration of the agent in the medium on one or more occasions during the predetermined time.
124. The method of any one of claims 119 to 123, wherein the predetermined rate is a rate of release of an agent from the gel into a medium that maintains, for a predetermined time, a concentration of the agent, in the medium, that is at or above a concentration of the agent in a subject which effects a desired therapeutic response in the subject.
125. The method of any one of claims 119 to 123, wherein the predetermined rate is 50% of the agent in the gel being released from the gel within up to about 1 day.
126. The method of any one of claims 119 to 123, wherein the predetermined rate is 50% of the agent in the gel being released from the gel within a range of about 1 day to about 30 days.
127. The method of any one of claims 119 to 123, wherein the predetermined rate is 50% of the agent in the gel being released from the gel within a range of about 1 week to about 26 weeks.
128. The method of any one of claims 119 to 123, wherein the predetermined rate is 50% of the agent in the gel being released from the gel within a range of up to about 1 month to about 12 months.
129. The method of any one of claims 119 to 128, wherein the agent is conjugated to an antibody.
130. The method of any one of claims 119 to 128, wherein the agent is conjugated to a microparticle, or a nanoparticle.
131. The method of any one of claims 119 to 130, wherein the agent is encapsulated by a microparticle, or a nanoparticle.
132. The method of any one of claims 119 to 128, wherein the agent is an antibody or a nucleic acid.
133. The method of any one of claims 119 to 132, wherein the agent is dispersed in the gel under sterile conditions.
134. A method for administering an antibody to a subject, the method comprising administering to the subject a composition comprising a effective amount of an antibody dispersed in a guanosine/GMP gel.
135. The method of claim 134, wherein the administering is performed is orally, intravenously, intrapleurally, intranasally, intramuscularly, subcutaneously, intraperitoneally, or as an aerosol.
136.. The method of claim 134 or 135, wherein the administering comprises implanting the gel in the subject.
137. The method of any one of claims 134 to 136, wherein the gel is implanted fully or partially within or adjacent to a diseased tissue of the subject.
138. The method of claim 137, wherein the diseased tissue is a cancer tissue.
139. The method of claim 138, wherein the antibody binds specifically to a cell of the cancer tissue.
140. The method of any one of claims 134 to 139, wherein the antibody is conjugated to a therapeutic agent.
141. The method of claim 137, wherein the diseased tissue is inflamed.
142. The method of claim 141, wherein the antibody binds to a receptor of an inflammatory pathway and inhibits inflammation in the subject.
143. The method of any one of claims 134 to 142, wherein the antibody is released from the guanosine/GMP gel at a predetermined rate.
144. The method of any one of claims 134 to 143, wherein the predetermined rate is 50% of the antibody in the gel being released within up to about 1 day.
145. The method of any one of claims 134 to 143, wherein the predetermined rate is 50% of the antibody in the gel being released within a range of about 1 day to about 30 days.
146. The method of any one of claims 134 to 143, wherein the predetermined rate is 50% of the antibody in the gel being released within a range of about 1 week to about 26 weeks.
147. The method of any one of claims 134 to 143, wherein the predetermined rate is 50% of the antibody in the gel being released within a range of about 1 month to about 12 months.
148. The method of any one of claims 134 to 147, wherein release of the antibody from the gel results in a concentration of the antibody in the subject that is sufficient to effect a desired therapeutic result.
149. The method of any one of claims 134 to 147, wherein the subject has or is suspected of having a disease treatable by the antibody, optionally wherein the disease is cancer.
150. The method of claim 149, wherein the antibody binds to a cell of the cancer.
151. The method of claim 150, wherein the antibody is conjugated with a therapeutic agent.
152. The method of claim 151, wherein the therapeutic agent is a toxin, a nucleic acid, an cytotoxic compound, or a radioisotope.
153. The method of any one of claims 134, 135 and 143 to 152, wherein the composition is administered intravenously and the guanosine/GMP gel is tuned to release the antibody at a predetermined rate at a pH between about 7.2 and 7.6.
154. The method of any one of claims 134, 135 and 143 to 152, wherein the composition is administered orally and the guanosine/GMP gel is tuned to release the antibody at a predetermined rate at a pH about equal to the pH of the intestines of the subject.
155. The method of claim 154, wherein the guanosine/GMP gel is tuned to release the antibody at substantially less than the predetermined rate at a pH about equal to the pH of the stomach of the subject.
156. A method for administering a nucleic acid to a subject, the method comprising administering to the subject a composition comprising an effective amount of a nucleic acid dispersed in a guanosine/GMP gel.
157. The method of claim 156, wherein the administering is performed is orally, intravenously, intrapleurally, intranasally, intramuscularly, subcutaneously, intraperitoneally, or as an aerosol.
158. The method of claim 156 or 157, wherein the administering comprises implanting the gel in the subject.
159. The method of claim 158, wherein the gel is implanted within, fully or partially, or adjacent to a diseased tissue of the subject.
160. The method of claim 159, wherein the diseased tissue is a cancer.
161. The method of any one of claims 156 to 160, wherein the nucleic acid is released from the guanosine/GMP gel at a predetermined rate.
162. The method of any one of claims 156 to 161, wherein the predetermined rate is 50% of the nucleic acid in the gel being released within up to about 1 day.
163. The method of any one of claims 156 to 161, wherein the predetermined rate is 50% of the nucleic acid in the gel being released within a range of about 1 day to about 30 days.
164. The method of any one of claims 156 to 161, wherein the predetermined rate is 50% of the nucleic acid in the gel being released within a range of about 1 week to about 26 weeks.
165. The method of any one of claims 156 to 161, wherein the predetermined rate is 50% of the nucleic acid in the gel being released within a range of about 1 month to about 12 months.
166. The method of any one of claims 156 to 165, wherein release of the nucleic acid from the gel results in a concentration of the nucleic acid in the subject that is sufficient to effect a desired therapeutic result.
167. The method of any one of claims 156 to 166, wherein the nucleic acid is a small interfering nucleic acid.
168. The method of claim 167, wherein the small interfering nucleic acid is a shRNA, microRNA, siRNA, antisense oligonucleotide, or a miRNA sponge.
169. The method of claim 168, wherein the small interfering nucleic acid inhibits expression of a gene associated with a condition for which the subject is being treated, optionally wherein the gene is an oncogene.
170. The method of any one of claims 156 to 166, wherein the nucleic acid is an plasmid vector.
171. The method of claim 166, wherein the plasmid vector is a vector that expresses a mRNA or a small interfering nucleic acid.
172. The method of any one of claims 156 to 166, wherein the nucleic acid is in a virus vector.
173. The method of claim 172, wherein the virus vector is an adenovirus, lentivirus, retrovirus, herpesvirus, or adeno-associated virus.
174. A method for administering a particle to a subject, the method comprising administering to the subject a composition comprising an effective amount of a particle dispersed in a guanosine/GMP gel.
175. The method of claim 174, wherein the administering is performed is orally, intravenously, intrapleurally, intranasally, intramuscularly, subcutaneously, intraperitoneally, or as an aerosol.
176. The method of claim 174, wherein the administering comprises implanting the gel in the subject.
177. The method of claim 176, wherein the gel is implanted within, fully or partially, or adjacent to a diseased tissue of the subject.
178. The method of claim 177, wherein the diseased tissue is a cancer.
179. The method of any one of claims 174 to 178, wherein the particle is released from the guanosine/GMP gel at a predetermined rate.
180. The method of any one of claims 174 to 179, wherein the predetermined rate is 50% of the particle in the gel being released within up to about 1 day.
181. The method of any one of claims 174 to 179, wherein the predetermined rate is 50% of the particle in the gel being released within a range of about 1 day to about 30 days.
182. The method of any one of claims 174 to 179, wherein the predetermined rate is 50% of the particle in the gel being released within a range of about 1 week to about 26 weeks.
183. The method of any one of claims 174 to 179, wherein the predetermined rate is 50% of the particle in the gel being released within a range of about 1 month to about 12 months.
184. The method of any one of claims 174 to 183, wherein release of the particle from the gel results in a concentration of the particle in the subject that is sufficient to effect a desired therapeutic result.
185. The method of any one of claims 174 to 184, wherein the particle is a microparticle or nanoparticle.
186. The method of any one of claims 174 to 185, wherein the particle is in a range of about 1 nM to less than 1 μM in diameter.
187. The method of any one of claims 174 to 185, wherein the particle is in a range of about 1 μM to less than 1 mM in diameter.
188. The method of any one of claims 174 to 185, wherein the particle is in a range of about 1 nM to about 10 nM in diameter.
189. The method of any one of claims 174 to 188, wherein the particle is conjugated to a therapeutic agent.
190. The method of any one of claims 174 to 189, wherein the particle encapsulates a therapeutic agent.
191. The method of any one of claims 174 to 188, wherein the particle is conjugated to a diagnostic agent, optionally an imaging agent, optionally a radiolabel substance.
192. The method of any one of claims 174 to 188 and 191, wherein the particle encapsulates a diagnostic agent, optionally an imaging agent, optionally a radiolabel substance.
193. The composition of any one of claims 174 to 192, further comprising a salt or a buffering agent.
194. A method for administering a ligand to a subject, the method comprising administering to the subject a composition comprising a effective amount of a ligand dispersed in a guanosine/GMP gel.
195. The method of claim 194, wherein the administering is performed is orally, intravenously, intrapleurally, intranasally, intramuscularly, subcutaneously, intraperitoneally, or as an aerosol.
196. The method of claim 194 or 195, wherein the administering comprises implanting the gel in the subject.
197. The method of any one of claims 194 to 196, wherein the ligand specifically binds to a receptor and activates or inhibits a pathway associated with a disease for which the subject is being treated.
198. The method of any one of claims 194 to 197, wherein the ligand is conjugated with a therapeutic or diagnostic agent.
199. A method for administering a cell to a subject, the method comprising administering to the subject a composition comprising a effective amount of a cell dispersed in a guanosine/GMP gel.
200. The method of claim 199, wherein the administering comprises implanting the gel in the subject.
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