US20030170216A1 - SYN3 compositions and methods - Google Patents

SYN3 compositions and methods Download PDF

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US20030170216A1
US20030170216A1 US10/329,043 US32904302A US2003170216A1 US 20030170216 A1 US20030170216 A1 US 20030170216A1 US 32904302 A US32904302 A US 32904302A US 2003170216 A1 US2003170216 A1 US 2003170216A1
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syn3
pharmaceutically acceptable
gene
pharmaceutical composition
delivery
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Peter Ihnat
Leonore Witchey-Lakshmanan
Varda Sandweiss
Sydney Ugwu
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Merck Sharp and Dohme LLC
Schering Plough Corp
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Schering Plough Corp
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Priority to US10/329,043 priority Critical patent/US20030170216A1/en
Assigned to SCHERING CORPORATION reassignment SCHERING CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WITSHEY-OAKSHMANAN, LEONORE C., SANDWEISS, VARDA, UGWU, SYDNEY O., IHNAT, PETER M.
Publication of US20030170216A1 publication Critical patent/US20030170216A1/en
Assigned to SCHERING CORPORATION reassignment SCHERING CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WITCHEY-LAKSHMANAN, LEONORE C., SANDWEISS, VARDA, UGWU, SYDNEY O., IHNAT, PETER M.
Priority to US12/838,795 priority patent/US9115374B2/en
Priority to US14/798,277 priority patent/US20150313926A1/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
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
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    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/10—Drugs for disorders of the urinary system of the bladder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
    • C07J41/0061—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives one of the carbon atoms being part of an amide group
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10351—Methods of production or purification of viral material

Definitions

  • the present invention is directed to compositions for treating cancer by gene therapy using a therapeutic gene, such as a tumor suppressor gene delivered by a gene delivery system, such as a recombinant viral vector delivery system, in combination with a transduction enhancing agent.
  • a therapeutic gene such as a tumor suppressor gene delivered by a gene delivery system, such as a recombinant viral vector delivery system
  • this invention relates to the delivery of a tumor suppressor gene (e.g., p53 or retinoblastoma (RB)) to cancerous epithelial tissues and organs, such as the bladder, using a recombinant adenoviral vector delivery system formulated in a stabilized buffer in combination with a transduction enhancing agent, such as SYN3.
  • a tumor suppressor gene e.g., p53 or retinoblastoma (RB)
  • a recombinant adenoviral vector delivery system formulated in a stabilized buffer in combination with a transduction
  • Epithelial Cancer is an insidious disease.
  • one type of epithelial cancer, carcinoma of the bladder represents a significant source of morbidity and mortality.
  • Bladder cancer reportedly ranks 10th in males and 12th in females in cancer related mortality.
  • Therapies available for the treatment of bladder cancer include adjuvant chemotherapy or immunotherapy, transurethral resection of superficial disease, radical cystectomy or radiotherapy which is often combined with systemic chemotherapy. Despite these therapeutic options, overall survival has not changed appreciably. Thus, new therapeutic modalities must be developed for the treatment of bladder cancer.
  • Gene therapy strategies have been reportedly developed as an alternative therapeutic approach. Distinct approaches have been developed to treat neoplasms based on gene transfer methods. Methods have been reportedly developed to correct specific lesions at defined genetic loci which give rise to neoplastic transformation and progression. Overexpression of dominant oncogenes can be addressed using techniques to inhibit the transforming gene or gene product. It has been reported that loss of tumor suppressor gene function may be approached using methods to reconstitute wild-type tumor suppressor gene. Besides these methods to achieve mutation compensation, genetic techniques have been reportedly developed to specifically and selectively eradicate tumor cells. These approaches of molecular chemotherapy reportedly rely on specific expression of toxin genes in neoplastic cells.
  • tumor suppressor genes such as p53 and RB
  • reversion of the neoplastic phenotype can be demonstrated with replacement of the corresponding wild-type tumor suppressor gene.
  • U.S. Pat. No. 5,789,244 claims a composition comprising a viral vector in which a nucleotide sequence encoding a transgene has been inserted, wherein the viral vector is formulated in a buffer comprising ethanol in a concentration range of about 1% to 50% (v/v).
  • 5,837,520 claims a method for purification of an intact viral particle from a cell lysate comprising treating the cell lysate which contains the intact viral particle with an enzymatic agent that selectively degrades both unencapsulated DNA and RNA; chromatographing the treated lysate from the first step on a first resin; and chromatographing the eluant from the second step on a second resin; wherein one resin is an anion exchange resin and the other is an immobilized metal ion affinity resin.
  • 5,932,210 describes a composition comprising a recombinant adenovirus expression vector and a pharmaceutically acceptable carrier, the vector comprising: (a) an insert of exogenous DNA comprising a gene encoding a foreign protein; and (b) adenovirus DNA in which all of the coding sequences of E1a, E1b, and protein IX, and at least part of E3 have been deleted.
  • U.S. Pat. No. 6,165,779 discloses a composition comprising a recombinant virus vector formulated in a buffer comprising a detergent.
  • 6,210,939 claims a recombinant adenovirus expression vector comprising (a) an insert of exogenous DNA comprising a gene encoding a foreign protein and (b) adenovirus DNA which has sustained a deletion beginning at nucleotide 357 and ending at nucleotide 4020 to 4050.
  • 6,312,681 discloses a method for delivering an adenoviral vector which comprises a transgene to a cancer cell in the epithehial membrane of a bladder, the method comprising administering to the epithelial membrane the adenoviral vector and between 1% and 50% (v/v) ethanol, wherein the adenoviral vector infects the cell and the transgene is expressed in infected cells. All of these references are hereby incorporated by reference thereto in their entirety.
  • the present invention provides a pharmaceutical composition comprising SYN3 in combination with a pharmaceutically acceptable carrier.
  • a further aspect of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising SYN3 in combination with a pharmaceutically acceptable nonaqueous carrier.
  • a further aspect of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising SYN3 in combination with a pharmaceutically acceptable aqueous carrier.
  • a further aspect of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising SYN3 in combination with a pharmaceutically acceptable carrier and at least one pharmaceutically acceptable solubilizer.
  • a further aspect of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising SYN3 in combination with a pharmaceutically acceptable nonaqueous carrier and at least one pharmaceutically acceptable solubilizer.
  • a further aspect of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising SYN3 in combination with a pharmaceutically acceptable aqueous carrier and at least one pharmaceutically acceptable solubilizer.
  • a further aspect of the invention is a lyophilized pharmaceutical composition
  • a lyophilized pharmaceutical composition comprising SYN3 in combination with a pharmaceutically acceptable carrier, at least one pharmaceutically acceptable solubilizer and a at least one pharmaceutically acceptable bulking agent.
  • a further aspect of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising SYN3 in combination with a pharmaceutically acceptable aqueous carrier, at least one pharmaceutically acceptable solubilizer, and at least one pharmaceutically acceptable bulking agent.
  • a further aspect of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising SYN3 in combination with a pharmaceutically acceptable aqueous carrier, at least one pharmaceutically acceptable solubilizer, at least one pharmaceutically acceptable bulking agent and at least one pharmaceutically acceptable buffering agent.
  • a further aspect of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising SYN3 in combination with a pharmaceutically acceptable carrier and an expression vector comprising a foreign DNA sequence inserted into the vector.
  • a further aspect of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising SYN3 in combination with a pharmaceutically acceptable nonaqueous carrier and an expression vector comprising a foreign DNA sequence inserted into the vector.
  • a further aspect of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising SYN3 in combination with a pharmaceutically acceptable aqueous carrier and an expression vector comprising a foreign DNA sequence inserted into the vector.
  • a further aspect of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising SYN3 in combination with a pharmaceutically acceptable carrier, an expression vector comprising a foreign DNA sequence inserted into the vector and at least one pharmaceutically acceptable solubilizer.
  • a further aspect of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising SYN3 in combination with a pharmaceutically acceptable nonaqueous carrier, an expression vector comprising a foreign DNA sequence inserted into the vector and at least one pharmaceutically acceptable solubilizer.
  • a further aspect of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising SYN3 in combination with a pharmaceutically acceptable aqueous carrier, an expression vector comprising a foreign DNA sequence inserted into the vector and at least one pharmaceutically acceptable solubilizer.
  • a further aspect of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising SYN3 in combination with a pharmaceutically acceptable aqueous carrier, an expression vector comprising a foreign DNA sequence inserted into the vector, at least one pharmaceutically acceptable solubilizer, and at least one pharmaceutically acceptable bulking agent.
  • a further aspect of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising SYN3 in combination with a pharmaceutically acceptable aqueous carrier, an expression vector comprising a foreign DNA sequence inserted into the vector, at least one pharmaceutically acceptable solubilizer, at least one pharmaceutically acceptable bulking agent and at least one pharmaceutically acceptable buffering agent.
  • a further aspect of the invention is a use of SYN3 in the preparation of a medicament for the treatment of bladder cancer.
  • a further aspect of the invention is a method of treating a disease in a mammal comprising administering a therapeutically effective amount of a pharmaceutical composition comprising SYN3 in combination with a pharmaceutically acceptable carrier.
  • FIG. 1 illustrates a chemical structural formula of SYN3.
  • FIG. 2 illustrates one method for the synthesis of SYN3.
  • one aspect of the invention is that a unique surfactant-like molecule SYN3 is formulated with excipients to maintain solubility and stability as well as compatibility with the adenovirus.
  • a further aspect of the invention is that the SYN3 formulations are nontoxic to tissues, e.g., the bladder with which it comes in contact at therapeutic levels. Indeed, surfactants which act as permeation enhancers often produce some toxicity due to membrane irritation. The use of SYN3 thus provides this further benefit of avoiding this toxicity. Connor, et al., Gene Therapy, Vol. 8, pp. 41-48 (2001).
  • a further aspect of the invention is that the stability of the vector is unaffected by combination with the SYN3. Often, surfactant levels required to improved transduction may impart instability to the vector. Combination of the adenovirus and SYN3 preparations produces a more potent admixture compared with adenovirus.
  • SYN3 is (N-(3-cholamidopropyl)-N-(3 (actobionamidopropyl))—cholamide (FIG. 1).
  • SYN3 exists in various optical, tautomeric, stereoisomeric and isomeric forms.
  • FIG. 1 illustrates a preferred isomer.
  • the compositions of the present invention encompass all such forms in any percentage or racemic mixture thereof.
  • SYN3 is a surfactant-like molecule that enhances transduction of recombinant adenovirus/therapeutic gene vectors for treatment of epithelial tissue and tumors, or, more specifically, in bladder tumors.
  • SYN3 can be present in a concentration of from about 0.001 mg/ml to about 150 mg/ml.
  • therapeutic transgene refers to a nucleotide sequence the expression of which in the target cell produces a therapeutic effect.
  • therapeutic transgene includes but is not limited to tumor suppressor genes, antigenic genes, cytotoxic genes, cytostatic genes, pro-drug activating genes, apoptotic genes, pharmaceutical genes or anti-angiogenic genes.
  • the vectors of the present invention may be used to produce one or more therapeutic transgenes, either in tandem through the use of IRES elements or through independently regulated promoters.
  • tumor suppressor gene refers to a nucleotide sequence, the expression of which in the target cell is capable of suppressing the neoplastic phenotype and/or inducing apoptosis.
  • tumor suppressor genes useful in the practice of the present invention include the p53 gene, the APC gene, the DPC-4 gene, the BRCA-1 gene, the BRCA-2 gene, the WT-1 gene, the retinoblastoma gene (Lee, et al., Nature, 329:642 (1987)), the MMAC-1 gene, the adenomatous polyposis coli protein (Albertsen, et al., U.S. Pat. No. 5,783,666 issued Jul.
  • the deleted in colon carcinoma (DCC) gene the MMSC-2 gene, the NF-1 gene, nasopharyngeal carcinoma tumor suppressor gene that maps at chromosome 3p21.3.
  • DCC colon carcinoma
  • MMSC-2 the NF-1 gene
  • NF-1 nasopharyngeal carcinoma tumor suppressor gene that maps at chromosome 3p21.3.
  • antigenic genes refers to a nucleotide sequence, the expression of which in the target cells results in the production of a cell surface antigenic protein capable of recognition by the immune system.
  • antigenic genes include carcinoembryonic antigen (CEA), p53 (as described in Levine, A. PCT International Publication No. WO94/02167 published Feb. 3, 1994).
  • CEA carcinoembryonic antigen
  • p53 as described in Levine, A. PCT International Publication No. WO94/02167 published Feb. 3, 1994.
  • the antigenic gene may be fused to the MHC class I antigen.
  • cytotoxic gene refers to nucleotide sequence, the expression of which in a cell produces a toxic effect.
  • examples of such cytotoxic genes include nucleotide sequences encoding pseudomonas exotoxin, ricin toxin, diptheria toxin, and the like.
  • cytostatic gene refers to nucleotide sequence, the expression of which in a cell produces an arrest in the cell cycle.
  • examples of such cytostatic genes include p21, the retinoblastoma gene, the E2F-Rb gene, genes encoding cyclin dependent kinase inhibitors such as P16, p15, p18 and p19, the growth arrest specific homeobox (GAX) gene as described in Branellec, et al. (PCT Publication WO97/16459 published May 9, 1997 and PCT Publication WO96/30385 published Oct. 3, 1996).
  • GX growth arrest specific homeobox
  • cytokine gene refers to a nucleotide sequence, the expression of which in a cell produces a cytokine.
  • cytokines include GM-CSF, the interleukins, especially IL-1, IL-2, IL-4, IL-12, IL-10, IL-19, IL-20, interferons of the alpha, beta and gamma subtypes especially interferon ⁇ -2b and fusions such as interferon ⁇ -2 ⁇ -1.
  • chemokine gene refers to a nucleotide sequence, the expression of which in a cell produces a cytokine.
  • chemokine refers to a group of structurally related low-molecular cytokines weight factors secreted by cells are structurally related having mitogenic, chemotactic or inflammatory activities. They are primarily cationic proteins of 70 to 100 amino acid residues that share four conserved cysteine. These proteins can be sorted into two groups based on the spacing of the two amino-terminal cysteines. In the first group, the two cysteines are separated by a single residue (C-x-C), while in the second group, they are adjacent (C—C).
  • C-x-C chemokines
  • chemokines include but are not limited to platelet factor 4 (PF4), platelet basic protein (PBP), interleukin-8 (IL-8), melanoma growth stimulatory activity protein (MGSA), macrophage inflammatory protein 2 (MIP-2), mouse Mig (ml 19), chicken 9E3 (or pCEF-4), pig alveolar macrophage chemotactic factors I and I (AMCF-I and -II), pre-B cell growth stimulating factor (PBSF),and IP10.
  • PF4 platelet factor 4
  • PBP platelet basic protein
  • IL-8 interleukin-8
  • MGSA melanoma growth stimulatory activity protein
  • MIP-2 macrophage inflammatory protein 2
  • mouse Mig mouse Mig (ml 19)
  • pig alveolar macrophage chemotactic factors I and I AMCF-I and -II
  • PBSF pre-B cell growth stimulating factor
  • members of the ‘C—C’ group include, but are not limited to, monocyte chemotactic protein 1 (MCP-1), monocyte chemotactic protein 2 (MCP-2), monocyte chemotactic protein 3 (MCP-3), monocyte chemotactic protein 4 (MCP-4), macrophage inflammatory protein 1 alpha (MIP-1-alpha), macrophage inflammatory protein 1 beta (MIP-1-beta), macrophage inflammatory protein I gamma (MIP-1-gamma), macrophage inflammatory protein 3 alpha (MIP-3-alpha, macrophage inflammatory protein 3 beta (MIP-3-beta), chemokine (ELC), macrophage inflammatory protein 4 (MIP-4), macrophage inflammatory protein 5 (MIP-5), LD78 beta, RANTES, SIS-epsilon (p500), thymus and activation-regulated chemokine (TARC), eotaxin, 1-309, human protein HCC-1/NCC-2, human protein
  • pharmaceutical protein gene refers to nucleotide sequence, the expression of which results in the production of protein have pharmaceutically effect in the target cell.
  • pharmaceutical genes include the proinsulin gene and analogs (as described in PCT International Patent Application No. WO98/31397, growth hormone gene, dopamine, serotonin, epidermal growth factor, GABA, ACTH, NGF, VEGF (to increase blood perfusion to target tissue, induce angiogenesis, PCT publication WO98/32859 published Jul. 30, 1998), thrombospondin etc.
  • pro-apoptotic gene refers to a nucleotide sequence, the expression thereof results in the programmed cell death of the cell.
  • pro-apoptotic genes include p53, adenovirus E3-11.6K, the adenovirus E4 or f4 gene, p53 pathway genes, and genes encoding the caspases.
  • pro-drug activating genes refers to nucleotide sequences, the expression of which, results in the production of protein capable of converting a nontherapeutic compound into a therapeutic compound, which renders the cell susceptible to killing by external factors or causes a toxic condition in the cell.
  • An example of a prodrug activating gene is the cytosine deaminase gene. Cytosine deaminase converts 5-fluorocytosine to 5 fluorouracil, a potent antitumor agent). The lysis of the tumor cell provides a localized burst of cytosine deaminase capable of converting 5FC to 5FU at the localized point of the tumor resulting in the killing of many surrounding tumor cells.
  • TK thymidine kinase
  • anti-angiogenic genes refers to a nucleotide sequence, the expression of which results in the extracellular secretion of anti-angiogenic factors.
  • Anti-angiogenesis factors include angiostatin, inhibitors of vascular endothelial growth factor (VEGF) such as Tie 2 (as described in PNAS(USA)(1998) 95:8795-8800), endostatin.
  • VEGF vascular endothelial growth factor
  • the reference to the p53 gene includes not only the wild type protein but also modified p53 proteins.
  • modified p53 proteins include modifications to p53 to increase nuclear retention as described in Wahl, et al., Nat. Cell Biol., 3(12):E277-86 (2001), deletions such as the delta13-19 amino acids to eliminate the calpain consensus cleavage site, modifications to the oligomerization domains (as described in Bracco, et al. PCT published application WO97/0492 or U.S. Pat. No. 5,573,925).
  • therapeutic genes may be secreted into the media or localized to particular intracellular locations by inclusion of a targeting moiety such as a signal peptide or nuclear localization signal(NLS).
  • a targeting moiety such as a signal peptide or nuclear localization signal(NLS).
  • fusion proteins of the therapeutic transgene with the herpes simplex virus type 1 (HSV-1) structural protein, VP22 are included in the definition of therapeutic transgene. Fusion proteins containing the VP22 signal, when synthesized in an infected cell, are exported out of the infected cell and efficiently enter surrounding noninfected cells to a diameter of approximately 16 cells wide. This system is particularly useful in conjunction with transciptionally active proteins (e.g.
  • a gene delivery system refers to any means of delivery of a therapeutic gene to a particular epithelial tissue or organ including, for example, recombinant viral vectors and nonviral vector systems.
  • nonvector systems include, but are not limited to, any lipid-based, lipid encapsulated DNA or cationic lipid/DNA complexes.
  • recombinant viral vectors include, but are not limited to, herpes virus, retrovirus, vaccinia virus, adenovirus, and adenoassociated viruses.
  • Recombinant refers to nucleic acids and protein encoded by them wherein the nucleic acids are constructed by methods of recombinant DNA technology, also termed “genetic engineering”.
  • a preferred recombinant viral vector is the adenoviral vector delivery system which has a deletion of the protein IX gene. See International patent Application WO 95/11984, which is herein incorporated by reference in its entirety.
  • the recombinant vector delivery system comprising a therapeutic gene, such as a tumor suppressor gene, is formulated in a buffer that stabilizes the vector and is combined with a delivery enhancing agent that is compatible with the vector.
  • a “delivery-enhancing agent” refers to any agent which enhances delivery of a therapeutic gene, such as a tumor suppressor gene to a cancerous tissue or organ. Such enhanced delivery may be achieved by various mechanisms. One such mechanism may involve the disruption of the protective glycosaminoglycan layer on the epithelial surface of the bladder.
  • Examples of such delivery-enhancing agents are detergents, alcohols, glycols, surfactants, bile salts, heparin antagonists, cyclooxygenase inhibitors, hypertonic salt solutions, and acetates.
  • Alcohols include, for example, the aliphatic alcohols such as ethanol, N-propanol, isopropanol, butyl alcohol, acetyl alcohol.
  • Glycols include, for example, glycerol, propyleneglycol, polyethyleneglycol, and thioglycerol.
  • Acetates such as acetic acid, gluconol acetate, and sodium acetate are further examples of delivery-enhancing agents.
  • Hypertonic salt solutions like 1M NaCl are also examples of delivery-enhancing agents.
  • surfactants are sodium dodecyl sulfate (SDS) and lysolecithin, polysorbate 80, nonylphenoxypolyoxyethylene, lysophosphatidylcholine, polyethylenglycol 400, polysorbate 20, polyoxyethylene ethers, and polyglycol ether surfactants.
  • Bile salts such as taurocholate, sodium tauro-deoxycholate, deoxycholate, chenodesoxycholate, glycocholic acid, glycochenodeoxycholic acid and other astringents like silver nitrate may be used.
  • Heparin-antagonists like quaternary amines such as protamine sulfate may also be used.
  • Cyclooxygenase inhibitors such as sodium salicylate, salicylic acid, and nonsteroidal antiinflammatory drug (NSAIDS) like indomethacin, naproxen, diclofenac may be used.
  • NSAIDS nonsteroidal antiinflammatory drug
  • the term “enhanced” describes the increased delivery of the therapeutic gene, such as a tumor suppressor gene, to the cancerous tissue or organ.
  • Increased delivery of a therapeutic gene, such as a tumor suppressor gene can be measured by various means, for example by measuring expression of the tumor suppressor gene compared to expression levels when the tumor suppressor gene is delivery in an adenoviral vector delivery system in a buffer lacking the delivery-enhancing agent.
  • therapeutic genes are tumor suppressor genes and the suicide gene thymidine kinase.
  • tumor suppressor genes include, but are not limited to, p53, the retinoblastoma gene, either full length, such as p110B, or fragments thereof such as p94RB or p56RB, Rb56, a functional variant of Rb gene, a functional variant of the p53 gene, and p16.
  • Other therapeutic genes include but are not limited to CFTR, genes encoding cytokines (such as the interferons alpha, beta, gamma, delta, interleukins (e.g., IL-4, IL-10, IL-2), GM-CSF, and any other genes encoding proteins which have therapeutic potential in the treatment of noncancerous diseases of the bladder such as cystitis.
  • the therapeutic gene encodes antisense RNA.
  • compositions of the invention comprise a therapeutically effective amount of a therapeutic gene, such as a tumor suppressor gene, contained in a recombinant viral vector delivery system in a buffer comprising a delivery-enhancing agent.
  • a therapeutically effective refers to the prevention of, reduction of, or curing of symptoms associated with a disease state.
  • Therapeutically effective amounts of the pharmaceutical composition comprising a therapeutic gene, such as p53, or the retinoblastoma tumor suppressor gene, in a recombinant viral vector delivery system formulated in a buffer comprising a delivery-enhancing agent will be administered in accord with the teaching of this invention.
  • therapeutically effective amounts of the p53 tumor suppressor gene in the recombinant adenoviral vector delivery system formulated in a buffer containing a delivery-enhancing agent are in the range of about 1 ⁇ 10 particles/ml to 2 ⁇ 10 12 particles/ml, more typically about 1 ⁇ 10 8 particles/ml to 9 ⁇ 10 11 particles/ml, most typically 1 ⁇ 10 10 particles/ml to 9 ⁇ 10 11 particles/ml.
  • P53 also known as ACN53, is a recombinant adenovirus type 5 encoding wild-type p53 tumor suppressor protein, and is described in, for example, PCT patent application WO95/11984.
  • the formulated SYN3 is combined with p53 injection and the admixture is instilled into the bladder. This preparation is intended to treat epithelial carcinomas.
  • p53 will be present in an amount of about 5 to about 8 ⁇ 10 13 particles.
  • Detergents for use within the scope of the present invention include, for example, anionic, cationic, zwitterionic, and nonionic detergents.
  • Exemplary detergents include, for example, but are not limited to taurocholate, deoxycholate, taurodeoxycholate, cetylpyridium, benalkonium chloride, ZWITTERGENT 3-14 detergent, CHAPS (3-[(3-Cholamidopropyl)dimethylammoniol]-1-propanesulfonate hydrate, available from Aldrich, Big CHAP, Deoxy Big CHAP, TRITON-X-100 detergent available from Union Carbide, C12E8, Octyl-B-D-Glucopyranoside, PLURONIC-F64, PLURONIC-F68, PLURONIC-F69 detergents available form BASF, TWEEN20 detergent, and TWEEN80 detergent available from ICI.
  • the delivery-enhancing agent is included in the buffer in which the recombinant adenoviral vector delivery system is formulated.
  • the delivery-enhancing agent may be administered prior to the recombinant virus or concomitant with the virus.
  • the delivery-enhancing agent is provided with the virus by mixing a virus preparation with a delivery-enhancing agent formulation just prior to administration to the patient.
  • the delivery-enhancing agent and virus are provided in a single vial to the caregiver for administration.
  • the pharmaceutical composition may be administered over time in the range of about 5 minutes to 3 hours, preferably about 10 minutes to 120 minutes, and preferably about 15 minutes to 90 minutes.
  • the delivery-enhancing agent may be administered prior to administration of the recombinant adenoviral vector delivery system containing the tumor suppressor gene.
  • the prior administration of the delivery-enhancing agent may be in the range of about 30 seconds to 1 hour, preferably about 1 minute to 10 minutes, and preferably about 1 minute to 5 minutes prior to administration of the adenoviral vector delivery system containing the tumor suppressor gene.
  • Solvents that may be used for the formulations of the present invention include, for example, aqueous solvents such as water for injection, and/or nonaqueous solvents, such as DMSO and N,N-Dimethyylacetamide, also known as DMA, and co-solvent mixtures, e.g., glycerol and water, as prepared preferably in accordance with USP standards.
  • aqueous solvents such as water for injection
  • nonaqueous solvents such as DMSO and N,N-Dimethyylacetamide, also known as DMA
  • co-solvent mixtures e.g., glycerol and water
  • the formulations preferably contain polysorbates, or polyoxyethylene sorbitan esters, a class of nonionic surfactants that included, e.g., polysorbate 80 and polysorbate 20, amongst others, Pluronics, or polyethylenepolypropylene glycol block copolymers, a class of nonionic surfactants, that include e.g. Pluronic L68 and L92, amongst others, and hydroxypropyl-beta-cyclodextrin, a polysubstituted hydroxyalkyl-beta-cyclodextrin, which is a class of nonionic complexing agents, that include, e.g., HP ⁇ CD and BigCHAP.
  • nonionic complexing agents that include, e.g., HP ⁇ CD and BigCHAP.
  • HP ⁇ CD HP ⁇ CD
  • BigCHAP Polysorbate 80
  • Polysorbate 20 Polysorbate 20
  • Pluronic L64 Pluronic L92
  • HP ⁇ CD HP ⁇ CD
  • BigCHAP can be present in a concentration of about 20 to about 360 mg/ml
  • Polysorbate 80 can be present in a concentration of about 1 to 36 mg/ml
  • the Pluronics can be present in concentrations of about 1 to about 150 mg/ml
  • the other ingredients may be present in concentrations as set forth below.
  • the concentration of the delivery-enhancing agent will depend on a number of factors known to one of ordinary skill in the art such as the particular delivery-enhancing agent being used, the buffer, pH, target tissue or organ and mode of administration.
  • the concentration of the delivery-enhancing agent will be in the range of 0.01% to 50% (w/v), preferably 10% to 40% (w/v), preferably 14% to 19% (w/v), and preferably 0.01% to 30% (w/v).
  • the detergent concentration will be about 1% to 12% (w/v) in the formulation prior to admixture, and preferably 0.1% (w/v) of the formulation when in admixture.
  • the detergent concentration in the final formulation administered to the patient is about 0.5-2 times the critical micellization concentration (CMC).
  • the CMC is equal to 0.001 mg/ml in the recombinant adenovirus formulation.
  • the lyophilized formulations of SYN3 preferably contain a citrate buffering system. More preferably, the citrate buffering system can comprise at least one citric buffer, such as citric acid monohydrate USP or sodium citrate dihydrate USP. More preferably, the citrate buffering system comprises a combination of citric acid monohydrate USP and sodium citrate dihydrate USP.
  • the amount of citric acid monohydrate USP can be present in a concentration of about 0.005 to about 2 mg/ml, more preferably 0.016 to about 1.35 mg/ml, preferably 0.016 to about 0.72 mg/ml, preferably about 0.005 to about 1.35, and the sodium citrate dihydrate USP can be present in a concentration of about 0.02 to about 5.37 mg/ml, preferably 0.05 to 3.00 mg/ml, preferably 0.05 to 2.31 mg/ml.
  • suitable buffering systems that are suitable include, for example, phosphate, glycine, either in place of the citrate buffering system or in combination therewith, and varying combinations of all of the above.
  • the buffering system will provide a pH of the lyophilized formulation such that there is improved stability.
  • the pH will be in a range of about 5 to about 6.
  • the admixture aqueous formulations of SYN3 are preferably buffered at about a pH of about 7 to about 8.5, preferably about 7.4, and SYN3 remains stable in the dehydrated powder for at least 3 months at 40° C.
  • the lyophilized formulations preferably contain glycine or mannitol as freeze-drying bulking agents.
  • suitable freeze-drying bulking agents include, for example, lactose, recombinant gelatin, and methylcellulose.
  • the freeze drying-bulking agent may be present in a concentration of from about 5 to 100 mg/ml when the agent is mannitol, and about 10 to 200 mg/ml when the agent is glycine.
  • the lyophilized formulations preferably contain ascorbic acid as an antioxidant.
  • suitable antioxidants include, for example, citric acid.
  • ascorbic acids When ascorbic acids is the antioxidant, it may be present in a concentration of about 0.001 to about 0.6 mg/ml.
  • compositions of this invention may additionally include, for example, a stabilizer, enhancer or other pharmaceutically acceptable carriers or vehicles.
  • a pharmaceutically acceptable carrier can contain a physiologically acceptable compound that acts, for example, to stabilize the recombinant adenoviral vector delivery system comprising the tumor suppressor gene
  • a physiologically acceptable compound can include, for example, carbohydrates, such as glucose, sucrose or dextrans, Hydroxypropyl- ⁇ -Cyclodextrin, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients.
  • physiologically acceptable compounds include, for example, wetting agents, emulsifying agents, dispersing agents or preservatives, which are particularly useful for preventing the growth or action of microorganisms.
  • Various preservatives are well known and include, for example, phenol and ascorbic acid.
  • pharmaceutically acceptable carrier depends on the route of administration and the particular physiochemical characteristics of the recombinant adenoviral vector delivery system and the particular tumor suppressor gene contained therein. Examples of carriers, stabilizers or adjuvants can be found in Gennaro, Remington's: The Science and Practice of Pharmacy, 19th Ed. (Mack Publishing. Co., Easton, Pa. 1995), incorporated herein by reference.
  • the recombinant viral vector delivery system comprising a therapeutic gene formulated in a buffer comprising a delivery-enhancing agent may be delivered to any cancerous tissue or organ using any delivery method known to the ordinarily skilled artisan for example, intratumoral or intravesical administration.
  • Cancerous tissues and organs include, for example, any tissue or organ having an epithelial membrane such as the gastrointestinal tract, the bladder, respiratory tract, and the lung.
  • Examples include but are not limited to carcinoma of the bladder and upper respiratory tract, vulva, cervix, vagina or bronchi; local metastatic tumors of the peritoneum; broncho-alveolar carcinoma; pleural metastatic carcinoma; carcinoma of the mouth and tonsils; carcinoma of the nasopharynx, nose, larynx, oesophagus, stomach, colon and rectum, gallbladder, or skin; or melanoma.
  • the delivery-enhancing agents of the invention can also be used to formulate other pharmaceutical agents, such as proteins, nucleic acids, antisense RNA, small molecules, etc., for administration to any tissue or organ having an epithelial membrane.
  • the delivery-enhancing agent is included in the buffer in which an expression vector is formulated.
  • the delivery-enhancing agent can be administered prior to the expression vector or concomitant with the expression vector.
  • the delivery-enhancing agent is provided with the expression vector by mixing an expression vector with a delivery-enhancing agent formulation just prior to administration to the patient.
  • the delivery-enhancing agent and the expression vector are provided in a single vial to the caregiver for administration.
  • the pharmaceutical composition can be administered over time in the range of about 5 minutes to 3 hours, preferably about 10 minutes to 120 minutes, and most preferably about 15 minutes to 90 minutes.
  • the delivery-enhancing agent may be administered prior to administration of the recombinant adenoviral vector delivery system containing the tumor suppressor gene.
  • the prior administration of the delivery-enhancing agent may be in the range of about 30 seconds to 1 hour, preferably about 1 minute to 10 minutes, and most preferably about 1 minute to 5 minutes prior to administration of the adenoviral vector delivery system containing the tumor suppressor gene.
  • the expression vector formulated in a buffer comprising a delivery-enhancing agent can be delivered to any tissue or organ, including neoplastic tissues such as cancer tissue, using any delivery method known to the ordinarily skilled artisan for example, intratumoral or intravesical administration.
  • Tissues and organs include any tissue or organ having an epithelial membrane such as the gastrointestinal tract, the bladder, respiratory tract, and the lung.
  • Examples include but are not limited to carcinoma of the bladder and upper respiratory tract, vulva, cervix, vagina or bronchi; local metastatic tumors of the peritoneum; broncho-alveolar carcinoma; pleural metastatic carcinoma; carcinoma of the mouth and tonsils; carcinoma of the nasopharynx, nose, larynx, oesophagus, stomach, colon and rectum, gallbladder, or skin; or melanoma.
  • an expression vector is formulated in mucosal, topical, and/or buccal formulations, particularly mucoadhesive gel and topical gel formulations.
  • exemplary permeation enhancing compositions, polymer matrices, and mucoadhesive gel preparations for transdermal delivery are disclosed in U.S. Pat. No. 5,346,701.
  • Such formulations are especially useful for the treatment of cancers of the mouth, head and neck cancers (e.g., cancers of the tracheobronchial epithelium) skin cancers (e.g., melanoma, basal and squamous cell carcinomas), cancers of the intestinal mucosa, vaginal mucosa, and cervical cancer.
  • a therapeutic agent is formulated in ophthalmic formulations for administration to the eye.
  • Such formulations are useful in the delivery of the retinoblastoma (RB) gene to the eye, optionally in conjunction with the delivery of p53.
  • RB retinoblastoma
  • composition of the invention are typically administered to enhance transfer of gene to a cell.
  • the cell can be provided as part of a tissue, such as an epithelial membrane, or as an isolated cell, such as in tissue culture.
  • the cell can be provided in vivo, ex vivo, or in vitro.
  • compositions can be introduced into the tissue of interest in vivo or ex vivo by a variety of methods.
  • the modulating agent is introduced to cells by such methods as microinjection, calcium phosphate precipitation, liposome fusion, or biolistics.
  • the therapeutic agent is taken up directly by the tissue of interest.
  • compositions of the invention are administered ex vivo to cells or tissues explanted from a patient, then returned to the patient.
  • ex vivo administration of therapeutic gene constructs include Arteaga et al., Cancer Research 56(5):1098-1103 (1996); Nolta et al., Proc Natl. Acad. Sci. USA 93(6):2414-9 (1996); Koc et al., Seminars in Oncology 23 (1):46-65 (1996); Raper et al., Annals of Surgery 223(2):116-26 (1996); Dalesandro et al., J. Thorac. Cardi. Surg., 11(2):416-22 (1996); and Makarov et al., Proc. Natl. Acad. Sci. USA 93(1):402-6 (1996).
  • the following table represents ranges of the ingredients for nonaqueous liquid formulations of the present invention.
  • the SYN3 solution Prior to administration (for bladder cancer), the SYN3 solution is combined with the recombinant adenovirus preparation in a 1:50 v/v ratio to form an admixture that is administered to the patient.
  • Stability testing was accomplished by HPLC. Solution formulations were placed in the indicated temperature conditions, incubated for specified times and concentrations were determined by HPLC and compared to initial concentrations ( ⁇ 80° C.). The nonaqueous, solution formulations of SYN3 remain stable for at least 1 month at 55° C. when SYN3 is dissolved in N,N-Dimethylacetamide (DMA).
  • DMA N,N-Dimethylacetamide
  • the following table represents ranges of the ingredients for lyophilized formulations of the present invention.
  • the compounded solution is filled as indicated into a 20-ml capacity Type II glass vial and lyophilized. Preparation for administration requires addition of 20 ml of WFI to the vial containing the freeze-dried cake to redissolve the SYN3.
  • the SYN3 solution is combined with p53, or any recombinant adenovirus preparation, in a v/v ratio of 1:5.
  • the admixture is then administered to the patient for, for instance, bladder cancer.
  • the volume of Water for Injection to be charged to the batch is to be determined according to the following formula:
  • the compounded batch may be stored at 2° C. to 8° C. for up to 24 hours in a sealed, sterilized, stainless steel pressure vessel prior to filling into the vials. The batch may be filtered more than once to assure sterility.
  • the product is a white to off-white cake.
  • the vials should be stored between 2° C. to 8° C. after inspection. For labeling and inspection purposes, the vials may be exposed to 19° C.-25° C. for up to 6 hours.
  • Stability testing was accomplished by HPLC. Lyophilized formulations were reconstituted (redissolved) with 19.5 ml WFI. Samples were placed in the indicated temperature conditions, incubated for specified times and concentrations were determined by HPLC and compared to initial concentrations.
  • Product temperature must remain at or above ⁇ 20° C. for at least 6 hours before proceeding. Heat the shelf to 25° C. in 1 hour and reduce pressure to approximately 50 mm Hg pressure. Maintain the shelf temperature at 25° C. at approximately 50 mm Hg pressure for 14 hours. Vent the chamber with sterile filtered nitrogen to approximately 950 mm Hg. Stopper the vials inside the lyophilizer. Remove the vials from the lyophilizer and crimp the vials with 20-mm aluminum. The vials should be stored at 2° C. to 8° C. until inspection is completed.
  • the product is a white to off-white cake.
  • the vials should be stored between 2° C. to 8° C. after inspection. For labeling and inspection purposes, the vials may be exposed to 19° C.-25° C. for up to 6 hours.
  • p53 (rAD/p53) remains stable when combined with the lyophilized formulations of SYN3 for at least 2 hours at 37° C. and 24 hours at 25° C. p53 remains stable when combined with the aqueous solution formulations of SYN3 for at least 4 hours at 37° C. and 24 hours at 25° C.
  • FIG. 2 The synthetic scheme for SYN3 is shown in FIG. 2, which is adapted from U.S. Pat. No. 6,392,069.
  • the lactone of lactobionic acid (II) was synthesized by dissolving one g (2.8 mmol) of lactobionic acid (I) in 50 ml of methanol, evaporating to dryness on a rotary evaporator, and repeating this process six times.
  • the resulting residue (II) was dissolved in 50 ml of isopropanol by heating to 50° C.
  • To this solution was added 1.2 ml (8.4 mmol) of N-3-aminopropyl)-1,2-propanediamene.

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