EP1812460A1 - Formulations, procedes de production et utilisations du fgf-20 - Google Patents

Formulations, procedes de production et utilisations du fgf-20

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Publication number
EP1812460A1
EP1812460A1 EP05851340A EP05851340A EP1812460A1 EP 1812460 A1 EP1812460 A1 EP 1812460A1 EP 05851340 A EP05851340 A EP 05851340A EP 05851340 A EP05851340 A EP 05851340A EP 1812460 A1 EP1812460 A1 EP 1812460A1
Authority
EP
European Patent Office
Prior art keywords
protein
formulation
arginine
seq
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05851340A
Other languages
German (de)
English (en)
Inventor
Cyrus Karkaria
Tracy Chen
Rajeev Chillakuru
Gan Wei
Saravanamoorthy Rajendran
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CuraGen Corp
Original Assignee
CuraGen Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US10/980,659 external-priority patent/US20050208514A1/en
Priority claimed from US10/980,695 external-priority patent/US20050215473A1/en
Priority claimed from US10/980,459 external-priority patent/US20050256042A1/en
Priority claimed from US10/980,458 external-priority patent/US20080287349A1/en
Priority claimed from US10/980,370 external-priority patent/US20060094651A1/en
Priority claimed from US10/980,764 external-priority patent/US20050164929A1/en
Priority claimed from PCT/US2005/010731 external-priority patent/WO2005113809A2/fr
Priority claimed from PCT/US2005/010732 external-priority patent/WO2006073417A2/fr
Priority claimed from PCT/US2005/010545 external-priority patent/WO2005112979A2/fr
Application filed by CuraGen Corp filed Critical CuraGen Corp
Publication of EP1812460A1 publication Critical patent/EP1812460A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to improved formulations comprising FGF-20, its fragments, derivatives, variants, homologs, analogs, or a combination thereof, improved methods for production, and methods of use thereof.
  • the fibroblast growth factor (“FGF”) family consists of more than 20 members, each containing a conserved amino acid core (see, e.g., Powers et al., Endocr. Relat. Cancer, 7(3):65- 197 (2000)). FGFs regulate diverse cellular functions such as growth, survival, apoptosis, motility, and differentiation (see, e.g., Szebenyi et al., Int. Rev. Cytol., 185:45-106 (1999)).
  • FGF FGF-like growth factor
  • FGF-20 a novel human FGF, FGF-20. See U.S. Patent Application Nos. 09/494,585, filed January 13, 2000, and 09/609,543, filed July 3, 2000, the disclosure of each references is incorporated herein by reference.
  • the amino acid sequence of FGF-20 shows close homology with human FGF-9 (70% identity) and FGF16 (64% identity).
  • FGF-20 Recombinant full length FGF-20 has been shown to induce a proliferative response in mesenchymal and epithelial cells, but not in human smooth muscle, erythroid, or endothelial cells (see, e.g., Jeffers et al, Cancer Res. 61(7):3131-3138 (2001)). FGF-20 and its variants or derivatives have also been shown to be effective in preventing and/or treating certain diseases, such as oral mucositis (see International Patent Application Publication No. WO 2003/099201 , filed May 9, 2003), inflammatory bowel disease ("IBD”) (see International Patent Application Publication No.
  • IBD inflammatory bowel disease
  • the present invention provides improved formulations comprising a fibroblast growth factor, preferably FGF-20, or its fragments, derivatives, variants, homologs, analogs, or a combination thereof.
  • the present invention also provides improved production methods for isolating one or more CG53135 proteins.
  • the present invention further provides methods of use of CG53135 proteins and the improved formulations comprising one or more CG53135 proteins.
  • the present invention provides a formulation comprising about 0.1-1 M arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose, about 0.01-0.1 M sodium phosphate monobasic (NaH2PO4 ⁇ 2O), about 0.01 % -0.1 % weight/volume (“w/v") polysorbate 80 or polysorbate 20, and an isolated fibroblast growth factor ("FGF").
  • the concentration of the FGF in the formulations of the invention is about 0.005 mg/ml to about 50 mg/ml.
  • the FGF protein is preferably a CG53135 protein.
  • the formulations of the present invention comprise one or more isolated proteins selected from the group consisting of: (a) a protein comprising an amino acid sequence of SEQ ID NOs:2, 4, 7, 10, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; (b) a protein with one or more amino acid substitutions to the protein of (a), wherein said substitutions are no more than 15% of the amino acid sequence of SEQ ID NOs:2, 4, 7, 10, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, and wherein said protein with one or more amino acid substitutions retains cell proliferation stimulatory activity; and (c) a fragment of the protein of (a) or (b), which fragment retains cell proliferation stimulatory activity.
  • the formulations of the invention comprise one or more isolated proteins, where the concentration of the proteins is of 0.5-30 mg/ml. In a specific embodiment, the concentration of the proteins is 10 mg/ml. In some embodiments, the formulations of the invention are lyophilized or spray dried.
  • the formulations of the invention comprise an arginine in a salt form, which is selected from the group consisting of arginine, arginine sulfate, arginine phosphate, and arginine hydrochloride.
  • the arginine in a salt form, sulfobutyl ether Beta- cyclodextrin sodium, or sucrose in the formulations of the invention has a concentration of 0.01-0.7 M, preferably 0.5 M.
  • the sodium phosphate monobasic in the formulations of the invention has a concentration of 0.05 M.
  • polysorbate 80 or polysorbate 20 of the formulations of the invention is 0.01% (w/v).
  • the formulations of the invention comprise polysorbate 80.
  • the formulations of the invention comprise polysorbate 20.
  • a formulation of the invention comprises about 10 mg/ml of an isolated protein comprising an amino acid sequence of SEQ ID NO:24, 0.5 M arginine sulfate, 0.05 M sodium phosphate monobasic, and 0.01 % (w/v) polysorbate 80.
  • a formulation of the invention comprises about 10 mg/ml of an isolated protein comprising an amino acid sequence of SEQ ID NO:2, 0.5 M arginine sulfate, 0.05 M sodium phosphate monobasic, and 0.01% (w/v) polysorbate 80.
  • a formulation of the invention comprises 0.5 M arginine sulfate, 0.05 M sodium phosphate monobasic, 0.01 % (w/v) polysorbate 80, and about 10 mg/ml of a mixture of isolated proteins, wherein said proteins comprise a first protein comprising an amino acid sequence of SEQ ID NO:24, and a second protein comprising an amino acid sequence of SEQ ID NO:2.
  • a formulation of the invention further comprises one or more isolated proteins, wherein said proteins comprise an amino acid sequence selected from the group consisting of SEQ ID NOs:26, 28, 30 and 32.
  • the formulations of the invention comprise one or more isolated proteins that are carbamylated.
  • the present invention provides methods of increasing solubility of a fibroblast growth factor ("FGF") in an aqueous solution by adding arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose, or a combination thereof to said solution to a final concentration of 0.01 - 1 M.
  • FGF fibroblast growth factor
  • the fibroblast growth factor is an isolated CG53135 protein.
  • the fibroblast growth factor is an isolated protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 7, 10, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40.
  • an arginine in a salt form is selected from the group consisting of arginine, arginine sulfate, arginine phosphate, and arginine hydrochloride.
  • the final concentration of arginine in a salt form is 0.01 - 0.7 M, preferably 0.5 M.
  • the methods of the invention further comprise adding acetate, succinate, tartrate, or a combination thereof to the solution to increase the solubility of the protein.
  • the acetate, succinate, tartrate, or a combination thereof has a final concentration of 0.01-0.2 M in the solution.
  • the present invention provides a method of producing an isolated protein comprising the steps of: (1) fermenting an E. coli cell containing a vector comprising SEQ ID NO:8; (2) chilling the fermented culture to 10-15 0 C; (3) diluting the chilled culture with a lysis buffer comprising 50-100 mM sodium phosphate, 60 mM ethylene diamine tetraacetic acid, 7.5 mM DTT, and 3.5-5 M urea; (4) lysing the cells in the diluted culture; (5) loading the resultant cell lysate onto a pre-equilibrated cation exchange column, and flushing the column with a buffer comprising 50-100 mM sodium phosphate, 40 mM EDTA, 10 mM sodium sulfate, and 3-5 M urea; (6) washing the flushed column with a buffer comprising 50-100 mM sodium phosphate, 5 mM EDTA, 10-25 mM sodium sulfate, and 2.22
  • the method further comprises the steps of: (11) concentrating the resultant eluate; (12) filtering the retentate obtained together with a solution comprising 50 mM sodium phosphate, 0.5 M arginine; (13) concentrating the filtered retentate; and (14) filtering the concentrated retentate.
  • the cells are fermented by a method comprising the steps of: (a) culturing E. coli cells containing a vector comprising SEQ ID NO:8 to exponential growth phase with 2.5 to 4.5 OD600 units in a chemically defined seed medium; (b) inoculating cells of step (a) to a seed medium and culturing the cells to an exponential growth phase with 3.0 to 5.0 OD600 units; (c) transferring the cells of step (b) to a chemically defined batch medium; (d) culturing the cells of step (c) to 25-35 units OD600, and adding additional chemically defined medium with a feeding rate of 0.7 g/kg broth/minute; (e) culturing the cells of step (d) to 135 to 165 units OD600; and (f) culturing the cells of step (e) for about four hours.
  • the present invention also provides isolated proteins produced by the methods of the invention.
  • compositions and formulations of the invention for preventing and/or treating a disease, e.g., alimentary mucositis, arthritis, a disorder or symptom associated with radiation exposure, a disorder of central nerve system or cardiovascular system, are also provided.
  • a disease e.g., alimentary mucositis, arthritis, a disorder or symptom associated with radiation exposure, a disorder of central nerve system or cardiovascular system.
  • the term "about" in the context of a given numerate value or range refers to a value or range that is within 20%, preferably within 10%, and more preferably within 5% of the given value or range.
  • CG531335 refers to a class of proteins (including peptides and polypeptides) or nucleic acids encoding such proteins or their complementary strands, where the proteins comprise an amino acid sequence of SEQ ID NO:2 (211 amino acids), or its fragments, derivatives, variants, homologs, or analogs.
  • a CG53135 protein retains at least some biological activity of FGF-20.
  • biological activity means that a CG53135 protein possesses some but not necessarily all the same properties of (and not necessarily to the same degree as) FGF-20.
  • a member (e.g., a protein and/or a nucleic acid encoding the protein) of the CG53135 family may further be given an identification name.
  • CG53135-01 SEQ ID NOs:1 and 2 represents the first identified FGF-20 (see U.S. Patent Application No.
  • CG53135-05 represents a codon-optimized, full length FGF-20 (Ae., the nucleic acid sequence encoding FGF-20 has been codon optimized, but the amino acid sequence has not been changed from the originally identified FGF-20);
  • CG53135-12 (SEQ ID NOs:21 and 22) represent a single nucleotide polymorphism ("SNP") of FGF-20 where one amino acid in CG53135-12 is different from SEQ ID NO:2 (the aspartic acid at position 206 is changed to asparagine, " 206 D ⁇ N").
  • Some members of the CG53135 family may differ in their nucleic acid sequences but encode the same CG53135 protein, e.g., CG53135-01 , CG53135-03, and CG53135-05 all encode the same CG53135 protein.
  • An identification name may also be an in-frame clone ("IFC") number, for example, IFC 250059629 (SEQ ID NOs:33 and 34) represents amino acids 63-196 of the full length FGF-20 (cloned in frame in a vector).
  • Table 1 A shows a summary of some of the CG53135 family members.
  • the invention includes a variant of FGF-20 protein, in which some amino acids residues, e.g., no more than 1 %, 2%, 3%, 5%, 10% or 15% of the amino acid sequence of FGF-20 (SEQ ID NO:2), are changed.
  • the invention includes nucleic acid molecules that can hybridize to FGF-20 under stringent hybridization conditions.
  • the term "effective amount” refers to the amount of a therapy (e.g., a formulation comprising a CG53135 protein) which is sufficient to reduce and/or ameliorate the severity and/or duration of a disease (e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases) or one or more symptoms thereof, prevent the advancement of a disease, cause regression of a disease, prevent the recurrence, development, or onset of one or more symptoms associated with a disease, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy.
  • a disease e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases
  • FGF-20 refers to a protein comprising an amino acid sequence of SEQ ID NO:2, or a nucleic acid sequence encoding such a protein or the complementary strand thereof.
  • hybridizes under stringent conditions describes conditions for hybridization and washing under which nucleotide sequences at least 30% (preferably, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%) identical to each other typically remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • stringent hybridization conditions comprise a salt concentration from about 0.1 M to about 1.0 M sodium ion, a pH from about 7.0 to about 8.3, a temperature is at least about 6O 0 C 1 and at least one wash in 0.2 X sodium chloride/sodium citrate (SSC), 0.01% bovine serum albumin (BSA).
  • stringent hybridization conditions are hybridization at 6X SSC at about 45°C, followed by one or more washes in 0.1 X SSC, 0.2% sodium dodecyl sulfate (SDS) at about 68°C.
  • stringent hybridization conditions are hybridization in 6XSSC at about 45°C, followed by one or more washes in 0.2 X SSC 1 0.1% SDS at 50-65 0 C ⁇ i.e., one or more washes at 5O 0 C, 55 0 C 1 6O 0 C or 65 0 C). It is understood that the nucleic acids of the invention do not include nucleic acid molecules that hybridize under these conditions solely to a nucleotide sequence consisting of only A or T nucleotides.
  • the term "isolated" in the context of a protein agent refers to a protein agent that is substantially free of cellular material or contaminating proteins from the cell or tissue source from which it is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • substantially free of cellular material includes preparations of a protein agent in which the protein agent is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • a protein agent that is substantially free of cellular material includes preparations of a protein agent having less than about 30%, 20%, 10%, or 5% (by dry weight) of host cell proteins (also referred to as a "contaminating proteins").
  • the protein agent When the protein agent is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein agent preparation.
  • culture medium represents less than about 20%, 10%, or 5% of the volume of the protein agent preparation.
  • the protein agent When the protein agent is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein agent. Accordingly, such preparations of a protein agent have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the protein agent of interest.
  • protein agents disclosed herein are isolated.
  • nucleic acid molecules refers to a nucleic acid molecule which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • nucleic acid molecules are isolated.
  • the terms "prevent,” “preventing,” and “prevention” refer to the prevention of the recurrence, onset, or development of a disease (e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases) or one or more symptoms thereof in a subject resulting from the administration of a therapy (e.g., a composition comprising a CG53135 protein), or the administration of a combination of therapies.
  • a disease e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases
  • a therapy e.g., a composition comprising a CG53135 protein
  • prophylactically effective amount refers to the amount of a therapy (e.g., a composition comprising a CG53135 protein) which is sufficient to result in the prevention of the development, recurrence, or onset of a disease (e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases) or one or more symptoms thereof, or to enhance or improve the prophylactic effect(s) of another therapy.
  • a therapy e.g., a composition comprising a CG53135 protein
  • a disease e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases
  • stability in the context of a protein formulation, refers to the ability of a particular protein formulation to maintain the native, active structure of a protein as the protein is exposed to thermo-mechanical stresses over time.
  • stability of a protein formulation generally refers to the tendency of a protein formulation to form biologically inactive and/or insoluble aggregates of the protein as a result of exposure of the protein to thermo- mechanical stresses, as well as the tendency of a protein formulation to form biologically inactive and/or insoluble aggregates of the protein as a result of interaction with interfaces and surfaces that are destabilizing, such as hydrophobic surfaces and interfaces.
  • a related parameter to the "stability" of a protein formulation is its solubility in that higher molecular weight aggregates and denatured forms of a protein, including partially denatured forms of a protein, which are generally less soluble than their non-aggregated, lower molecular weight counterparts and native forms of the protein.
  • Another related parameter to the "stability" of a protein formulation is the protein concentration in that physically stable formulations may become less physically stable as the concentration of the protein is increased or decreased.
  • the terms “subject” and “subjects” refer to an animal, preferably a mammal, including a non-primate (e.g., a cow, pig, horse, cat, or dog), a primate (e.g., a monkey, chimpanzee, or human), and more preferably a human.
  • a non-primate e.g., a cow, pig, horse, cat, or dog
  • a primate e.g., a monkey, chimpanzee, or human
  • patient a mammal
  • the terms “treat,” “treatment,” and “treating” refer to the reduction of the progression, severity, and/or duration of a disease (e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases) or amelioration of one or more symptoms thereof, wherein such reduction and/or amelioration result from the administration of one or more therapies (e.g., a composition comprising a CG53135 protein).
  • a disease e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases
  • therapies e.g., a composition comprising a CG53135 protein
  • the term "therapeutically effective amount” refers to the amount of a therapy (e.g., a composition comprising a CG53135 protein), which is sufficient to reduce the severity or duration of a disease (e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases), prevent the advancement of a disease (e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases), cause regression of a disease (e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases), ameliorate one or more symptoms associated with a disease (e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases), or enhance or improve the therapeutic effect(s) of another therapy.
  • a disease e.g., alimentary mucositis, IBD, Irrit
  • FIG. 1 shows an example of manufacturing a drug product comprising one or more CG53135 proteins.
  • FIG. 2 shows SDS-PAGE analysis (gel code blue) of CG53135 produced by Process 1 and Process 2 (as described in Section 6), respectively.
  • Lane 1 molecular weight markers (kDa); lane 2-4: purified CG53135 (10 ⁇ g), reduced.
  • Lane 5-8 Process 1 reference standard DEV10 (720, 380, 45, and 28 ng) reduced.
  • (B) shows SDS-PAGE analysis (silver stain) of CG53135.
  • Lane 1 molecular weight markers (kDa) are shown on the left; lane 3: purified CG53135 by Process 2 (5 ⁇ g); lane 5: purified CG53135 reference standard Process 1 (5 ⁇ g); lane 7: purified CG53135 Process 2 (10 ⁇ g); lane 9: purified CG53135 reference standard Process 1 (10 ⁇ g).
  • FIG. 3 shows RP-HPLC analysis of CG53135 purified by Process 1 and Process 2, respectively (Process 1 is represented by the solid line).
  • FIG. 4 shows SEC-HPLC analysis of CG53135 purified by Process 1 and Process 2, respectively.
  • FIG. 5 shows host cell protein analysis of CG53135 E. coli purified product (by Process 1 and Process 2, respectively).
  • FIG. 6 shows Western Blot analysis of CG53135 purified by Process 1 and Process 2, respectively. Western blot was probed with anti-CG53135-05 antibody. Lane 1 : molecular weight marker (kDa); lane 3: CG53135 purified by Process 2 (10 ⁇ g); lane 5: CG53135 purified by Process 1 (10 ⁇ g).
  • FIG. 7 shows RP-HPLC identification analysis of CG53135 purified by Process 1 and Process 2, respectively.
  • Process 2 is represented by the dashed line.
  • FIG. 8 shows tryptic map of CG53135 purified by Process 1 and Process 2, respectively.
  • FIG. 9 shows circular dichroism spectroscopy analysis of CG53135 produced by Process 1 and Process 2, respectively.
  • the lower (grey) trace represents Process 1
  • the upper (black) trace represents Process 2.
  • FIG. 10 shows near UV circular dichroism spectroscopy analysis of CG53135 purified by Process 1 and Process 2, respectively.
  • the upper (grey) trace is the near UV CD spectrum of Process 1 and the black trace (lower) is the near UV CD spectrum of Process 2.
  • FIG. 11 shows second derivative absorbance spectra for Process 1 (grey trace) and Process 2 (black trace).
  • FIG. 12 shows temperature melting curves for Process 1 and Process 2, respectively, by differential scanning calorimetry.
  • FIG. 13 displays the biological activity of a truncated form of recombinant FGF-20 (CG53135-17, denoted by (d1-23)FGF20 in the Figure) as represented by its effects on DNA synthesis, compared to that of full length FGF-20 (denoted FGF20 in the Figure).
  • NIH 3T3 mouse fibroblasts were serum-starved, incubated with the indicated factor for 18 hours, and analyzed by a BrdU incorporation assay.
  • FIG. 14 shows dose response of CG53135-induced DNA synthesis in NIH 3T3 Fibroblasts. Serum starved NIH 3T3 cells were treated with purified CG53135-01 (CG53135 in figure), 10% serum or vehicle only (control). DNA synthesis was measured in triplicate for each sample, using a BrdU incorporation assay. Data points represent average BrdU incorporation and bars represent standard error (SE).
  • SE standard error
  • Y- axis identifies cell number, which is the average of 4 cell counts (treatment duplicates x duplicate counts) and standard error (SE).
  • C CG53135 induces DNA synthesis in 786-0 Kidney Epithelial cells. Serum starved 786-0 cells were left untreated or treated with partially purified CG53135-01 (from 5 ng/ ⁇ L stock), or with vehicle control (mock). DNA synthesis was measured in triplicate for each sample, using a BrdU incorporation assay. Data points represent average BrdU incorporation and bars represent standard error (SE).
  • FIG. 15 shows effect of Mucositis on the duration of mucositis induced by chemotherapy. The number of days with mucositis scores > 3 was evaluated.
  • FIG. 16 shows the cell positions in the crypt.
  • FIG. 17 (A) shows the crypt survival curve comparing prophylactic administration of CG53135-05 E. coli purified product treatment to PBS control group following different radiation dosages. (B) Shows the effect of prophylactic administration of CG53135-05 E. coli purified product on mice intestinal crypt survival after radiation insult.
  • FIG. 18 show the mean daily mucositis scores following treatment with CG53135-05 E. coli purified product. Mean group mucositis scores were obtained. Error bars represent the standard error of the means (SEM). A comparison of the untreated control group and the groups that received CG53135-05 12 mg/kg IP on days 1 and 2, with the groups that received CG53135-05 on day -1 only was performed. (A) shows groups that received CG53135-05 at 6 mg/kg or 12 mg/kg; and (B) shows groups that received CG53135-05 at 24 mg/kg or 48 mg/kg.
  • FIG. 19 shows mean daily mucositis scores following treatment with CG53135-05 E. coli purified product once, twice, thrice or four times. Mean group mucositis scores were obtained. Error bars represent the standard error of the means (SEM). A comparison of the untreated and vehicle control groups with the groups that received CG53135-05 E. coli purified product 12 mg/kg IP was performed. (A) Groups that received CG53135-05 E. coli purified product for one or two days; (B) Groups that received CG53135-05 purified product for three or four days.
  • FIG. 20 shows percent weight gain in animals with mucositis treated with CG53135-05 purified product.
  • Animals were weighed daily, the percent weight change from day -4 was calculated, and group means and standard errors of the mean (SEM) calculated for each day.
  • SEM standard errors of the mean
  • FIG. 21 shows Weight change represented as Area Under the Curve (AUC) gain in animals with mucositis treated with CG53135-05 E. coli purified product.
  • the area under the curve (AUC) was calculated for the percent weight change exhibited by each animal in the study. This calculation was made using the trapezoidal rule transformation. Group means were calculated and are shown with error bars representing SEM for each group. A One Way ANOVA was performed to compare groups.
  • FIG. 22 shows mean daily mucositis scores following treatment with CG53135-05 E. coli purified product once, twice, thrice or four times. Mean group mucositis scores were obtained. Error bars represent the standard error of the means (SEM).
  • FIG. 23 shows weight change represented as Area Under the Curve (AUC) for animals treated with single dose of CG53135-05 E. coli purified product for one, two, three or four days.
  • AUC Area Under the Curve
  • the area under the curve (AUC) was calculated for the percent weight change exhibited by each animal in the study. This calculation was made using the trapezoidal rule transformation. Group means were calculated and are shown with error bars representing SEM for each group. A One Way ANOVA was performed to compare groups.
  • FIG. 24 (A) and (B) show effects of CG53135 on body weight in animals with gastrointestinal injury induced by whole body irradiation as analyzed by one-way ANOVA and Dunnett's Multiple Comparison Test, respectively.
  • FIG. 25 (A) and (B) show effects of CG53135 on diarrhea score in mice with gastrointestinal injury induced by whole body irradiation as analyzed by one-way ANOVA and Tukey's Multiple Comparison Test, respectively.
  • FIG. 26 shows analysis of diarrhea score for each day of observation.
  • FIG. 27 shows the effect of Phosphate Buffered Saline (PBS) control on mice survival after exposure to radiation doses of 484 cGy, 534 cGy, 570 cGy, 606 cGy, or 641 cGy.
  • PBS Phosphate Buffered Saline
  • FIG. 28 shows the effect of prophylactic administration of CG53135 (day-1) on survival of mice after exposure to radiation doses of 484 cGy, 534 cGy, 570 cGy, 606 cGy, or 641 cGy.
  • B shows Kaplan-Meier plots for survival at 570 cGy and 606 cGy, with statistically significant differences between CG53135-treated and PBS-treated control animals.
  • C Probit analysis for survival over the range of radiation doses.
  • FIG. 29 shows the effect of prophylactic administration of CG53135 (day-2 and -1) on survival of mice after exposure to radiation doses of 484 cGy, 534 cGy, 570 cGy, 606 cGy, or 641 cGy.
  • FIG. 30 shows the effect of CG53135 multiple-dose administration prior to irradiation on crypt survival curves.
  • the plot represents the radiation dose-response for crypt survival.
  • Data points represent crypt survival in individual animals analyzed using a multi-target (Puck) analysis model, DRFIT.
  • FIG. 31 shows effect of CG53135 multiple-dose administration on crypt survival curves.
  • FIG. 32 (A)and (B) show CG53135 induced expression of scavengers, cycloxegenase, trefoil factor, and transcription factors in NIH 3T3 cells.
  • C shows CG53135 induced expression of scavengers, cycloxegenase, trefoil factor, and transcription factors in CCD1070sk cells.
  • D shows CG53135 induced expression of scavengers, cycloxegenase, trefoil factor, and transcription factors in CCDI8C0 cells.
  • E shows activation of ERK and AKT kinases by CG53135.
  • F shows CG53135 induced expression of scavengers, cycloxegenase, trefoil factor, and transcription factors in human umbilical vein endothelial cells (HUVEC).
  • FIG. 33 shows the effect of CG53135 on the survival of IEC 18 cells irradiated with different X-ray doses.
  • FIG. 34 shows the effect of CG53135 on the survival of HUVEC irradiated with different X- ray doses.
  • FIG. 35 shows survival curves for irradiated cells.
  • Cells of various types hematopoietic - 32D; mesenchymal - CCDI8-C0 and NIH3T3; epithelial - IEC18, IEC6 and bone - U2OS and Saos-2) were irradiated at the indicated doses then plated in complete growth media either with or without (untreated) 100 ng/ml CG53135-05 E. coli purified product and allowed to form colonies for 10-14 days until the colonies grew to an average diameter of 2 mm. The colonies were stained with crystal violet and counted. The natural log (Ln) of the surviving fraction is represented on the Y axis, and bars represent standard error.
  • FIG. 36 shows the effect of CG53135 on the release of cytokine in NIH 3T3 cells.
  • FIG. 37 shows dose response of CM-H 2 DCFDA fluorescence from IEC18 cells treated with CG53135 after 4Gy irradiation.
  • B shows response of CIvI-H 2 DCFDA fluorescence from IEC18 cells treated with CG53135 after 2Gy and 4Gy irradiation.
  • C shows dose response of CM- H 2 DCFDA fluorescence from CCD-I8C0 cells treated with CG53135 after 4Gy irradiation.
  • FIG. 38 shows dose response of Red CC-1 fluorescence from IEC18 cells treated with CG53135 after 4Gy irradiation.
  • B shows response of Red CC-1 fluorescence from IEC18 cells treated with CG53135 after 4Gy and 6Gy irradiation.
  • C shows response of Red CC-1 fluorescence from CCD-I8C0 cells treated with CG53135 before and after 10Gy irradiation.
  • FIG. 39 shows in vitro radioprotection of the myeloid cell line 32D by CG53135.
  • FlG. 40 shows effect of CG53135 on repopulation of thymus following bone marrow ablation and subsequent bone marrow transplant.
  • FIG. 41 shows the relative loss in body weight per group for study 439.
  • FIG. 42 shows the average diarrhea score over 3 days.
  • (B) shows the mean diarrhea severity.
  • FIG.43 shows effect of CG53135 in in vitro wound repair in CaCo2, HT-29, IEC-6 human cell lines.
  • FIG. 44 shows effect of CG53135 on COX-2 gene expression in HT-29 cells.
  • RT-PCR analysis was carried out to detect the expression of COX-2 gene in HT-29 cell line, in the presence of various concentration of CG53135 (0.1 , 1.0, 10, 100ng/ml).
  • COX-2 expression was also analyzed at various time points (1 , 3, 6, 24 hrs) after the addition of 10Ong/ml of CG53135.
  • FIG. 45 shows effect of CG53135 on COX-2 gene expression in Caco2 cells.
  • RT-PCR analysis was carried out to detect the expression of COX-2 gene in Caco2 cell line, in the presence of various concentration of CG53135 (0.1 , 1.0, 10, 100ng/ml).
  • COX-2 expression was also analyzed at various time points (1 , 3, 6, 24hrs) after the addition of 100ng/ml of CG53135.
  • FIG.46 shows effect of CG53135 on COX-2 gene expression in IEC-6 cells.
  • RT-PCR analysis was carried out to detect the expression of COX-2 gene in IEC-6 cell line, in the presence of various concentration of CG53135 (0.1 , 1.0, 10, 100ng/ml).
  • COX-2 expression was also analyzed at various time points (1 , 3, 6, 24hrs) after the addition of 100ng/ml of CG53135.
  • FIG. 47 shows effect of CG53135 on ITF gene expression in HT-29 and Caco2 cells.
  • ITF gene was detected by mRNA expression in HT-29 and Caco2 cells, in the presence of various concentration of CG53135 (0.1 , 1.0, 10, 100ng/ml).
  • ITF gene expression was also analyzed at various time points (1 , 3, 6, 24hrs) after the addition of 100ng/ml of CG53135.
  • FIG. 48 shows effect of CG53135 in inducing COX-2, TGF- ⁇ , ITF, PPAR- ⁇ in HT-29 mRNA expression analysis revealed that, upon induction with 100ng/ml of CG53135 for48hours, mammalian cells expressed COX-2, TGF- ⁇ , ITF, PPAR- ⁇ genes.
  • FIG.49 shows mechanism of Epithelial Restitution by CG53135.
  • TGF- ⁇ mediates epithelial restitution by FGF-20.
  • wound repair test was performed. Caco2 cells were incubated with CG53135-01 E. coli purified product (100ng/ml) and anti TGF- ⁇ (20 ⁇ g/ml) and percent closure was measured.
  • FIG. 50 shows Effect of CG53135 in stimulation of kinases in Caco2 cells. Expression of signal transducing kinases was analyzed after incubation of Caco2 cells with CG53135 E. coli purified product (100ng/ml) for different time points (10, 30, 60 minutes).
  • B Effect of kinase inhibitors in the expression of COX-2 gene in Caco2 cells. Caco2 cells were incubated with CG53135-01 E. coli purified product (100ng/ml) in the presence of 40 ⁇ M of PD098059 and 20 ⁇ M of SB203580 and COX-2 expression was analyzed.
  • C Effect of CG53135 in stimulation of kinases in THP- 1 cells.
  • Macrophage cell line THP-1 was cultured with CG53135-01 E. coli purified product (100ng/ml) for various time periods. Expression of signal transducing kinases was analyzed at different time points (10, 30, 60 mins).
  • D Effect of CG53135 on expression of kinases in intestinal epithelial cells. Caco2 cells were incubated with FGF-20 (100ng/ml) for 10, 30, 60 minutes and expression of p-Elk-1 , p-ATF-2 and p-PKC was analyzed. Similarly, HT-29 cells were incubated with FGF-20 (100ng/ml) for 10, 30, 60 minutes and expression of C-Fos and C-Jun was analyzed.
  • FIG. 17 also shows ITF expression in HT-29 cells in the presence of CG53135-01 E. coli purified product.
  • FlG. 51 shows presents the change in mean body weight from day 0 upon treating mice with varying doses of AB020258 (CG53135).
  • B presents the percent change in mean body weight from day 0 upon treating mice with varying doses of AB020258.
  • C presents mean colon blood content score upon treating mice with varying doses of AB020258.
  • FIG. 52 presents mean distal colon inflammation score upon treating mice with varying doses of AB020258.
  • B presents mean distal colon gland loss score upon treating mice with varying doses of AB020258.
  • C presents mean distal colon erosion score upon treating mice with varying doses of AB020258.
  • D presents mean sums of histopathology scores upon treating mice with varying doses of AB020258.
  • FlG. 53 presents mean splenic lymphoid atrophy score upon treating mice with varying doses of AB020258.
  • FlG. 54 presents mean splenic extramedullar hematopoiesis score upon treating mice with varying doses of AB020258.
  • FlG. 55 presents the effect of CG53135 Treatment on Small Intestine Weight in Indomethacin-treated rats.
  • FIG. 56 presents effect of CG53135 Treatment on Histopathology Scores in Indomethacin- treated rats. Five sections of affected intestine were evaluated and scored for necrosis and inflammation as described in the methods.
  • FIG. 57 presents images showing the protective effect of CG53135 on intestinal architecture.
  • Panel A Small intestine from normal control animal treated iv with vehicle (BSA).
  • Panel. B Small intestine from indomethacin- treated rat, further treated with vehicle (BSA) iv.
  • Panel C Small intestine from indomethacin-treated rat further treated with CG53135, 0.2 mg/kg iv. Sections were stained with H&E and visualized at a magnification of 25).
  • FIG. 60 shows the protective Effect of CG53135 on Intestinal Architecture in indomethacin treated rats.
  • Panel A normal control
  • Panel B disease control (indomethacin treated)
  • Panel C disease model animal treated with 0.2 mg/kg iv CG53135. Photomicrographs were obtained on sections stained with hemotoxylin and eosin, at 25X magnification.
  • FIG. 58 shows the effect of CG53135 treatment on BrdU Labeling in the Intestine. BrdU incorporation was detected by Immunoperoxidase staining.
  • Panel A Small intestine from normal control animal (100X).
  • Panel B Small intestine from indomethacin + vehicle (BSA) treated animal (50X).
  • Panel C Small intestine from indomethacin + CG53135 0.2 mg/kg iv treated rat (50X).
  • FIG. 59 shows effect of therapeutically-administered CG53135 on survival in the DSS model of colitis.
  • Female Balb/c mice were exposed to 4% DSS in drinking water for 7 days (day 0 to day 6) and then switched to normal drinking water for 4 additional days (day 7 to day 10).
  • CG53135 is identified as FGF-20 in FIG. 64.
  • CG53135 groups (n 9) received daily SC injections of the indicated concentrations of CG53135 on day 4 to day 9.
  • FIG. 60 shows weight change and histopathology in prophylactic group (IL-10KO mice). IL-10 KO mice were treated with various concentrations of CG53135 E. coli purified product (0.2, 1 , 5mg/kg) and weight change and histopathology was assessed.
  • B shows Total Cecal Histologic Score in prophylactic group (IL-10 KO mice). IL-10 KO mice were treated with various concentrations of FGF-20 (0.2, 1 , 5mg/kg) and total cecal histology was scored as described as described in the Example.
  • FIG. 61 shows IL-12 production in prophylactic group. IL-12 production was assayed by ELISA as described in Example 40, in MLN and colonic strip culture established.
  • B shows IFN- ⁇ production in prophylactic group. IFN- ⁇ production was assayed by ELISA, in MLN and colonic strip culture established.
  • C shows PGE2 production in prophylactic group. PGE2 production was assayed by ELISA in MLN prepared.
  • FIG. 62 shows FACS analysis (prophylactic group). FACS analysis was performed to get the total MLN number as well as number of CD4+, CD8+ and CD4+CD69+ cells.
  • FIG. 63 shows weight change in treatment group. Weight change in the treatment group was assessed.
  • FIG. 64 shows Histology of Cecum (treatment) was analyzed in vehicle control as well as CG53135 treated animals.
  • FIG. 65 shows Histology of Rectum (treatment) was analyzed in vehicle control as well as CG53135 treated animals.
  • FIG. 66 shows Total Cecal Histologic Score in treatment group (IL-10 KO mice). IL-10 KO mice were treated with CG53135 E. coli purified product (5mg/kg) and total cecal histology was scored.
  • FIG. 67 shows PGE2 and TNF- ⁇ production in treatment group.
  • PGE2 and TNF- ⁇ production was assayed by ELlSA, gut culture and unseparated splenocytes of CG53135 treated IL- 10 KO mice.
  • FIG. 68 shows the results of Forelimb Placing Test.
  • the mean and standard error of the score for groups receiving vehicle (diamonds), 1.0 ⁇ g/injection CG53135-05 (square), and 2.5 ⁇ g/injection CG53135-05 (triangles) are represented overtime. Asterisks indicate significant difference from vehicle control as assessed by one-way ANOVA.
  • (B) shows the results of Hindlimb Placing Test.
  • the mean and standard error of the score for groups receiving vehicle (diamonds), 1.0 ⁇ g/injection CG53135 (square), and 2.5 ⁇ g/injection CG53135 (triangles) are represented over time. Asterisks indicate significant difference from vehicle control as assessed by one-way ANOVA.
  • (C) shows the results of Body Swing Test.
  • the mean and standard error of the score for groups receiving vehicle (diamonds), 1.0 ⁇ g/injection CG53135 (square), and 2.5 ⁇ g/injection CG53135 (triangles) are represented over time.
  • a score range of ⁇ 50% swings to the right indicates no impairment, whereas 0% swings to the right swing indicates maximal impairment.
  • Asterisks indicate significant difference from vehicle control as assessed by one-way ANOVA.
  • (D) shows the results of Cylinder Test.
  • the mean and standard error of the score for groups receiving vehicle (diamonds), 1.0 ⁇ g/injection CG53135 (square), and 2.5 ⁇ g/injection CG53135 (triangles) are represented over time.
  • (E) shows the results of Body Weight.
  • the mean and standard errors of the weights for groups receiving vehicle (diamonds), 1.0 ⁇ g/injection CG53135 (square), and 2.5 ⁇ g/injection CG53135 (triangles) is represented overtime.
  • FIG. 69 shows the effect of CG53135 on Pro-MMP production in SW1353 cells in the presence of IL-1 beta.
  • B shows the effect of CG53135 on Pro-MMP production in SW1353 cells in the presence of TNF-alpha.
  • C shows the effect of CG53135 on TIMP production in SW1353 cells.
  • FIG. 70 shows the effect of intra-articular injection of CG53135 in the Meniscal Tear Model of Rat Osteoarthritis (Prophylactic Dosing): Mean Tibial Cartilage Degeneration.
  • B shows results of intra-articular injection of CG53135 in the Meniscal Tear Model of Rat Osteoarthritis: (Prophylactic Dosing): Total Cartilage Degeneration Width.
  • C shows results of intra-articular injection of CG53135 in the Meniscal Tear Model of Rat Osteoarthritis: (Prophylactic Dosing): Significant Tibial Cartilage Degeneration Width.
  • FIG. 71 shows results of intra-articular injection CG53135 in the Meniscal Tear Model of Rat Osteoarthritis (Therapeutic Dosing): Mean Tibial Degeneration.
  • B shows results of intra- articular injection of CG53135 in the Meniscal Tear Model of Rat Osteoarthritis (Therapeutic Dosing): Total Cartilage Degeneration Width.
  • C shows results of intra-articular injection of CG53135 in Meniscal Tear Model of Rat Osteoarthritis (therapeutic Dosing): Significant Tibial Cartilage Degeneration Width.
  • FIG. 72 shows trophic action of EGF, NGF and CG53135;
  • B shows the time course of CG53135-inhibited serum withdrawal-induced apoptosis.
  • FIG. 73 shows CG53135 inhibits serum withdrawal-induced caspase activation.
  • FIG. 74 shows neuritogenic action of CG53135 as compared to NGF.
  • FIG. 75 shows activation of MAPK by NGF and CG53135, and the inhibition of activity by PD98059, a MAPKK inhibitor.
  • the present invention provides improved formulations comprising one or more CG53135 proteins, which are more stable and soluble, and can be easily lyophilized by commercial equipments.
  • the improved formulations comprise 0.01-1 M of a stabilizer, such as arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose, 0.01-0.1 M sodium phosphate monobasic (NaH 2 PO 4 -H 2 O), 0.01 %-0.1 % weight/volume (“w/v”) polysorbate 80 or polysorbate 20, and one or more isolated CG53135 proteins.
  • a stabilizer such as arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose
  • 0.01 %-0.1 % weight/volume (“w/v”) polysorbate 80 or polysorbate 20 0.01
  • the concentration of CG53135 protein(s) in the improved formulations of the invention is less than 50 mg/ml, less than 30 mg/ml, less than 10 mg/ml, less than 5 mg/ml, or less than 1 mg/ml. In another embodiment, the concentration of CG53135 protein(s) in the improved formulations of the invention is between 0.005- 50 mg/ml. In a preferred embodiment, the formulation is lyophilized.
  • the present invention also provides methods for increasing solubility of a FGF protein in a solution (e.g., an aqueous solution).
  • a solution e.g., an aqueous solution.
  • the present invention provides a method for increasing solubility of a FGF protein in a solution by adding arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose to the solution.
  • the present invention provides a method for increasing solubility or stability of a FGF protein in a solution by adding buffering salts such as acetate, succinate, tartrate, phosphate, or a combination thereof to the solution.
  • buffering salts such as acetate, succinate, tartrate, phosphate, or a combination thereof is added in combination with arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose to the solution to increase the solubility of a FGF protein.
  • the arginine in a salt form can be, but is not limited to, arginine, arginine sulfate, arginine phosphate, and arginine hydrochloride. In a preferred embodiment, arginine sulfate is used.
  • the final concentration of the arginine in a salt form, sulfobutyl ether Beta- cyclodextrin sodium, or sucrose is between 0.01 M to 1 M. In one embodiment, the final concentration of the arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose is 0.5 M. In some embodiment, the final concentration of the buffering salts such as acetate, succinate, tartrate, phosphate, or a combination thereof is 0.05 M. In a preferred embodiment, the FGF protein is a FGF-20 protein, a fragment, a derivative, a variant, a homolog, or an analog of FGF-20, or a combination thereof.
  • the present invention further provides improved production methods for CG53135 proteins and/or formulations comprising one or more CG53135 proteins.
  • the improved production methods allow for commercial scale production of CG53135 proteins and/or formulations comprising one or more CG53135 proteins.
  • the improved production methods also allow for purifying CG53135 proteins to a high degree of purity.
  • the purity of the CG53135 purified by the improved production methods is at least 97%, at least 98%, at least 99%.
  • the purity of CG53135 purified by the improved production methods is from 99% up to 100% (including 100%).
  • the present invention also provides methods of use of the improved formulations and compositions of the invention for preventing or treating a disease (e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases) or one or more symptoms thereof, and dosage regiments.
  • a disease e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases
  • CG531335 refers to a class of proteins (including peptides and polypeptides) or nucleic acids encoding such proteins or their complementary strands, where the proteins comprise an amino acid sequence of SEQ ID NO:2 (211 amino acids, "FGF-20”), or its fragments, derivatives, variants, homologs, or analogs.
  • a CG53135 protein is a variant of FGF-20.
  • DNA sequence polymorphisms that lead to changes in the amino acid sequences of the FGF-20 protein may exist within a population (e.g., the human population).
  • Such genetic polymorphism in the FGF-20 gene may exist among individuals within a population due to natural allelic variation.
  • Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the FGF-20 gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in the FGF-20 protein, which are the result of natural allelic variation of the FGF-20 protein, are intended to be within the scope of the invention.
  • a CG53135 is CG53135-12 (SEQ ID NOs:21 and 22), which is a single nucleotide polymorphism ("SNP") of FGF-20 ⁇ i.e., 206 D ⁇ N).
  • SNP single nucleotide polymorphism
  • Other examples of SNPs of FGF-20 are also described in U.S. Patent Application No. 10/435,087, the content of which is incorporated herein by reference.
  • CG53135 refers to a nucleic acid molecule encoding a FGF-20 protein from other species or the protein encoded thereby, and thus has a nucleotide or amino acid sequence that differs from the human sequence of FGF-20.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues of the FGF-20 cDNAs of the invention can be isolated based on their homology to the human FGF-20 nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • CG53135 refers to a fragment of an FGF-20 protein, including fragments of variant FGF-20 proteins, mature FGF-20 proteins, and variants of mature FGF-20 proteins, as well as FGF-20 proteins encoded by allelic variants and single nucleotide polymorphisms of FGF-20 nucleic acids.
  • An example of an FGF-20 protein fragment includes, but is not limited to, residues 2-211 , 3-211 , 9-211 , 12-211 , 15-211 , 24-211 , 54-211 , or 55-211 of FGF-20 (SEQ ID NO:2).
  • CG53135 refers to a nucleic acid encodes a protein fragment that includes residues 2-211 , 3-211 , 9-211 , 12-211 , 15-211 , 24-211 , 54-211 , or 55-211 of SEQ ID NO:2.
  • the invention also encompasses derivatives and analogs of FGF-20.
  • the production and use of derivatives and analogs related to FGF-20 are within the scope of the present invention.
  • the derivative or analog is functionally active, i.e., capable of exhibiting one or more functional activities associated with a full-length, wild-type FGF-20.
  • Derivatives or analogs of FGF-20 can be tested for the desired activity by procedures known in the art, including but not limited to, using appropriate cell lines, animal models, and clinical trials.
  • FGF-20 derivatives can be made via altering FGF-20 sequences by substitutions, insertions or deletions that provide for functionally equivalent molecules.
  • alteration of an FGF-20 sequence is done in a region that is not conserved in the FGF protein family. Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence as FGF-20 may be used in the practice of the present invention. These include, but are not limited to, nucleic acid sequences comprising all or portions of FGF-20 which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.
  • a wild-type FGF-20 nucleic acid sequence is codon optimized to the nucleic acid sequence of SEQ ID NO:8 (CG53135-05).
  • the FGF-20 derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of FGF-20 including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • FGF-20 derivatives of the invention also include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of FGF-20 including altered sequences in which amino acid residues are substituted for residues with similar chemical properties. In a specific embodiment, 1 , 2, 3, 4, or 5 amino acids are substituted.
  • FGF-20 include, but are not limited to, those proteins which are substantially homologous to FGF-20 or fragments thereof, or whose encoding nucleic acid is capable of hybridizing to the FGF-20 nucleic acid sequence.
  • the FGF-20 derivatives and analogs of the invention can be produced by various methods known in the art.
  • the manipulations which result in their production can occur at the gene or protein level.
  • the cloned FGF-20 gene sequence can be modified by any of numerous strategies known in the art (e.g., Maniatis, T., 1989, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
  • the sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro.
  • the FGF-20-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
  • Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson, C. et a/., 1978, J. Biol. Chem 253:6551), use of TAB.RTM. linkers (Pharmacia), etc.
  • Manipulations of the FGF-20 sequence may also be made at the protein level. Included within the scope of the invention are FGF-20 fragments or other derivatives or analogs which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.
  • any of numerous chemical modifications may be carried out by known techniques, including but not limited to, reagents useful for protection or modification of free NH2- groups, free COOH- groups, OH- groups, side groups of Trp-, Tyr-, Phe-, His-, Arg-, or Lys-; specific chemical cleavage by cyanogen bromide, hydroxylamine, BNPS-Skatole, acid, or alkali hydrolysis; enzymatic cleavage by trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
  • analogs and derivatives of FGF-20 can be chemically synthesized.
  • a protein corresponding to a portion of FGF-20 which comprises the desired domain, or which mediates the desired aggregation activity in vitro, or binding to a receptor can be synthesized by use of a peptide synthesizer.
  • nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the FGF-20 sequence.
  • Non- classical amino acids include, but are not limited to, the D-isomers of the common amino acids, ⁇ - amino isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t- butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, designer amino acids such as ⁇ -methyl amino acids, C ⁇ -methyl amino acids, and N ⁇ -methyl amino acids.
  • the FGF-20 derivative is a chimeric or fusion protein comprising FGF-20 or a fragment thereof fused via a peptide bond at its amino- and/or carboxy-terminus to a non-FGF-20 amino acid sequence.
  • the non-FGF-20 amino acid sequence is fused at the amino-terminus of an FGF-20 or a fragment thereof.
  • such a chimeric protein is produced by recombinant expression of a nucleic acid encoding the protein (comprising an FGF-20-coding sequence joined in-frame to a non-FGF-20 coding sequence).
  • Such a chimeric product can be custom made by a variety of companies (e.g., Retrogen, Operon, etc.) or made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art.
  • a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer.
  • a chimeric nucleic acid encoding FGF-20 with a heterologous signal sequence is expressed such that the chimeric protein is expressed and processed by the cell to the mature FGF- 20 protein.
  • FGF-20 and non-FGF-20 gene may also be used to predict tertiary structure of the molecules using computer simulation (Hopp and Woods, 1981 , Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); the chimeric recombinant genes could be designed in light of correlations between tertiary structure and biological function.
  • chimeric genes comprising an essential portion of FGF-20 molecule fused to a heterologous (non-FGF-20) protein-encoding sequence may be constructed.
  • such chimeric construction can be used to enhance one or more desired properties of an FGF-20, including but not limited to, FGF-20 stability, solubility, or resistance to proteases.
  • chimeric construction can be used to target FGF-20 to a specific site.
  • chimeric construction can be used to identify or purify an FGF-20 of the invention, such as a His-tag, a FLAG tag, a green fluorescence protein (GFP), ⁇ -galactosidase, a maltose binding protein (MaIE), a cellulose binding protein (CenA) or a mannose protein, etc.
  • a CG53135 protein is carbamylated.
  • a CG53135 protein can be modified so that it has improved solubility and/or an extended half-life in vivo using any methods known in the art.
  • Fc fragment of human IgG, or inert polymer molecules such as high molecular weight polyethyleneglycol (PEG) can be attached to a CG53135 protein with or without a multifunctional linker either through site- specific conjugation of the PEG to the N- or C-terminus of the protein or via epsilon-amino groups present on lysine residues. Linear or branched polymer derivatization that results in minimal loss of biological activity will be used.
  • the degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the CG53135 protein. Unreacted PEG can be separated from CG53135-PEG conjugates by size-exclusion or by ion- exchange chromatography. PEG-derivatized conjugates can be tested for in vivo efficacy using methods known to those of skill in the art.
  • a CG53135 protein can also be conjugated to albumin in order to make the protein more stable in vivo or have a longer half life in vivo.
  • the techniques are well known in the art, see e.g., International Publication Nos. WO 93/15199, WO 93/15200, and WO 01/77137; and European Patent No. EP 413, 622, all of which are incorporated herein by reference.
  • CG53135 refers to CG53135-01 (SEQ ID NOs:1 and 2), CG53135- 02 (SEQ ID NOs:3 and 4), CG53135-03 (SEQ ID NOs:5 and 2), CG53135-04 (SEQ ID NOs:6 and 7), CG53135-05 (SEQ ID NOs:8 and 2), CG53135-06 (SEQ ID NOs:9 and 10), CG53135-07 (SEQ ID NOs:11 and 12), CG53135-08 (SEQ ID NOs:13 and 14), CG53135-09 (SEQ ID NOs:15 and 16), CG53135-10 (SEQ ID NOs:17 and 18), CG53135-11 (SEQ ID NOs:19 and 20), CG53135-12 (SEQ ID NOs:21 and 22), CG53135-13 (SEQ ID NOs:23 and 24), CG53135-14 (SEQ ID NOs:25 and 26), CG53135-01 (
  • CG53135 proteins employed in a formulation of the invention or produced by the production methods of the invention can have a purity in the range of 80 to 100 percent, or at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.
  • one or more CG53135 proteins employed in a formulation of the invention or produced by the production methods of the invention have a purity of at least 99%.
  • CG53135 is purified to apparent homogeneity, as assayed, e.g., by sodium dodecyl sulfate polyacrylamide gel electrophoresis.
  • any techniques known in the art can be used in purifying a CG53135 protein, including but are not limited to, separation by precipitation, separation by adsorption (e.g., column chromatography, membrane adsorbents, radial flow columns, batch adsorption, high-performance liquid chromatography, ion exchange chromatography, inorganic adsorbents, hydrophobic adsorbents, immobilized metal affinity chromatography, affinity chromatography), or separation in solution (e.g., gel filtration, electrophoresis, liquid phase partitioning, detergent partitioning, organic solvent extraction, and ultrafiltration).
  • separation by precipitation e.g., column chromatography, membrane adsorbents, radial flow columns, batch adsorption, high-performance liquid chromatography, ion exchange chromatography, inorganic adsorbents, hydrophobic adsorbents, immobilized metal affinity chromatography, affinity chromatography
  • separation in solution e.g.,
  • CG53135 may be monitored by one or more in vitro or in vivo assays as described in Section 5.3, infra.
  • the purity of CG53135 can be assayed by any methods known in the art, such as but not limited to, gel electrophoresis. See Scopes, supra.
  • a nucleic acid sequence encoding a CG53135 protein can be inserted into an expression vector for propagation and expression in host cells.
  • An expression construct refers to a nucleic acid sequence encoding a CG53135 protein operably associated with one or more regulatory regions which enable expression of a CG53135 protein in an appropriate host cell.
  • "Operably-associated” refers to an association in which the regulatory regions and the CG53135 sequence to be expressed are joined and positioned in such a way as to permit transcription, and ultimately, translation.
  • the regulatory regions necessary for transcription of CG53135 can be provided by the expression vector.
  • a translation initiation codon may also be provided if a CG53135 gene sequence lacking its cognate initiation codon is to be expressed.
  • cellular transcriptional factors such as RNA polymerase, will bind to the regulatory regions on the expression construct to effect transcription of the modified CG53135 sequence in the host organism.
  • the precise nature of the regulatory regions needed for gene expression may vary from host cell to host cell. Generally, a promoter is required which is capable of binding RNA polymerase and promoting the transcription of an operably-associated nucleic acid sequence.
  • Such regulatory regions may include those 5' non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • the non- coding region 3' to the coding sequence may contain transcriptional termination regulatory sequences, such as terminators and polyadenylation sites.
  • linkers or adapters providing the appropriate compatible restriction sites may be ligated to the ends of the cDNAs by techniques well known in the art (see e.g., Wu ef a/., 1987, Methods in Enzymol, 152:343-349). Cleavage with a restriction enzyme can be followed by modification to create blunt ends by digesting back or filling in single-stranded DNA termini before ligation.
  • a desired restriction enzyme site can be introduced into a fragment of DNA by amplification of the DNA using PCR with primers containing the desired restriction enzyme site.
  • An expression construct comprising a CG53135 sequence operably associated with regulatory regions can be directly introduced into appropriate host cells for expression and production of a CG53135 protein without further cloning. See, e.g., U.S. Patent No. 5,580,859.
  • the expression constructs can also contain DNA sequences that facilitate integration of a CG53135 sequence into the genome of the host cell, e.g., via homologous recombination. In this instance, it is not necessary to employ an expression vector comprising a replication origin suitable for appropriate host cells in order to propagate and express CG53135 in the host cells.
  • a variety of expression vectors may be used, including but are not limited to, plasmids, cosmids, phage, phagemids or modified viruses.
  • host-expression systems represent vehicles by which the coding sequences of a CG53135 gene may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express CG53135 in situ.
  • microorganisms such as bacteria (e.g., E.coli and S.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing CG53135 coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing CG53135 coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing CG53135 coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing CG53135 coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NSO, and 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian
  • bacterial cells such as Escherichia coli and eukaryotic cells are used for the expression of a recombinant CG53135 molecule.
  • mammalian cells such as Chinese hamster ovary cells (CHO) can be used with a vector bearing promoter element from major intermediate early gene of cytomegalovirus for effective expression of a CG53135 sequence (Foecking et a/., 1986, Gene 45:101; and Cockett et al., 1990, Bio/Technology 8:2).
  • a number of expression vectors may be advantageously selected depending upon the use intended for the CG53135 molecule being expressed.
  • vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Such vectors include, but are not limited to, the E.coli expression vector pCR2.1 TOPO (Invitrogen); plN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509) and the like.
  • pFLAG Sigma
  • pMAL NEB
  • pET Novagen
  • GST glutathione 5-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • a CG53135 coding sequence may be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).
  • a number of viral-based expression systems may be utilized.
  • a CG53135 coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination.
  • Insertion in a non-essential region of the viral genome will result in a recombinant virus that is viable and capable of expressing CG53135 in infected hosts (see, e.g., Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 8 1 :355- 359).
  • Specific initiation signals may also be required for efficient translation of inserted CG53135 coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
  • the efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153:51-544).
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript and post-translational modification of the gene product, e.g., glycosylation and phosphorylation of the gene product, may be used.
  • Such mammalian host cells include, but are not limited to, PC12, CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O and HsS78Bst cells.
  • Expression in a bacterial or yeast system can be used if post-translational modifications turn to be non-essential for a desired activity of CG53135.
  • E. coli is used to express a CG53135 sequence.
  • Cell lines that stably express CG53135 may be engineered by using a vector that contains a selectable marker.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the expression construct confers resistance to the selection and optimally allows cells to stably integrate the expression construct into their chromosomes and to grow in culture and to be expanded into cell lines. Such cells can be cultured for a long period of time while CG53135 is expressed continuously.
  • a number of selection systems may be used, including but not limited to, antibiotic resistance (markers like Neo, which confers resistance to geneticine, or G-418 (Wu and Wu, 1991 , Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem.
  • mutant cell lines including, but not limited to, tk-, hgprt- or aprt- cells, can be used in combination with vectors bearing the corresponding genes for thymidine kinase, hypoxanthine, guanine- or adenine phosphoribosyltransferase. Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al.
  • the recombinant cells may be cultured under standard conditions of temperature, incubation time, optical density and media composition. However, conditions for growth of recombinant cells may be different from those for expression of CG53135. Modified culture conditions and media may also be used to enhance production of CG53135. Any techniques known in the art may be applied to establish the optimal conditions for producing CG53135.
  • CG53135 or a fragment thereof is peptide synthesis.
  • an entire CG53135, or a protein corresponding to a portion of CG53135, can be synthesized by use of a peptide synthesizer. Conventional peptide synthesis or other synthetic protocols well known in the art may be used. Proteins having the amino acid sequence of CG53135 or a portion thereof may be synthesized by solid-phase peptide synthesis using procedures similar to those described by Merrifield, 1963, J. Am. Chem. Soc, 85:2149.
  • N- ⁇ -protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C-terminal and to an insoluble polymeric support, i.e., polystyrene beads.
  • the proteins are synthesized by linking an amino group of an N- ⁇ -deprotected amino acid to an ⁇ -carboxyl group of an N- ⁇ -protected amino acid that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide. The attachment of a free amino group to the activated carboxyl leads to peptide bond formation.
  • the most commonly used N- ⁇ -protecting groups include Boc which is acid labile and Fmoc which is base labile.
  • CG53135 Purification of the resulting CG53135 is accomplished using conventional procedures, such as preparative HPLC using gel permeation, partition and/or ion exchange chromatography.
  • preparative HPLC using gel permeation, partition and/or ion exchange chromatography.
  • the choice of appropriate matrices and buffers are well known in the art and so are not described in detail herein.
  • the present invention provides improved manufacturing processes for producing compositions comprising one or more CG53135 proteins.
  • the improved manufacturing processes provide benefits such as more stable and more pure drug product, and are also suitable for commercial scale production of a composition comprising one or more CG53135 proteins.
  • the present invention provides methods of isolating a protein, where the methods comprises the steps of: (1) fermenting a host cell, such as E. coli, that containing a vector, where the vector comprises a nucleotide sequence encoding a CG53135 protein.
  • the vector comprises a codon-optimized, full length CG53135-05 (SEQ ID NO:8); (2) lysing the cultured cells.
  • Cells may be lysed by any methods known in the art. In one embodiment, cells are lysed by homogenization.
  • the fermented cultured cells are chilled, and diluted with cell lysis buffer comprising 50-100 mM sodium phosphate, 60 mM EDTA, 7.5 mM DTT, 3.5-5 M urea, pH 7.2, and then lysed by, e.g., homogenization.
  • cell lysis buffer comprising 50-100 mM sodium phosphate, 60 mM EDTA, 7.5 mM DTT, 3.5-5 M urea, pH 7.2, and then lysed by, e.g., homogenization.
  • PEI polyethyleneimine
  • a pre-equilibrated expanded bed cation exchanger such as STREAMLINE SPTM is used.
  • the column is flushed with additional equilibration buffer comprising 50-100 mM sodium phosphate, 40 mM EDTA, 10 mM sodium sulfate, 3-5 M urea, pH 7.0.
  • additional equilibration buffer comprising 50-100 mM sodium phosphate, 40 mM EDTA, 10 mM sodium sulfate, 3-5 M urea, pH 7.0.
  • the column may be further washed with a buffer comprising 50-100 mM sodium phosphate, 5 mM EDTA, 10-25 mM sodium sulfate, 2.22 M dextrose, pH 7.0.
  • the elution buffer to elute the protein from the cation exchange column comprises, e.g., 50-100 mM sodium phosphate, 5 mM EDTA, 150-250 mM sodium sulfate, 0.5-1 M L-arginine, pH 7.0; and (4) further purification by using a hydrophobic interaction chromatography column (e.g., PPG 650M).
  • a hydrophobic interaction chromatography column e.g., PPG 650M
  • the hydrophobic interaction chromatography column e.g., PPG 650M, is equilibrated and washed with 50-100 mM sodium phosphate, 150-250 mM sodium sulfate, 5 mM EDTA, 1 M arginine, pH 7.0.
  • the column is further washed with 100-250 mM sodium phosphate, 5 mM EDTA, 0.8-1 M arginine, pH 7.0.
  • the protein is eluted with 50-100 mM sodium phosphate, 5 mM EDTA, and 0.1-0.3 M arginine, pH 7.0.
  • the eluted protein from step (4) described above may be further purified by either one or both of the following steps: (5) further purification by filtering the eluted protein.
  • a charged endotoxin binding filter e.g., CUNOTM 30 ZA depth filter
  • the filter is first flushed with water for injection, and then with 50- 100 mM sodium phosphate, 5 mM EDTA, 0.1-03 M arginine, pH 7.0; and (6) further purification by using a hydrophobic interaction chromatography column (e.g., Phenyl Sepharose HP Chromatography).
  • the column is equilibrated and washed with 50-100 mM sodium phosphate, 10-100 mM ammonium sulfate, 800-1000 mM sodium chloride, 0.5-1 M arginine, pH 7.0.
  • the protein is eluted with 50-100 mM sodium phosphate, 0.5-1 M arginine, pH 7.0.
  • a protein isolated by the methods of the present invention may be further concentrated and filtered to produce a drug product.
  • Pharmaceutical carriers may be added to produce a desired formulation, such as formulations provided by the present invention.
  • compositions for use in therapy can be tested in suitable animal model systems prior to testing in humans, including but not limited to in rats, mice, chicken, cows, monkeys, rabbits, etc (examples of such tests can be found in U.S. Patent Application Nos. 09/992,840, 10/011 ,364, 10/321,962, 10/435,087, 10/842,206, and 10/842,179, the disclosure of each is incorporated herein by reference).
  • a product of the manufacturing processes of the instant invention is at least 97%, at least 98%, or at least 99% pure by densitometry.
  • a product of the manufacturing processes of the instant invention is more than 97%, more than 98%, or more than 99% pure by densitometry.
  • Methods known in the art such as but not limited to, Western Blot, sequencing (e.g., N- terminal Edman sequencing), liquid chromatography (e.g., HPLC, RP-HPLC with both UV and electrospray mass spectrometric detection), mass spectrometry, total amino acid analysis, peptide mapping, SDS-PAGE, can be used to determine the identity of the product of the manufacturing processes of the instant invention.
  • the secondary, tertiary and/or quaternary structure of a product of the manufacturing processes of the instant invention can analyzed by any method know in the art, for example, far UV circular dichroism spectrum can be used to analyze the secondary structure, near UV circular dichroism spectroscopy and second derivative UV absorbance spectroscopy can be used to analyze the tertiary structure, and light scattering SEC-HPLC can be used to analyze quaternary structure.
  • Potency of a product of the manufacturing processes of the instant invention can be measured by methods known in the art or any bioassays that measuring one or more biological activities of a CG53135 protein. In one embodiment, potency of a product of the manufacturing processes of the instant invention is measured by the ability of the product to stimulate cell growth of NIH 3T3 cells (for an example of such assay, see Section 6.5).
  • a CG53135 protein reference standard which is representative of the bulk drug substance from the improved manufacturing process, is prepared and characterized.
  • this reference standard is characterized for its purity, identity (e.g., molecular weight, amino acid sequence), potency, structure (e.g., secondary, tertiary and quaternary structures), safety (e.g., endotoxin, bioburden), and other characters, such as but not limited to, pH, osmolality, and sulfyhydryl content.
  • identity e.g., molecular weight, amino acid sequence
  • structure e.g., secondary, tertiary and quaternary structures
  • safety e.g., endotoxin, bioburden
  • other characters such as but not limited to, pH, osmolality, and sulfyhydryl content.
  • a CG53245 protein reference standard should be clear and colorless.
  • the present invention provides methods of preventing and/or treating a disease (e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases) or one or more symptoms thereof comprising administering to a subject in need thereof an effective amount of a composition comprising one or more isolated CG53135 proteins.
  • a disease e.g., alimentary mucositis, IBD, Irritable Bowel Syndrome, arthritis, stroke, radiation-related diseases
  • the present invention provides methods of preventing and/or treating alimentary mucositis.
  • Alimentary mucositis that can be prevented and/or treated by the methods of the invention includes, but is not limited to, oral mucositis, esophagitis, stomatitis, enteritis, and proctitis.
  • the methods of the invention comprise administering an effective amount of a composition comprising one or more isolated CG53135 proteins to a subject with mucositis at more than one area in the alimentary canal (e.g., a subject with both oral mucositis and enteritis).
  • the methods of the invention comprise administering an effective amount of a composition comprising one or more isolated CG53135 proteins to a subject with mucositis at only one area in the alimentary canal (e.g., a subject with only oral mucositis, or a subject with only enteritis).
  • the alimentary mucositis that can be prevented and/or treated by the methods of the invention is oral mucositis.
  • the alimentary mucositis that can be prevented and/or treated by the methods of the invention is not an oral mucositis.
  • Alimentary mucositis may be induced by, e.g., chemical insult, radiation insult, biological insult (e.g., bacteria), or a combination thereof.
  • the present invention provides methods of preventing and/or treating alimentary mucositis in patient populations with alimentary mucositis and populations at risk to develop alimentary mucositis. In one embodiment, the present invention provides methods of preventing and/or treating alimentary mucositis in a subject who has been treated with radiation therapy and/or chemotherapy. In another embodiment, the present invention provides methods of preventing alimentary mucositis by administering a composition comprising one or more CG53135 proteins to a subject who is going to be treated with radiation therapy and/or chemotherapy.
  • the present invention provides methods of preventing and/or treating alimentary mucositis in a subject who has been treated with conditioning myeloablative radiation therapy and /or chemotherapy in preparation for autologous or allogenic hematopoietic stem cell transplant.
  • the present invention provides methods of preventing and/or treating alimentary mucositis in a subject who has received or is receiving mucosatoxic chemotherapy with mucositis-inducing agents (e.g., leukemia patients treated with cytarabine).
  • the present invention provides methods of preventing and/or treating alimentary mucositis in a subject who has head and/or neck cancer treated with radiation therapy with or without adjuvant chemotherapy.
  • the present invention provides a method of preventing alimentary mucositis comprising administering a composition comprising one or more CG53135 proteins prior to an insult (e.g., a chemical insult, a radiation insult, a biological insult, or a combination thereof) that may induce alimentary mucositis occurs to a subject.
  • an insult e.g., a chemical insult, a radiation insult, a biological insult, or a combination thereof
  • the present invention provides a method of preventing alimentary mucositis comprising administering a composition comprising one or more CG53135 proteins after an insult (e.g., a chemical insult, a radiation insult, a biological insult, or a combination thereof) that may induce alimentary mucositis occurs to a subject, but prior to the development of alimentary mucositis in the subject.
  • the present invention provides a method of treating alimentary mucositis comprising administering a composition comprising one or more CG53135 proteins after alimentary mucositis developed in a subject.
  • the present invention provides a method of preventing and/or treating alimentary mucositis comprising cyclically administering a composition comprising one or more CG53135 proteins.
  • cycling therapy involves the administration of a first therapy for a period of time, followed by the administration of a second therapy for a period of time and repeating this sequential administration, i.e., the cycle, in order to, e.g., to avoid or reduce the side effects of one of the therapies and/or to improve the efficacy of the therapies.
  • cycling therapy involves the administration of a therapy for a period of time, stop the therapy for a period of time, and repeat the administration of the therapy.
  • a composition comprising one or more CG53135 proteins can be administered to a subject prior to, during, or after the administration of a radiation therapy and/or chemotherapy, where such radiation therapy and/or chemotherapy is a cycling therapy.
  • a composition comprising one or more isolated CG53135 proteins can also be used in combination with other therapies to prevent and/or treat alimentary mucositis.
  • a composition comprising one or more isolated CG53135 proteins is administered in combination with one or more other agents that have prophylactic and/or therapeutic effect(s) on alimentary mucositis and/or have amelioration effect(s) on one or more symptoms associated with alimentary mucositis to a subject to prevent and/or treat alimentary mucositis.
  • Non-limiting examples of such agents are: mucosal protective agents (e.g., sucralfate, colloidal bismuth), antibiotics, antifungal agents (e.g., fluconazole, amphotericin B), antiviral agents (e.g., acyclovir), antiemetic agents (e.g., phenothiazines, butyrophenones, benzodiazepines, corticosteroids, cannabinoids, 5-HT3 serotonin receptor blockers), antidiarrhea agents (e.g., diphenoxylate, loperamide, kaolin, pectin, methylacellulose, activated attapulgite, magnesium aluminum silicate, non-steroidal anti-inflammatory agents (NSAIDs)), transforming growth factor (TGF), interleukin-11 (IL-11), granulocyte-macrophage colony stimulating factor (GM-CSF), keratinocyte growth factor (KGF), L-glutamine, Amifostene,
  • a composition comprising one or more isolated CG53135 proteins is administered in combination with one or more other therapies that have palliative effect on alimentary mucositis.
  • therapies are: application of topical analgesics such as lidocaine and/or systemic administration of narcotics and antibiotics, topical fluoride application with or without calcium phosphate, mechanical plaque removal, tooth sponges, sucking ice chips resulting in oral cooling, oral rinses with various anti-infective agents, oral mouthwashes with local anesthetics.
  • the present invention provides methods of preventing and/or treating one or more disorders associated with (e.g., caused by) radiation exposure, chemotherapy, chemical/biological warfare agents, and/or any other insults affecting rapidly proliferating tissues in the body, or one or more symptoms thereof by administering to a subject a prophylactically or therapeutically effective amount of a composition comprising one or more isolated CG53135 proteins.
  • the present invention provides methods of preventing and/or treating a pathology of epithelial cells and/or mesenchymal cells comprising administering to a subject in need thereof a composition comprising one or more CG53135 proteins.
  • the present invention provides methods of stimulating proliferation, differentiation or migration of epithelial cells and/or mesenchymal cells comprising administering to a subject in need thereof an effective amount of a composition comprising one or more CG53135 proteins.
  • Epithelial membranes are continuous sheets of cells with contiguous cell borders that have characteristic specialized sites of close contact called cell junction. Such membrane, which can be one or more cells thick, contain no capillaries. Epithelia are attached to the underlying connective tissue by a component known as a basement membrane, which is a layer of intercellular material of complex composition that is distributed as a thin layer between the epithelium and the connective tissue.
  • Stratified squamous nonkeratinizing epithelium is common on wet surfaces that are subject to considerable wear and tear at sites where absorptive function is not required.
  • the secretions necessary to keep such surfaces wet have to come from appropriately situated glands.
  • Sites lined by this type of epithelium include the esophagus and the floor and sides of the oral cavity.
  • Simple columnar epithelium is made up of a single layer of tall cells that again fit together in a hexagonal pattern.
  • the columnar cells are all specialized to secret mucus in addition to being protective. Sites of this type of epithelium is present include the lining of the stomach.
  • Mesenchymal cells are stem cells that can differentiate into, e.g., osteoblasts, chondrocytes, myocytes, and adipocytes. Mesenchymal-epithelial interactions play an important role in the physiology and pathology of epithelial tissues. Mesenchymal cells may associate with epithelium basement membrane (e.g., pericytes and perivascular monocyte-derived cells (MDCs)), or reside within epithelium (MDCs and T cells).
  • epithelium basement membrane e.g., pericytes and perivascular monocyte-derived cells (MDCs)
  • mesenchymal cells and tissue-specific cells may depend on the tissue type (e.g., brain versus epidermis), or on the prevention or allowance/stimulation of differentiation of cells into the suicidal state (apoptosis) by mesenchymal cells in a given epithelium.
  • Specialized mesenchymal cells such as pericytes, MDCs, and T lymphocytes, may significantly influence the differentiation and aging of epithelial cells.
  • the stromal compartment of the cavities of bone is composed of a net-like structure of interconnected mesenchymal cells. Stromal cells are closely associated with bone cortex, bone trabecule and to the hemopoietic cells.
  • the bone marrow-stromal micro- environment is a complex of cells, extracellular matrix (ECM) with growth factors and cytokines that regulate osteogenesis and hemopoiesis locally throughout the life of the individual.
  • ECM extracellular matrix
  • the role of the marrow stroma in creating the microenvironment for bone physiology and hemopoiesis lies in a specific subpopulation of the stroma cells. They differentiate from a common stem cell to the specific lineage each of which has a different role. Their combined function results in orchestration of a 3-D-architecture that maintains the active bone marrow within the bone.
  • the myeloid cell line includes, e.g., the following: (1) Immature cells called erythrocytes that later develop into red blood cells; (2) Blood clotting agents ( platelets); (3) Some white blood cells, including macrophages (which act as scavengers for foreign particles), eosinophils (which trigger allergies and also defend against parasites), and neutrophils (the main defenders against bacterial infections).
  • the lymphoid cell line includes, e.g., the lymphocytes, which are the body's primary infection fighters.
  • lymphocytes are responsible for producing antibodies, factors that can target and attack specific foreign agents (antigens). Lymphocytes develop in the thymus gland or bone marrow and are therefore categorized as either B-cells (bone marrow-derived cells) or T-cells (thymus gland-derived cells).
  • CG53135 can regulate proliferation, differentiation, and/or migration of epithelial cells and/or mesenchymal cells, and thus have prophylactic and/or therapeutic effects on a disorder associated with a pathology of epithelial cells and/or mesenchymal cells.
  • a composition used in accordance to the methods of the invention comprises a FGF-20 protein, a fragment, a derivative, a variant, a homolog, or an analog of FGF-20, or a combination thereof.
  • a composition used in accordance to the methods of the invention comprises CG53135-01 (SEQ ID NO:2), CG53135-02 (SEQ ID NO: 4), CG53135-03 (SEQ ID NO:2), CG53135-04 (SEQ ID NO:7), CG53135-05 (SEQ ID NO: 2), CG53135-06 (SEQ ID NO: 10), CG53135-07 (SEQ ID NO:12), CG53135-08 (SEQ ID NO:14), CG53135-09 (SEQ ID NO:16), CG53135-10 (SEQ ID NO:18), CG53135-11 (SEQ ID NO:20), CG53135-12 (SEQ ID NO:22), CG53135-13 (SEQ ID NO
  • compositions used in accordance to the methods of the present invention comprise (1) a protein comprising an amino acid sequence of SEQ ID NO:2, and (2) a protein comprising an amino acid sequence of SEQ ID NO:24.
  • compositions used in accordance to the methods of the present invention comprise (1) a protein comprising an amino acid sequence of SEQ ID NO:2, (2) a protein comprising an amino acid sequence of SEQ ID NO:24, (3) a protein comprising an amino acid sequence of SEQ ID NO:26, (4) a protein comprising an amino acid sequence of SEQ ID NO:28, (5) a protein comprising an amino acid sequence of SEQ ID NO:30, and (6) a protein comprising an amino acid sequence of SEQ ID NO:32.
  • compositions used in accordance to the methods of the present invention comprise (1) a protein comprising an amino acid sequence of SEQ ID NO:2, (2) a protein comprising an amino acid sequence of SEQ ID NO:24, (3) a protein comprising an amino acid sequence of SEQ ID NO:28, (4) a protein comprising an amino acid sequence of SEQ ID NO:30, and (5) a protein comprising an amino acid sequence of SEQ ID NO:32.
  • compositions used in accordance to the methods of the present invention comprise (1) a protein comprising an amino acid sequence of SEQ ID NO:32; (2) a protein comprising an amino acid sequence of SEQ ID NO:30, (3) a protein comprising an amino acid sequence of SEQ ID NO:28; and (4) a protein comprising an amino acid sequence of SEQ ID NO:24.
  • compositions used in accordance to the methods of the present invention comprise (1) a protein comprising an amino acid sequence of SEQ ID NO:2, (2) a protein comprising an amino acid sequence of SEQ ID NO:24, (3) a protein comprising an amino acid sequence of SEQ ID NO:28, (4) a protein comprising an amino acid sequence of SEQ ID NO:30, (5) a protein comprising an amino acid sequence of SEQ ID NO:32, (6) a carbamylated protein comprising an amino acid sequence of SEQ ID NO:24, and (7) a carbamylated protein comprising an amino acid sequence of SEQ ID NO:2.
  • an insult affecting rapidly proliferating tissues is radiation exposure.
  • the insult is ionizing radiation.
  • the insult may be one or more chemotherapies or one or more chemical/biological warfare agents (such as a vesicant agent or bacteria), or a combination thereof.
  • chemotherapy and chemical/biological warfare agent are alkylating agents, vesicant agents (e.g., mustard agents) and microorganisms.
  • an insult affecting rapidly proliferating tissues is one or more radiation exposures, one or more chemotherapies, one or more chemical/biological warfare agents, or a combination thereof.
  • Organs and body systems most sensitive to the effects of insult such as ionizing radiation include, but are not limited to, skin, hematopoietic and lymphatic systems, gonads, lungs, nerve tissues, and the Gl tract.
  • the insult are particularly damaging to hematopoietic and/or gastrointestinal tissues of a subject.
  • the disorder to be prevented or treated is a disorder of hematopoiesis, including but not limited to, anemia, leukopenia (e.g., neutropenia), thrombocytopenia, pancytopenia, and a clotting disorder.
  • the disorder to be prevented or treated is alimentary mucositis, including but not limited to, oral mucositis, esophagitis, stomatitis, enteritis, and proctitis.
  • the disorder to be prevented or treated is a cerebrovascular syndrome.
  • the symptoms associated with an insult affecting rapidly proliferating tissues include, but are not limited to, diarrhea, skin burn, sores, fatigue, dehydration, inflammation, hair loss, ulceration of oral mucosa, xerostomia, and bleeding (e.g., from the nose, mouth or rectum).
  • the present invention also provides methods of upregulating oxygen scavenging pathways in a subject, where the methods comprise administering to the subject a composition comprising one or more CG53135 proteins.
  • the oxygen scavenging pathways comprise one or more superoxide dismutases ("SOD"), including but are not limited to, intracellular CuZnSOD and MnSOD, and extracellular-SOD ("EC-SOD”).
  • the oxygen scavenging pathways comprise genes selected from the group consisting of ERK 1 AKT, a superoxide dismutase, cyclooxygenase-2 ("COX-2”), and Nrf-2.
  • CG53135 proteins Administering one or more CG53135 proteins to a subject increases transcription of enzymes, like superoxide, that scavenge ROS and convert them to less reactive intermediates, like hydrogen peroxide. Administering one or more CG53135 proteins to a subject also reduces the load of reactive oxygen species induced by an insult, such as radiation.
  • the present invention further provides methods of stimulating secretion of one or more endogenous cytokines and/or endogenous chemokines from cells of a subject comprising administering to the subject a composition comprising one or more CG53135 proteins.
  • the endogenous cytokines secreted can be, but are not limited to, interleukin ("IL") - 1 b, IL-6, IL-7, IL-8, IL-11 , and granulocyte-colony forming factor ("G-CSF").
  • the endogenous chemokines secreted can be, but are not limited to, chemokine (C-X-C motif) ligand 1 ("CXCL1”) and monocyte chemoattractant protein (“MCP-1").
  • the present invention provides methods of stimulating proliferation of hematopoietic stem cells and/or gastrointestinal stem cells of a subject, where the methods comprise administering to the subject a composition comprising one or more CG53135 proteins.
  • administration of one or more CG53135 proteins to a subject stimulates fibroblast cells within the bone marrow stroma to secret factors that facilitate the health and proliferative capacity of hematopoietic stem cells.
  • administration of one or more CG53135 proteins to a subject leads to a rapid proliferative burst of gastrointestinal stem cells, which is followed by a counter-regulatory inhibition in proliferation 24 hours later. This leads to a synchronization of the cell cycle at the tissue level, which is more radio-resistant.
  • the present invention also provides methods of optimizing engraftment of hematopoietic stem cells in a subject, where the methods comprise administering to the subject a composition comprising one or more CG53135 proteins.
  • administration of one or more CG53135 proteins improves successful engraftment or repopulation of T-cells following a bone marrow transplant after marrow radioablation.
  • administration of one or more CG53135 proteins increases the speed of T-cell reconstitution within the thymus after bone marrow transplant.
  • the patient population that can be targeted using the methods of the present invention include, but are not limited to, subjects who have been exposed to an insult affecting rapidly proiiferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents), subjects who are suspected to have been exposed an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents), subjects who will be exposed to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents), and subjects who are at risk to be exposed to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents).
  • an insult affecting rapidly proiiferating tissues such as radiation, chemotherapy, and chemical/biological warfare agents
  • subjects who are suspected to have been exposed an insult affecting rapidly proliferating tissues such as radiation, chemotherapy, and chemical/biological warfare agents
  • subjects who will be exposed to an insult affecting rapidly proliferating tissues such as radiation, chemotherapy, and chemical/biological warfare agents
  • a composition comprising one or more CG53135 proteins is administered to a subject prior to the subject's exposure to an insult affecting rapidly proliferating tissue in a body.
  • a composition comprising one or more CG53135 proteins is administered to a subject after the subject's exposure to an insult affecting rapidly proliferating tissue in a body but prior to a disorder associated with the insult or a symptom thereof developed in the subject.
  • a composition comprising one or more CG53135 proteins is administered to a subject after a disorder associated with an insult affecting rapidly proliferating tissue in a body or a symptom thereof developed in the subject.
  • a composition comprising one or more CG53135 proteins is administered to a subject who is at risk for exposure to an insult affecting rapidly proliferating tissues.
  • compositions comprising one or more CG53135 proteins can also be administered in combination with one or more other therapies to prevent, treat, or ameliorate a disorder or one or more symptoms associated with an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents).
  • a composition comprising one or more CG53135 proteins is administered in combination with one or more other therapies known to be used in preventing, treating, or ameliorating a disorder or one or more symptoms associated with an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents).
  • Examples of such other therapies include, but are not limited to, Mesna (sodium 2-mercaptoethene sulfonate) and other analogues with free thiol moieties, dimesna (disodium 2,2'-dithiobis ethane sulfonate) and other disulfides, and compounds such as, for example, described in U.S. Application Publication No. 20030092681 , and KGF (see e.g., U.S. Patent No. 6,743,422). Examples of other agents that can be used in combination with a composition comprising CG53135 is shown in Table 1 B.
  • a CG53135 protein and/or another therapy are administered in a sub-optimal amount, e.g., an amount that does not manifest detectable therapeutic benefits when administered alone, as determined by methods known in the art.
  • co-administration of a CG53135 protein and another therapy results in an overall improvement in effectiveness of treatment.
  • a composition comprising one or more CG53135 proteins and one or more other therapies are administered within the same patient visit.
  • a composition comprising one or more CG53135 proteins is administered prior to the administration of one or more other therapies.
  • a composition comprising one or more CG53135 proteins is administered subsequent to the administration of one or more other therapies.
  • a composition comprising one or more CG53135 proteins and one or more other therapies are cyclically administered to a subject. Cycling therapy involves the administration of a composition comprising one or more CG53135 proteins for a period of time, followed by the administration of one or more other therapies for a period of time and repeating this sequential administration. Cycling therapy can reduce the development of resistance to one or more of the therapies, avoid or reduce the side effects of one of the therapies, and/or improve the efficacy of the treatment.
  • the present invention provides methods of preventing and/or treating inflammatory bowel disease or irritable bowel syndrome comprising administering to a subject in need thereof an effective amount of a composition comprising one or more isolated CG53135 proteins.
  • Inflammatory bowel disease that can be prevented and/or treated by the methods of the invention includes, but is not limited to, ulcerative colitis and Crohn's disease.
  • the present invention provides methods of preventing and/or treating inflammatory bowel disease in patient populations with inflammatory bowel disease and populations at risk to develop inflammatory bowel disease.
  • the present invention also provides methods of preventing and/or treating irritable bowel syndrome in patient populations with irritable bowel syndrome and populations at risk to develop irritable bowel syndrome.
  • the present invention provides a method of preventing or treating inflammatory bowel disease or irritable bowel syndrome comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of an isolated protein selected from the group consisting of: (a) a protein comprising an amino acid sequence of SEQ ID NOs:2, 4, 7, 10, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40; (b) a protein with one or more amino acid substitutions to the protein of (a), wherein said substitutions are no more than 15% of the amino acid sequence of SEQ ID NOs:2, 4, 7, 10, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, and wherein said protein with one or more amino acid substitutions retains cell proliferation stimulatory activity; and (c) a fragment of the protein of (a) or (b), which fragment retains cell proliferation stimulatory activity.
  • an isolated protein selected from the group consisting of: (a) a protein comprising an amino acid sequence of SEQ ID NOs:2, 4, 7, 10, 22, 24, 26, 28, 30, 32, 34,
  • the present invention provides a method of preventing and/or treating inflammatory bowel disease or irritable bowel syndrome comprising cyclically administering a composition comprising one or more CG53135 proteins.
  • cycling therapy involves the administration of a first therapy for a period of time, followed by the administration of a second therapy for a period of time and repeating this sequential administration, i.e., the cycle, in order to, e.g., to avoid or reduce the side effects of one of the therapies and/or to improve the efficacy of the therapies.
  • cycling therapy involves the administration of a therapy for a period of time, stop the therapy for a period of time, and repeat the administration of the therapy.
  • a composition comprising one or more isolated CG53135 proteins can also be used in combination with other therapies to prevent and/or treat inflammatory bowel disease or irritable bowel syndrome.
  • a composition comprising one or more isolated CG53135 proteins is administered in combination with one or more other therapies (e.g., therapeutic agents) that have prophylactic and/or therapeutic effect(s) on inflammatory bowel disease and/or have amelioration effect(s) on one or more symptoms associated with inflammatory bowel disease to a subject to prevent and/or treat inflammatory bowel disease.
  • therapies e.g., therapeutic agents
  • Non-limiting examples of such therapies are: 5-aminosalicylates, antibiotics, corticosteroids, immunomodulators (e.g., 6-mercaptoputine, azathioprine, methotrexate, cyclosporine), and biological response modifiers (e.g., infliximab).
  • a composition comprising one or more isolated CG53135 proteins is administered in combination with one or more other therapies (e.g., therapeutic agents) that have prophylactic and/or therapeutic effect(s) on irritable bowel syndrome and/or have amelioration effect(s) on one or more symptoms associated with irritable bowel syndrome to a subject to prevent and/or treat inflammatory bowel disease.
  • Non-limiting examples of such therapies are: laxatives; antidiarrheals (e.g., diphenoxylate (e.g., Lomotil, Lomocot); loperamide (e.g., Imodium, Pepto Diarrhea), cholestyramine (e.g., Questran, Cholybar)); antispasmodics (e.g., dicyclomine, hyoscyamine, and clidinium (in combination with chlordiazepoxide hydrochloride)); peppermint oil; direct smooth muscle relaxants; antidepressants; 5-HT3 antagonists (e.g., Alosetron (Lotronex), cilansetron); 5-HT4 agonists (e.g., tegaserod (Zelnorm/Zelmac) and prucalopride); M3 receptor antagonists (e.g., zamifenacin and darifenacin).
  • antidiarrheals
  • the present invention provides methods of preventing and/or treating a disease (e.g., a joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, or neurodegenerative diseases) comprising administering to a subject in need thereof an effective amount of a composition comprising one or more isolated CG53135 proteins.
  • a disease e.g., a joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, or neurodegenerative diseases
  • the present invention provides methods of preventing and/or treating arthritis (e.g., osteoarthritis or rheumatic arthritis) comprising administering to a subject in need thereof a composition comprising one or more CG53135 proteins.
  • arthritis e.g., osteoarthritis or rheumatic arthritis
  • the present invention provides methods of reducing cartilage degeneration comprising administering to a subject in need thereof a composition comprising one or more CG53135 proteins.
  • the present invention provides methods of stimulating cartilage repair comprising administering to a subject in need thereof a composition comprising one or more CG53135 proteins.
  • the present invention provides methods of stimulating cartilage healing after surgery in a subject comprising administering to a subject a composition comprising one or more CG53135 proteins.
  • the present invention provides methods of preventing and/or treating a cardiovascular disease, such as stroke (e.g., ischemic stroke, hemorrhagic stroke), comprising administering to a subject a composition comprising one or more CG53135 proteins.
  • a cardiovascular disease such as stroke
  • the present invention provides methods of preventing and/or treating a cardiovascular disease, such as stroke, comprising administering to a subject a composition comprising an isolated protein comprising an amino acid sequence of SEQ ID NO: 4, 7, 10, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, or a combination thereof.
  • the present invention provides methods of preventing and/or treating a neurodegenerative disease (e.g., Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis, Huntington's disease) comprising administering to a subject in need thereof a composition comprising one or more CG53135 proteins.
  • a neurodegenerative disease e.g., Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis, Huntington's disease
  • the present invention provides methods of preventing and/or treating a neurodegenerative disease comprising administering to a subject in need thereof a composition comprising an isolated protein comprising an amino acid sequence of SEQ ID NO: 4, 7, 10, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, or a combination thereof.
  • the present invention provides a method of preventing and/or treating a disease (e.g., a joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, or neurodegenerative diseases) comprising cyclically administering a composition comprising one or more CG53135 proteins.
  • a disease e.g., a joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, or neurodegenerative diseases
  • cycling therapy involves the administration of a first therapy for a period of time, followed by the administration of a second therapy for a period of time and repeating this sequential administration, i.e., the cycle, in order to, e.g., to avoid or reduce the side effects of one of the therapies and/or to improve the efficacy of the therapies.
  • cycling therapy involves the administration of a therapy for a period of time, stop the therapy for a period of time, and repeat the administration of the therapy.
  • a composition comprising one or more CG53135 proteins can be administered to a subject prior to, during, or after the administration of a radiation therapy and/or chemotherapy, where such radiation therapy and/or chemotherapy is a cycling therapy.
  • composition comprising one or more isolated CG53135 proteins can also be used in combination with other therapies to prevent and/or treat a disease (e.g., a joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, or neurodegenerative diseases).
  • a disease e.g., a joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, or neurodegenerative diseases.
  • a composition comprising one or more isolated CG53135 proteins is administered in combination with one or more other agents that have prophylactic and/or therapeutic effect(s) on a disease (e.g., a joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, or neurodegenerative diseases) and/or have amelioration effect(s) on one or more symptoms associated with the disease to a subject to prevent and/or treat the disease.
  • a disease e.g., a joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, or neurodegenerative diseases
  • any other agents or therapies that are known in the art that can be used to prevent and/or treat a disease can be used in combination with a composition comprising one or more CG53135 proteins in accordance to the methods of the present invention.
  • a disease such as a joint disease, ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning, or neurodegenerative diseases
  • the present invention provides methods of stimulating cartilage healing after surgery in a subject comprising administering to a subject a composition comprising one or more CG53135 proteins.
  • Toxicity and efficacy of the prophylactic and/or therapeutic protocols of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 5O /ED 5 o.
  • Prophylactic and/or therapeutic agents that exhibit large therapeutic indices are preferred. While prophylactic and/or therapeutic agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage of the prophylactic and/or therapeutic agents for use in humans.
  • the dosage of such agents lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • composition of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
  • dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.
  • the dosage of a composition comprising one or more G53135 proteins for administration in a human patient provided by the present invention is at least 0.001 mg/kg, at least 0.005 mg/kg, at least 0.01 mg/kg, at least 0.03 mg/kg, at least 0.05 mg/kg, at least 0.1 mg/kg, at least 0.2 mg/kg, at least 0.3 mg/kg, at least 0.4 mg/kg, at least 0.5 mg/kg, at least 0.6 mg/kg, at least 0.7 mg/kg, at least 0.8 mg/kg, at least 0.9 mg/kg, at least 1 mg/kg, at least 2 mg/kg, at least 3 mg/kg, at least 4 mg/kg, at least 5 mg/kg, at least 6 mg/kg, at least 7 mg/kg, at least 8 mg/kg, at least 9 mg/kg, or at least 10 mg/kg (as measured by UV assay).
  • the dosage of a composition comprising one or more CG53135 proteins for administration in a human patient provided by the present invention is between 0.001-10 mg/kg, between 0.005-5 mg/kg, between 0.005-1 mg/kg, between 0.005-0.9 mg/kg, between 0.005-0.8 mg/kg, between 0.005-0.7 mg/kg, between 0.005-0.6 mg/kg, between 0.005-0.5 mg/kg, or between 0.005-0.3 mg/kg, between 0.01-1 mg/kg, between 0.01-0.9 mg/kg, between 0.01-0.8 mg/kg, between 0.01-0.7 mg/kg, between 0.01-0.6 mg/kg, between 0.01-0.5 mg/kg, or between 0.01-0.3 mg/kg (as measured by UV assay).
  • Protein concentration can be measured by methods known in the art, such as Bradford assay or UV assay, and the concentration may vary depending on what assay is being used.
  • the protein concentration in a pharmaceutical composition of the instant invention is measured by a UV assay that uses a direct measurement of the UV absorption at a wavelength of 280 nm, and calibration with a well characterized reference Standard of CG53135 protein (instead of IgG). Test results obtained with this UV method (using CG53135 reference standard) are three times lower than test results for the same sample(s) tested with the Bradford method (using IgG as calibrator).
  • a dosage of a composition comprising one or more CG53135 proteins for administration in a human patient provided by the present invention is between 0.001-10 mg/kg measured by UV assay, then the dosage is 0.003-30 mg/kg as measured by Bradford assay.
  • each patient prior to administering the first full dose, preferably receives a subcutaneous injection of a small amount (e.g., 1/100 to 1/10 of the prescribed dose) of a composition of the invention to detect any acute intolerance.
  • a small amount e.g., 1/100 to 1/10 of the prescribed dose
  • the injection site is examined one and two hours after the test. If no reaction is detected, then the full dose is administered.
  • the present invention encompasses pharmaceutical compositions (including formulations) comprising one or more CG53135 proteins.
  • the pharmaceutical compositions can be administered to a subject at a prophylactically or therapeutically effective amount to prevent and/or treat one or more diseases, such as inflammatory bowel disease ("IBD"), irritable bowel syndrome, alimentary mucositis (including oral mucositis), arthritis, diseases associated with the central nerve system or cardiovascular system, and symptoms associated with radiation exposure.
  • IBD inflammatory bowel disease
  • alimentary mucositis including oral mucositis
  • arthritis diseases associated with the central nerve system or cardiovascular system
  • diseases associated with the central nerve system or cardiovascular system such as a central nerve system or cardiovascular system.
  • Various delivery systems are known and can be used to administer a composition comprising one or more CG53135 proteins.
  • Such delivery systems include, but are not limited to, encapsulation in liposomes, microparticles, microcapsules, expression by recombinant cells, receptor-mediated endocytosis, construction of the nucleic acids of the invention as part of a retroviral or other vectors, etc.
  • Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intrathecal, intracerebroventricular, epidural, intravenous, subcutaneous, intranasal, intratumoral, transdermal, rectal, and oral routes.
  • compositions of the invention may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, virginal mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. Administration can be systemic or local.
  • the present invention comprises using single or double chambered syringes, preferably equipped with a needle-safety device and a sharper needle, that are pre-filled with a composition comprising one or more CG53135 proteins.
  • dual chambered syringes e.g., Vetter Lyo-Ject dual-chambered syringe by Vetter Pharmar-Fertist
  • Vetter Lyo-Ject dual-chambered syringe by Vetter Pharmar-Fertist
  • Such systems are desirable for lyophilized formulations, and are especially useful in an emergency setting.
  • compositions of the invention may be desirable to administer locally to the area in need of treatment. This may be achieved by, for example, topical application, by injection, by infusion pump, by means of a suppository, or by means of an implant (the implant being of a reservoir with a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers).
  • a CG53135 nucleic acid can be administered in vivo Xo promote expression of their encoded proteins, by constructing the nucleic acid as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector, or by direct intramuscular or intradermal injection, or by use of microparticle bombardment (e.g., a gene gun), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus, etc.
  • a CG53135 nucleic acid can be introduced intracellular ⁇ and incorporated within host cell DNA for expression, by homologous recombination.
  • compositions of the invention are a pharmaceutical composition.
  • Such compositions comprise a prophylactically or therapeutically effective amount of CG53135, and a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions are formulated to be suitable for the route of administration to a subject.
  • the term "pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally regarded as safe for use in humans (GRAS).
  • carrier refers to a diluent, adjuvant, bulking agent (e.g., arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose), excipient, or vehicle with which CG53135 is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils (e.g., oils of petroleum, animal, vegetable or synthetic origins, such as peanut oil, soybean oil, mineral oil, sesame oil and the like), or solid carriers, such as one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, or encapsulating material.
  • oils e.g., oils of petroleum, animal, vegetable or synthetic origins, such as peanut oil, soybean oil, mineral oil, sesame oil and the like
  • solid carriers such as one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, or encapsulating material.
  • Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include, but are not limited to, starch or its synthetically modified derivatives such as hydroxyethyl starch, stearate salts, glycerol, glucose, lactose, sucrose, trehalose, gelatin, sulfobutyl ether Beta-cyclodextrin sodium, sodium chloride, glycerol, propylene, glycol, water, ethanol, or a combination thereof.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • compositions comprising CG53135 may be formulated into any of many possible dosage forms such as, but not limited to, liquid, suspension, microemulsion, microcapsules, tablets, capsules, gel capsules, soft gels, pills, powders, enemas, sustained-release formulations and the like.
  • the compositions comprising CG53135 may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the composition can also be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch or its synthetically modified derivatives such as hydroxyethyl starch, stearate salts, sodium saccharine, cellulose, magnesium carbonate, etc.
  • a pharmaceutical composition comprising CG53135 is formulated to be compatible with its intended route of administration.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal, intratumoral or topical administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic or hypertonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as benzyl alcohol or lidocaine to ease pain at the site of the injection.
  • composition comprising CG53135
  • the composition can be formulated in the form of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Coated condoms, gloves and the like may also be useful.
  • compositions of the invention are in admixture with a topical delivery agent, such as but not limited to, lipids, liposomes, micelles, emulsions, sphingomyelins, lipid-protein or lipid peptide complexes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.
  • a topical delivery agent such as but not limited to, lipids, liposomes, micelles, emulsions, sphingomyelins, lipid-protein or lipid peptide complexes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.
  • the compositions comprising CG53135 may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, the compositions comprising CG53135 may be complexed to lipids, in particular to cationic lipids.
  • viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity preferably greater than water are typically employed.
  • suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as Freon or hydrofluorocarbons) or in a squeeze bottle.
  • a pressurized volatile e.g., a gaseous propellant, such as Freon or hydrofluorocarbons
  • Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well-known in the art.
  • a composition comprising CG53135 can be formulated in an aerosol form, spray, mist or in the form of drops or powder if intranasal administration is preferred.
  • a composition comprising CG53135 can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, other hydrofluorocarbons, carbon dioxide or other suitable gas).
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, other hydrofluorocarbons, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Microcapsules (composed of, e.g., polymerized surface) for use
  • One or more CG53135 proteins may also be formulated into a microcapsule with one or more polymers (e.g., hydroxyethyl starch) form the surface of the microcapsule.
  • polymers e.g., hydroxyethyl starch
  • Such formulations have benefits such as slow-release.
  • a composition comprising CG53135 can be formulated in the form of powders, granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets if oral administration is preferred. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc, or silica
  • disintegrants e.g., potato starch or sodium starch
  • Liquid preparations for oral administration may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • the preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate.
  • Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of a prophylactic or therapeutic agent(s).
  • compositions of the invention are orally administered in conjunction with one or more penetration enhancers, e.g., alcohols, surfactants and chelators.
  • Preferred surfactants include, but are not limited to, fatty acids and esters or salts thereof, bile acids and salts thereof.
  • combinations of penetration enhancers are used, e.g., alcohols, fatty acids/salts in combination with bile acids/salts.
  • sodium salt of lauric acid, capric acid is used in combination with UDCA.
  • Further penetration enhancers include, but are not limited to, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.
  • compositions of the invention may be delivered orally in granular form including, but is not limited to, sprayed dried particles, or complexed to form micro or nanoparticles.
  • Complexing agents that can be used for complexing with the compositions of the invention include, but are not limited to, poly-amino acids, polyimines, polyacrylates, polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates, cationized gelatins, albumins, acrylates, polyethyleneglycols (PEG), DEAE-derivatized polyimines, pollulans, celluloses, and starches.
  • Particularly preferred complexing agents include, but are not limited to, chitosan, N-trimethylchitosan, poly-L-Iysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g.
  • a composition comprising CG53135 can be delivered to a subject by pulmonary administration, e.g., by use of an inhaler or nebulizer, of a composition formulated with an aerosolizing agent.
  • a composition comprising CG53135 is formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion).
  • Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as benzyl alcohol or lidocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a sealed container, such as a vial, ampoule or sachette, indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion container containing sterile pharmaceutical grade water or saline.
  • an ampoule or vial of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • a composition comprising CG53135 can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include, but are not limited to, those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • compositions comprising CG53135 may also be formulated as a depot preparation.
  • Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
  • the compositions may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • suitable polymeric or hydrophobic materials for example, as an emulsion in an acceptable oil
  • ion exchange resins for example, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophilic drugs.
  • Toxicity and efficacy of the prophylactic and/or therapeutic protocols of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 ZED 50 .
  • Prophylactic and/or therapeutic agents that exhibit large therapeutic indices are preferred. While prophylactic and/or therapeutic agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage of the prophylactic and/or therapeutic agents for use in humans.
  • the dosage of such agents lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (Ae., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 the concentration of the test compound that achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • composition of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
  • dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.
  • the dosage of a composition comprising one or more G53135 proteins for administration in a human patient provided by the present invention is at least 0.001 mg/kg, at least 0.01 mg/kg, at least 0.1 mg/kg, at least 0.5 mg/kg, at least 1mg/kg, at least 2 mg/kg, at least 3 mg/kg, at least 4 mg/kg, at least 5 mg/kg, at least 6 mg/kg, at least 7 mg/kg, at least 8 mg/kg, at least 9 mg/kg, at least 10 mg/kg, at least 15 mg/kg, at least 20 mg/kg, at least 25 mg/kg, at least 30 mg/kg, at least 35 mg/kg, at least 40 mg/kg, at least 45 mg/kg, at least 50 mg/kg, at least 60 mg/kg, at least 70 mg/kg, at least 80 mg/kg, at least 90 mg/kg, at least 100 mg/kg, at least 150 mg/kg, or at least 200 mg/kg (as measured by Bradford assay).
  • the dosage of a composition comprising one or more CG53135 proteins for administration in a human patient provided by the present invention is between 0.001-300 mg/kg, between 0.01-300 mg/kg, between 0.1-300 mg/kg, between 0.5-250 mg/kg, between 1-200 mg/kg, between 1-150 mg/kg, between 1- 125 mg/kg, between 1-100 mg/kg, between 1-90 mg/kg, between 1-80 mg/kg, between 1-70 mg/kg, between 1-60 mg/kg, between 1-50 mg/kg, between 1-40 mg/kg, between 1-35 mg/kg, between 1- 30 mg/kg, between 1-25 mg/kg, between 1-20 mg/kg, between 1-15 mg/kg, between 1-10 mg/kg, or between 1-5 mg/kg (as measured by Bradford assay).
  • Protein concentration can be measured by methods known in the art, such as Bradford assay or UV assay, and the concentration may vary depending on what assay is being used.
  • the protein concentration in a pharmaceutical composition of the instant invention is measured by a UV assay that uses a direct measurement of the UV absorption at a wavelength of 280 nm, and calibration with a well characterized reference standard of CG53135 protein (instead of IgG). Test results obtained with this UV method (using CG53135 reference standard) are three times lower than test results for the same sample(s) tested with the Bradford method (using IgG as calibrator).
  • a dosage of a composition comprising one or more CG53135 proteins for administration in a human patient provided by the present invention is between 0.1-300 mg/kg measured by Bradford assay, then the dosage is 0.033 - 100 mg/kg as measured by a UV assay.
  • each patient prior to administering the first full dose, each patient preferably receives a bolus injection of a small amount (e.g., 1/100 to 1/10 of the prescribed dose) of a composition of the invention to detect any acute intolerance.
  • a small amount e.g., 1/100 to 1/10 of the prescribed dose
  • the injection site is examined one and two hours after the test. If no reaction is detected, then the full dose is administered.
  • kits for carrying out the therapeutic regimens of the invention comprise in one or more containers prophylactically or therapeutically effective amounts of the composition of the invention in pharmaceutically acceptable form.
  • the composition in a vial of a kit of the invention may be in the form of a pharmaceutically acceptable solution, e.g., in combination with sterile saline, dextrose solution, or buffered solution, or other pharmaceutically acceptable sterile fluid.
  • the composition may be lyophilized or desiccated; in this instance, the kit optionally further comprises in a container a pharmaceutically acceptable solution (e.g., saline, dextrose solution, etc.), preferably sterile, to reconstitute the composition to form a solution for injection purposes.
  • a pharmaceutically acceptable solution e.g., saline, dextrose solution, etc.
  • a kit of the invention further comprises a needle or syringe (single or dual chambered), preferably packaged in sterile form, for injecting the formulation, and/or a packaged alcohol pad.
  • a kit of the invention comprises pre-filled needles or syringes (single or dual chambered) that are pre-filled with a composition comprising one or more CG53135 proteins. Instructions are optionally included for administration of the formulations of the invention by a clinician or by the patient.
  • kits comprising a plurality of containers each comprising a pharmaceutical formulation or composition comprising a dose of the composition of the invention sufficient for a single administration.
  • the present invention provides improved formulations comprising one or more FGFs 1 preferably one or more CG53135 proteins, and methods for increasing solubility of FGF proteins.
  • the improved formulations are more stable and more favorable for commercial scale productions.
  • the improved formulations are based partially on the discovery that high concentrations of arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, sucrose, acetate, succinate, or tartrate or a combination thereof can increase solubility of a growth factor, including FGF proteins.
  • the present invention provides a method of increasing solubility of a FGF protein in a solution (e.g., an aqueous solution) by adding arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose to the solution.
  • the present invention provides a method for increasing solubility of a FGF protein in a solution by adding acetate, succinate, tartrate, or a combination thereof to the solution.
  • acetate, succinate, tartrate or a combination thereof is added in combination with arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose to the solution to increase the solubility of a FGF protein.
  • the arginine in a salt form can be, but is not limited to, arginine, arginine sulfate, arginine phosphate, and arginine hydrochloride. In a preferred embodiment, arginine sulfate is used.
  • the final concentration of the arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose is between 0.01 M to 1 M. In one embodiment, the final concentration of the arginine in a salt form is 0.5 M. In some embodiment, the final concentration of the acetate, succinate, tartrate or a combination thereof is 0.01 to 0.2 M.
  • the FGF protein in the formulation is a FGF-20 protein, a fragment, a derivative, a variant, a homolog, or an analog of FGF-20, or a combination thereof.
  • the FGF protein in the formulation is CG53135-01 (SEQ ID NO:2), CG53135- 02 (SEQ ID NO: 4), CG53135-03 (SEQ ID NO:2), CG53135-04 (SEQ ID NO:7), CG53135-05 (SEQ ID NO: 2), CG53135-06 (SEQ ID NO: 10), CG53135-07 (SEQ ID NO:12), CG53135-08 (SEQ ID NO:14), CG53135-09 (SEQ ID NO:16), CG53135-10 (SEQ ID NO:18), CG53135-11 (SEQ ID NO:20), CG53135-12 (SEQ ID NO:22), CG53135-13 (SEQ ID NO:24), CG53135-14 (
  • the FGF proteins in the formulation comprise (1) a protein comprising an amino acid sequence of SEQ ID NO:2, and (2) a protein comprising an amino acid sequence of SEQ ID NO:24.
  • the FGF proteins in the formulation comprise (1) a protein comprising an amino acid sequence of SEQ ID NO:2, (2) a protein comprising an amino acid sequence of SEQ ID NO:24, (3) a protein comprising an amino acid sequence of SEQ ID NO:26, (4) a protein comprising an amino acid sequence of SEQ ID NO:28, (5) a protein comprising an amino acid sequence of SEQ ID NO:30, and (6) a protein comprising an amino acid sequence of SEQ ID NO:32.
  • the FGF proteins in the formulation comprise (1) a protein comprising an amino acid sequence of SEQ ID NO:2, (2) a protein comprising an amino acid sequence of SEQ ID NO:24, (3) a protein comprising an amino acid sequence of SEQ ID NO:28, (4) a protein comprising an amino acid sequence of SEQ ID NO:30, and (5) a protein comprising an amino acid sequence of SEQ ID NO:32.
  • a formulation of the invention comprises (1) a protein comprising an amino acid sequence of SEQ ID NO:32; (2) a protein comprising an amino acid sequence of SEQ ID NO:30, (3) a protein comprising an amino acid sequence of SEQ ID NO:28; and (4) a protein comprising an amino acid sequence of SEQ ID NO:24.
  • the FGF proteins in the formulation comprise (1) a protein comprising an amino acid sequence of SEQ ID NO:2, (2) a protein comprising an amino acid sequence of SEQ ID NO:24, (3) a protein comprising an amino acid sequence of SEQ ID NO:28, (4) a protein comprising an amino acid sequence of SEQ ID NO:30, (5) a protein comprising an amino acid sequence of SEQ ID NO:32, (6) a carbamylated protein comprising an amino acid sequence of SEQ ID NO:24, and (7) a carbamylated protein comprising an amino acid sequence of SEQ ID NO:2.
  • the present invention provides improved formulations comprising arginine in a salt form, sodium phosphate monobasic (NaH 2 PO 4 -H 2 O), a surfactant, and one or more CG53135 proteins.
  • the present invention provides improved formulations comprising 0.1-1 M arginine in a salt form, 0.01-0.1 M sodium phosphate monobasic (NaH 2 PO 4 H 2 O), 0.01%-0.1% weight/volume (“w/v”) polysorbate 80 or polysorbate 20, and 0.005-50 mg/ml of one or more CG53135 proteins.
  • the arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose thereof can be, but is not limited to, arginine, arginine sulfate, arginine phosphate, and arginine hydrochloride. In a preferred embodiment, arginine sulfate is used. In some embodiments, the final concentration of the arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose thereof is 0.01-0.7 M.
  • the final concentration of the arginine in a salt form, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose thereof is 0.5 M.
  • the concentration of sodium phosphate monobasic in the formulations is between 0.02-0.09 M, 0.03- 0.08 M, or 0.04-0.06M.
  • the sodium phosphate monobasic is 0.05M.
  • the improved formulations comprise a surfactant, which may be added, e.g., during the diafiltration and/or ultrafiltration step, to minimize the formation of aggregates.
  • the surfactant can be, but is not limited to, polysorbate 80 and polysorbate 20.
  • the concentration of polysorbate 80 or polysorbate 20 is 0.01% (weight/volume).
  • the improved formulations of the present invention comprise one or more CG53135 proteins.
  • the improved formulations of the invention comprise one or more proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 7, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40.
  • the improved formulations of the invention comprise one or more proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 4, 7, 10, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40.
  • the improved formulations of the invention comprise a protein comprising an amino acid sequence of SEQ ID NO:2.
  • the improved formulations of the invention comprise a protein comprising an amino acid sequence of SEQ ID NO:24.
  • the improved formulations of the invention comprise (1) a protein comprising an amino acid sequence of SEQ ID NO:2, and (2) a protein comprising an amino acid sequence of SEQ ID NO:24.
  • the improved formulations of the invention comprise (1) a protein comprising an amino acid sequence of SEQ ID NO:2, (2) a protein comprising an amino acid sequence of SEQ ID NO:24, (3) a protein comprising an amino acid sequence of SEQ ID NO:26, (4) a protein comprising an amino acid sequence of SEQ ID NO:28, (5) a protein comprising an amino acid sequence of SEQ ID NO:30, and (6) a protein comprising an amino acid sequence of SEQ ID NO:32.
  • the improved formulations of the invention comprise one or more proteins produced by any of the processes described in Section 5.2, supra.
  • the concentration of one or more CG53135 proteins in the improved formulations of the instant invention is at least 2 mg/ml, at least 10 mg/ml, at least 15 mg/ml, at least 20 mg/ml, at least 25 mg/ml, at least 30 mg/ml, at least 35 mg/ml, at least 40 mg/ml, at least 45 mg/ml, or at least 50 mg/ml.
  • the concentration of one or more CG53135 proteins in the improved formulations of the instant invention is no more than 50 mg/ml, no more than 30 mg/ml, no more than 10 mg/ml, no more than 5 mg/ml, no more than 1 mg/ml, or no more than 0.5 mg/ml. In some embodiments, the concentration of one or more CG53135 proteins in the improved formulations of the instant invention is 0.0005- 60 mg/ml, 0.005- 50 mg/ml, 0.05-50 mg/ml, 0.5-50 mg/ml, 1-60 mg/ml, 1-50 mg/ml, 5-40 mg/ml, 5-30 mg/ml, or 5-20 mg/ml. In a specific embodiment, the concentration of one or more CG53135 proteins in the improved formulations of the instant invention is 10 mg/ml.
  • the improved formulations of the invention can be lyophilized or spray dried, which results more stable products with longer shelf life and the ease of handling and shipment.
  • the process of lyophilization is very well known in the art and is not described in detail herein. Briefly, lyophilization is the process by which the moisture content of the product is reduced by freezing and subsequent sublimation under vacuum. The lyophilization process primarily consists of three stages. The first stage involves freezing the product and creating a frozen matrix suitable for drying. This step impacts the drying characteristics in the next two stages. The second stage is primary drying. Primary drying involves the removal of the ice by sublimation by reducing the pressure (to typically around 50-500 ⁇ m Hg) of the product's environment while maintaining the product temperature at a low, desirable level. The third stage in the process is called secondary drying where the bound water is removed until the residual moisture content reaches below the target level. Any lyophilization process known in the art can be used to lyophilize the formulations of the invention.
  • the objective of a lyophilization process is to achieve a freeze-dried protein cake with acceptable appearance, biological potency, ease of reconstitution, and long-term storage stability.
  • a prudently designed lyophilization cycle is one that is robust, consumes less time and energy, and maintains product quality. Both formulation-related and cycle-related factors contribute to achieving this goal.
  • a lyophilization excipient in the processes described herein may be necessary.
  • One or more excipients may be added.
  • the lyophilization excipients contemplated for use in the present processes include, but are not limited to, sucrose, lactose, mannitol, dextran, sucrose, heparin, glycine, glucose, glutamic acid, gelatin, sorbitol, histidine, dextrose, trehalose, methocel, hydroxy ethyl cellulose, hydroxy ethyl starch, poly(ethylene glycol), polyvinyl pyrolidone), sulfobutyl ether Beta-cyclodextrin sodium and polyvinyl alcohol, or various combinations thereof, as well as other buffers, protein stabilizers, cryoprotectants, and cryopreservatives commonly used by those skilled in the art.
  • the active ingredient of the improved formulations of the invention is a FGF protein, preferably one or more CG53135 protein
  • the improved formulations of the invention can be used accordingly in any situation that a FGF protein, preferably a CG53135 protein, is known to be effective.
  • the improved formulations of the invention can be used in prevention and/or treatment of disorders such as alimentary mucositis, inflammatory bowel disease, osteoarthritis, disorders of the central nerve system or cardiovascular system, and disorders associated with radiation exposure or symptoms thereof.
  • NIH 3T3 cells were transfected with pFGF-20, which incorporates an epitope tag (V5) and a polyhistidine tag into the carboxy-terminus of the CG53135-01 protein in the pcDNA3.1 vector (Invitrogen
  • CG53135-01 (the full-length CG53135 gene) was cloned as a BgI Il-Xho I fragment into the Bam Hl-Xho I sites in mammalian expression vector, pcDNA3.1 V5His (Invitrogen Corporation, Carlsbad, CA).
  • the resultant construct, pFGF-20 (construct 1a) has a 9 amino acid V5 tag and a 6 amino acid histidine tag (His) fused in-frame to the carboxy-terminus of CG53135-01. These tags aid in the purification and detection of CG53135-01 protein.
  • CG53135-01 protein was detected in the conditioned medium using an anti-V5 antibody (Invitrogen, Carlsbad, CA).
  • the full-length CG53135-01 gene was also cloned as a BgI Il-Xho I fragment into the Bam Hl-Xho I sites of mammalian expression vector pCEP4/Sec (CuraGen Corporation).
  • the resultant construct, plgK-FGF-20 construct 1 b
  • construct 1 b has a heterologous immunoglobulin kappa (IgK) signal sequence that could aid in secretion of CG53135-01.
  • IgK immunoglobulin kappa
  • CG53135-01 protein was detected in the soluble fraction of the cells.
  • CG53135-05 a codon-optimized, full-length FGF- 20 gene
  • CG53135-02 a codon-optimized deletion construct of FGF-20, with the N-terminal amino acids 2-54 removed
  • CAATG Nde I restriction site
  • TAA stop codons
  • CCGAG Xho restriction site
  • the synthesized gene was cloned into pCRScript (Stratagene, La JoIIa, CA) to generate pCRScript-CG53135-05.
  • An Nde i-Xho I fragment containing the codon-optimized CG53135-05 gene was isolated from the pCRscript-CG53135-05 and subcloned into Nde I-Xho l-digested pET24a to generate pET24a-CG53135-05 (construct 3).
  • the full-length, codon-optimized version of FGF-20 is referred to as CG53135-05.
  • oligonucleotide primers were designed to amplify the truncated FGF-20 gene from pCRScript-CG53135-05.
  • the forward primer contained an Nde I site (CATATG) followed by coding sequence starting at amino acid 55.
  • the reverse primer contained a Hindlll restriction site.
  • a single PCR product of approximately 480 base pairs was obtained and cloned into pCR2.1 vector (Invitrogen) to generate pCR2.1- CG53135del.
  • An Nde I-Hind III fragment was isolated from pCR2.1-53135del and subcloned into Nde I-Hind Ill-digested pET24a to generate pET24a-CG53135-02 (construct 4).
  • the plasmids, pET24a-CG53135-05 (construct 3) and pET24a-CG53135-02 (construct 4) have no tags.
  • Each vector was transformed into E.coli BLR (DE3), induced with isopropyl thiogalactopyranoside. Both the full-length and the N-terminally truncated CG53135 protein was detected in the soluble fraction of cells.
  • PROCESS 1 PRODUCTION PROCESS AND PHARMACEUTICAL FORMULATIONS
  • CG53135-05 was expressed in Escherichia coli BLR (DE3) using a codon-optimized construct, purified to homogeneity, and characterized by standard protein chemistry techniques.
  • the isolated CG53135-05 protein migrated as a single band (23 kilodalton) using standard SDS- PAGE techniques and stained with Coommassie blue.
  • the CG53135-05 protein was electrophoretically transferred to a polyvinylidenefluoride membrane and the stained 23 kD band was excised from the membrane and analyzed by an automated Edman sequencer (Procise, Applied Biosystems, Foster City, CA); the N-terminal amino acid sequence of the first 10 amino acids was confirmed as identical to the predicted protein sequence.
  • CG53135-05 was expressed using Escherichia coli BLR (DE3) cells (Novagen). These cells were transformed with full length, codon optimized CG53135-05 using pET24a vector (Novagen). A Manufacturing Master Cell Bank (MMCB) of these cells was produced and qualified. The fermentation and primary recovery processes were performed at the 100 L ⁇ i.e., working volume) scale reproducibly.
  • Seed preparation was started by thawing and pooling of 1 - 6 vials of the MMCB and inoculating 4 - 7 shake flasks each containing 750 mL of seed medium. At this point, 3-6 L of inoculum was transferred to a production fermentor containing 60-80 L of start-up medium. The production fermentor was operated at a temperature of 37 0 C and pH of 7.1. Dissolved oxygen was controlled at 30% of saturation concentration or above by manipulating agitation speed, air sparging rate and enrichment of air with pure oxygen. Addition of feed medium was initiated at a cell density of 30-40 AU (600 nm) and maintained until end of fermentation.
  • the cells were induced at a cell density of 40-50 AU (600 nm) using 1mM isopropyl-beta-D-thiogalactoside (IPTG) and CG53135-05 protein was produced for 4 hours post-induction.
  • IPTG isopropyl-beta-D-thiogalactoside
  • the fermentation was completed in 10-14 hours and about 100-110 L of cell broth was concentrated using a continuous centrifuge.
  • the resulting cell paste was stored frozen at -70 0 C.
  • the frozen cell paste was suspended in lysis buffer (containing 3M urea, final concentration) and disrupted by high-pressure homogenization.
  • the cell lysate was clarified using continuous flow centrifugation.
  • the resulting clarified lysate was directly loaded onto a SP-sepharose Fast Flow column equilibrated with SP equilibration buffer (3 M urea, 100 mM sodium phosphate, 20 mM sodium chloride, 5 mM EDTA, pH 7.4).
  • CG53135-05 protein was eluted from the column using SP elution buffer (100 mM sodium citrate, 1 M arginine, 5 mM EDTA, pH 6.0).
  • the collected material was then diluted with an equal volume of SP elution buffer.
  • the SP Sepharose FF pool was filtered through a 0.2 ⁇ m PES filter and frozen at -8O 0 C.
  • the SP-sepharose Fast Flow pool was precipitated with ammonium sulfate. After overnight incubation at 4°C, the precipitate was collected by bottle centrifugation and subsequently solubilized in Phenyl loading buffer (100 mM sodium citrate, 500 mM L-arginine, 750 mM NaCI, 5 mM EDTA, pH 6.0). The resulting solution was filtered through a 0.45 uM PES filter and loaded onto a Phenyl- sepharose HP column. After washing the column, the protein was eluted with a linear gradient with Phenyl elution buffer (100 mM sodium citrate, 500 mM L-arginine, 5 mM EDTA, pH 6.0). The Phenyl-sepharose HP pool was filtered through a 0.2 ⁇ m PES filter and frozen at -8O 0 C in 1.8 L aliquots.
  • the formulated drug product was sterile-filtered into a sterile tank and aseptically filled (at 10.5 ml_ per 20 ml_ vial) and sealed.
  • the filled and sealed vials were inspected for fill accuracy and visual defects.
  • a specified number of vials were drawn and labeled for release assays, stability studies, safety studies, and retained samples. The remaining vials were labeled for the clinical study, and finished drug product was stored at -80 ⁇ 15°C.
  • the finished drug product is a sterile, clear, colorless solution in single-use sterile vials for injection.
  • CG53135-05 E. coli purified product was formulated at a final concentration of 8.2 mg/mL (Table 3).
  • coli purified product resulted in slow absorption (maximum plasma level at 10 hours) and plasma levels of 40-80 ng/mL up to 48 hours after dosing; some accumulation in plasma was seen following the third daily dose.
  • Intraperitoneal administration of CG53135-05 E. coli purified product resulted in slow absorption (maximum plasma level at 2-4 hours) and plasma levels of 40-70 ng/mL up to 10 hours after dosing; decreased exposure was seen following third daily dose. No significant gender differences were observed by any route of administration.
  • the SDS-PAGE, RP-HPLC, and Bradford assays are indicative of protein degradation or gross aggregation.
  • the SEC-HPLC assay detects aggregation of the protein or changes in oligomerization, and the bioassay detects loss of biological activity of the protein.
  • the stability studies for the purified drug substance were conducted at -80 to 15 0 C with samples tested at intervals of 3, 6, 9, 12, and 24 months.
  • Stability data collected after 1 month indicate that finished drug product is stable for at least 1 month when stored at -80 ;fc15 0 C or at -20 ⁇ 5 0 C (Table 5).
  • Table 5 Stability Data for Drug Product after 1 -month interval
  • samples of finished drug product were stored at -80 ⁇ 15 0 C or stressed at 5 ⁇ 3°C, 25 ⁇ 2°C, or 37 ⁇ 2°C and tested at various intervals for 1 month. Stability data indicate that finished drug product showed no significant instability after 1 month of storage at -80 ⁇ 15OC or 5 ⁇ 3°C.
  • finished drug product When stressed at 25 ⁇ 2°C, finished drug product was stable for at least 48 hours; degradation was apparent after 1 week at this temperature. When stressed at 37 ⁇ 2°C, degradation of finished drug product was apparent within 4 hours.
  • FIG. 1 shows the steps involved in the improved manufacturing process of CG53135.
  • the codon-optimized, full-length, untagged molecule of CG53135-05 construct 3 in Example 1. The process steps for the improved manufacture process are described below.
  • MMCB Manufacturing Master Cell Bank
  • MWCB Manufacturing Working Cell Bank
  • Seed and Final Fermentation the shake flasks with cells in exponential growth phase (2.5 - 4.5 OD 600 units) are used to inoculate a single 25 L ⁇ i.e., working volume) seed fermenter containing the seed medium.
  • the cells upon reaching exponential growth phase (3.0 - 5.0 OD 600 units) in the 25 L seed fermenter are transferred to a 1500 L production fermenter with 780 - 820 L of chemically defined batch medium.
  • the temperature is controlled at 37 ⁇ 2°C, pH at 7.1 ⁇ 0.1 , agitation at 150 - 250 rpm and sparging with 0.5 - 1.5 (vvm) of air or oxygen- enriched air to control dissolved oxygen at 25% or above.
  • Antifoam agent (Fermax adjuvant 27) is used as needed to control foaming in the fermenter.
  • additional chemically defined medium is fed at 0.7 g/kg broth/min initially and then with feed rate adjustment as needed.
  • the induction for expression of the CG53135-05 protein is started when OD at 600 nm reaches 135 - 165 units. After 4 hours post-induction the fermentation is completed. The final fermentation broth volume is approximately 1500 L.
  • the culture is then chilled to 10 - 15 0 C.
  • the chilled culture is diluted with cell lysis buffer at the ratio of one part of fermentation broth to two parts of cell lysis buffer (50 mM sodium phosphate, 60 mM EDTA, 7.5 mM DTT, 4.5 M urea, pH 7.2.
  • Polyethyleneimine (PEI) a flocculating agent is added to the diluted fermentation broth to a final PEI concentration at 0.033% (W/V).
  • the cells are lysed at 10 - 15 0 C with 3 passages through a high-pressure homogenizer at 750 - 850 bar.
  • the chilled cell lysate is directly loaded in the upflow direction onto a pre-equilibrated Streamline SP expanded bed cation exchange column. During the loading, the bed expansion factor is maintained between 2.5 - 3.0 times the packed bed column volume. After loading, the column is flushed with additional Streamline SP equilibration buffer (100 mM sodium phosphate, 40 mM EDTA, 10 mM sodium sulfate, 3 M urea, pH 7.0) in the upflow direction.
  • additional Streamline SP equilibration buffer 100 mM sodium phosphate, 40 mM EDTA, 10 mM sodium sulfate, 3 M urea, pH 7.0
  • the column is then washed further with SP Streamline wash buffer (100 mM sodium phosphate, 5 mM EDTA, 25 mM sodium sulfate, 2.22 M dextrose, pH 7.0) in the downflow direction.
  • SP Streamline wash buffer 100 mM sodium phosphate, 5 mM EDTA, 25 mM sodium sulfate, 2.22 M dextrose, pH 7.0
  • the protein is eluted from the column with Streamline SP elution buffer (100 mM sodium phosphate, 5 mM EDTA, 200 mM sodium sulfate, 1 M L-arginine, pH 7.0) in the downflow direction.
  • PPG 650M Chromatography the SP Streamline eluate is loaded on to a pre-equilibrated PPG 650 M, hydrophobic interaction chromatography column.
  • the column is equilibrated and washed with 100 mM sodium phosphate, 200 mM sodium sulfate, 5 mM EDTA, 1 M Arginine pH 7.0.
  • the column is further washed with 100 mM sodium phosphate, 5 mM EDTA, 0.9 M Arginine, pH 7.0.
  • the product is eluted with 100 mM sodium phosphate, 5 mM EDTA, 0.2 M Arginine, pH 7.0.
  • CUNO Filtration the PPG eluate is passed through an endotoxin binding CUNO 30ZA depth filter.
  • the filter is flushed first with water for injection (WFI) and then with 100 mM sodium phosphate, 5 mM EDTA, 0.2 M Arginine, pH 7.0 (PPG eluate buffer). After flushing, the PPG eluate is passed through the filter. Air pressure is used to push the final liquid through the filter and its housing.
  • WFI water for injection
  • PPG eluate buffer 100 mM sodium phosphate, 5 mM EDTA, 0.2 M Arginine, pH 7.0
  • Air pressure is used to push the final liquid through the filter and its housing.
  • Phenyl Sepharose Chromatography the CUNO filtrate is then loaded on to a pre- equilibrated Phenyl Sepharose hydrophobic interaction chromatography column. The column is equilibrated and washed with 100 mM sodium phosphate, 50 mM ammonium sulfate, 800 mM sodium chloride, 0.5 M Arginine, pH 7.0. The product is eluted with 50 mM sodium phosphate, 0.5 M Arginine, pH 7.0.
  • Concentration and Diafiltration a 1 % Polysorbate 80 is added to the Phenyl Sepharose eluate so that the final concentration in the drug substance is 0.01 % (w/v).
  • the eluate is then concentrated in an ultrafiltration system to about 2 - 3 g/L.
  • the retentate is then diafiltered with 7 diafiltration volumes of 50 mM sodium phosphate, 0.5 M Arginine, pH 7.0 (Phenyl Sepharose elution buffer). After diafiltration the retentate is concentrated between 12 - 15 g/L.
  • the retentate is filtered through a 0.22 ⁇ m filter and subsequently diluted to 10 g/L.
  • the Frozen Drug Substance is used for the manufacture of the Drug Product.
  • the bottles of frozen Drug Substance are thawed at ambient temperature. After the Drug Substance is completely thawed, it is pooled in a sterile container, filtered, filled into vials, partially stoppered, and lyophilized. After completion of the freeze-drying process, the vials are stoppered and capped.
  • the lyophilized Drug Product is stored at 2-8 0 C.
  • a protein reference standard was prepared using a 140L scale manufacturing process that was representative of the bulk drug substance manufacturing process as described in Section 6.2.2 (Example 2).
  • the reference standard was stored as 1 mL aliquots in 2 mL cryovials at -80 0 C ⁇ 15 0 C.
  • the proposed specifications for the reference standard are listed in Table 6.
  • Example 2 The drug products produced by the manufacturing process as described in Section 6.2 (Example 2) are analyzed for purity by the experiments described in this section.
  • Purified protein using the improved manufacturing process as described in Section 6.2.2 (“Process 2”) and the purified protein using manufacturing process as described in Section 6.2.1 (“Process 1”) were analyzed by loading increasing amounts of protein on a 4-12% gradient Bis-Tris NuPAGE gel and stained with the gel code blue stain to detect trace impurities (FIG. 2A).
  • Purified protein (from both Process 1 and Process 2, respectively) migrated as a single major band under reducing conditions ( ⁇ 23 kDa). No impurities above the LOD ( ⁇ 28 ng) were detected.
  • Purified protein of Process 1 and Process 2 was also analyzed by loading increasing amounts of protein on a 4-12% gradient Bis-Tris NuPAGE gel and using silver stain to detect trace impurities (FIG. 2B).
  • Purified protein (from both Process 1 and Process 2, respectively) migrated as a single major band ( ⁇ 23 kDa) at all loads under reducing conditions.
  • Purified drug product was analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC).
  • Purified protein from Process 1 and Process 2 was loaded onto a Protein C4 column (Vydac, 5 ⁇ m, 150 mm X 4.6 mm) using a standard HPLC system in a mobile phase containing water, acetonitrile and trifluoroacetic acid.
  • Purified protein from Process 1 elutes as a major peak at 24.0 min and additional peaks at 24.3 and 24.7 minutes. These represent isoforms of CG53135-05.
  • CG53135-05 obtained using Process 2 elutes as a major peak with a retention time of 24.0 minutes (FIG. 3). Characterization of these peaks is discussed further below in Section 6.7 (Example 7).
  • Purified protein (from both Process 1 and Process 2, respectively) was analyzed by size exclusion chromatography (SEC-HPLC) with UV detection at 280 nm. Analysis was performed by injecting the protein onto a size exclusion HPLC column (Bio-Sil SEC-250, 0.78 cm X 30 cm, Bio- Rad) using a standard HPLC system with a mobile phase containing 100 mM sodium phosphate, 1 M arginine-HCI, pH 7.0. Purified protein eluted isocratically as a single mono-disperse peak with a retention time of 20.5 minutes (FIG. 4) for Process 1 and 2. This retention time corresponds to an apparent molecular weight of approximately 45 kilodaltons (when compared against a set of calibration standards run under identical conditions), which suggests that FGF-20 exists as a non- covalently linked dimer.
  • SEC-HPLC size exclusion chromatography
  • the levels of host cell protein impurities in purified drug product were assessed qualitatively by Western blot analysis.
  • the purified CG53135 protein was resolved by SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane.
  • the membrane was incubated with a primary antibody (rabbit anti-E. coli, Dako Systems) followed by a secondary antibody (goat anti- rabbit Alkaline Phosphatase conjugated, Bio-Rad) and developed using standard techniques. No host cell protein impurities were visible for Process 1 and only one band (-7OkDa) is apparent from Process 2 (FIG. 5).
  • Purified protein (from both Process 1 and Process 2, respectively) was identified by Western blot using rabbit polyclonal anti-CG53135 sera (FIG. 6). Purified CG53135-05 was resolved by loading 10 ⁇ g of protein on a 4-12% gradient Bis-Tris NuPAGE gel and electrophoretically transferred to a nitrocellulose membrane. The membrane was incubated with a primary antibody (polyclonal anti-CG53135 sera) followed by a secondary antibody (goat anti-rabbit Alkaline Phosphatase conjugated, Bio-Rad) and then developed using standard techniques. Purified protein (from both Process 1 and Process 2, respectively), which migrates as a single band of the expected molecular weight (molecular weight of FGF-20) under reducing and nonreducing conditions, is immunoreactive with CG53135-specific antiserum.
  • the potency was measured by cell growth of NIH 3T3 cells in response to the purified protein from Process 1 and Process 2. Cell growth was measured indirectly using fluorescence by the conversion of resazurin (CellTiter Blue Reagent) into resorufin. Using DEV-10 (Process 1) as the reference standard, the Process 2 interim reference standard was found to have comparable potency at 101 %. Several lots manufactured by Process 2 were analyzed. These results are shown in Table 7.
  • the average potency for all of the lots tested is 106.4 ⁇ 10.3. This indicates that the potency of lots from Process 2 are equivalent to lot DEV-10 made with Process 1. Residual DNA, endotoxin and bioburden in the drug substance can also be tested using qualified assays.
  • the biological activity of CG53135-05 related species collected from the 4 peaks identified by LC and MS was measured by treatment of serum-starved cultured NIH 3T3 murine embryonic fibroblast cells with various doses of the isolated CG53135-05 related species and measurement of incorporation of bromodeoxyuridine (BrdU) during DNA synthesis.
  • BrdU bromodeoxyuridine
  • cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were grown in 96-well plates to confluence at 37°C in 10% CO 2 /air and then starved in Dulbecco's modified Eagle's medium for 24 - 72 hours.
  • CG53135-05-related species were added and incubated for 18 hours at 37°C in 10% CO 2 /air.
  • BrdU (10 mM final concentration) was added and incubated with the cells for 2 hours at 37°C in 10% CO 2 /air. Incorporation of BrdU was measured by enzyme-linked immunosorbent assay according to the manufacturer's specifications (Roche Molecular Biochemicals, Indianapolis, IN).
  • Peak 4 was not included in this assay since insufficient material was collected (Peak 4 is less than 3% of the total peak area for CG53135-05).
  • CG53135-05 and material collected from all 3 remaining fractions i.e., Peak 1 , 2, and 3 induced DNA synthesis in NIH 3T3 mouse fibroblasts in a dose-dependent manner (Table 4).
  • the Pl 2 oo was defined as the concentration of protein that resulted in incorporation of BrdU at 2 times the background.
  • CG53135-05 and CG53135-05 related species recovered from all 3 measurable peaks demonstrated similar biological activity with a PI 20O of O.7 - 11 ng/mL (Table 8).
  • Purified drug substance (by both Process 1 and Process 2, respectively) was further analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) with both UV and electrospray mass spectrometric detection.
  • Purified protein from either Process 1 or Process 2 was loaded onto a Protein C4 column (Vydac, 5 ⁇ m, 150 mm X 4.6 mm) using a standard HPLC system in a mobile phase containing water, acetonitrile and trifluoroacetic acid.
  • the elution gradient for this method was modified to resolve four distinct chromatographic peaks eluting at 26.6, 27.3, 28.5 and 30.0 min respectively (FIG. 7). These peaks were characterized by electrospray mass spectrometry.
  • Table 10 Identity of peaks from the RP-HPLC separation of CG53135-05 based upon accurate molecular weight determination.
  • CG53135-05 E. coli purified product is used hereon to refer to a purified protein product from E. coli expressing a CG53135-05 construct.
  • coli purified product may contain a mixture of the full length CG53135-05 protein (SEQ ID NO:2), CG53135-13 (SEQ ID NO:24), CG53135-15 (SEQ ID NO:28), CG53135-16 (SEQ ID NO:30), and CG53135-17 (SEQ ID NO:32), with the majority of the content being CG53135-13 (SEQ ID NO:24).
  • the experimental N-terminal amino acid sequence of the Process 1 reference standard, DEV10, and the Process 2 interim reference standard were determined qualitatively.
  • the reference standards were resolved by SDS-PAGE and electrophoretically transferred to a polyvinylidenefluoride membrane; the Coomassie-stained ⁇ 23 kDa major band corresponding to each reference standard was excised from the membrane and analyzed by an automated Edman sequencer (Procise, Applied Biosystems, Foster City, CA). A comparison of the two major sequences is shown in Table 11 below.
  • the predominant sequence for each reference standard were identical and corresponded to residues 3-20 in the theoretical N-terminal sequence of CG53135-05.
  • Table 11 Edman sequencing data for the first 20 amino acids of CG53135-05 for Process 1 and 2.
  • the experimental amino acid composition of the DEV10 reference standard and the PX3536G001-H reference standard were determined in parallel. Quadruplicate samples of each reference standard were hydrolyzed for 16 hours at 115°C in 100 ⁇ L of 6 N HCl, 0.2% phenol containing 2 nmol norleucine as an internal standard. Samples were dried in a Speed Vac Concentrator and dissolved in 100 ⁇ L sample buffer containing 2 nmol homoserine as an internal standard. The amino acids in each sample were separated on a Beckman Model 7300 amino acid analyzer. The amino acid composition of both reference standards showed no significant differences as shown in Table 8 below. Note that Cys and trp are destroyed during acid hydrolysis of the protein.
  • Purified drug substance from Process 1 and 2 was reduced and alklated with iodoacetic acid and then digested with sequencing grade trypsin.
  • the tryptic peptides were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) using both UV and electrospray mass spectrometric detection.
  • RP-HPLC reversed-phase high-performance liquid chromatography
  • the tryptic digest from either Process 1 or Process 2 was loaded onto an ODS-1 nonporous silica column (Micra, 1.5 ⁇ m; 53 x 4.6 mm) using a standard HPLC system in a mobile phase containing water, acetonitrile and trifluoroacetic acid.
  • the eluting peptides were detected by UV at 214 nm (FIG.
  • EXAMPLE 8 CHARACTERIZATION OF SECONDARY STRUCTURE: CIRCULAR DICHOROISM SPECTROSCOPY OF THE PURIFIED PROTEIN
  • the far UV circular dichroism spectrum of the purified protein (from both Process 1 and Process 2, respectively) is characterized by a broad maximum at 226-227 nm and a sharp minimum at approximately 206 nm. Both features are common in other fibroblast growth factors and suggest a secondary structure dominated by ⁇ -sheet and ⁇ -turns.
  • the far UV circular dichroism spectra of the DEV10 reference standard and the PX3536G001-H reference standard both display these features and are nearly identical (FIG. 9). The small differences in the spectra are attributable to experimental error. 6.9 EXAMPLE 9: CHARACTERIZATION OF TERTIARY STRUCTURE
  • the near UV Circular Dichroism (CD) spectrum of a protein reflects the number and orientation of the protein's aromatic amino acids. For proteins having identical numbers of aromatic amino acids any differences in their near UV CD spectra represent differences in the position and orientation of the aromatic amino acids. The position and orientation of the aromatic amino acids are a measure of a protein's tertiary structure. Hence, differences in the near UV CD spectra for proteins represent differences in tertiary structure.
  • the near UV CD spectra of the DEV10 reference standard and the PX3536G001-H reference standard are shown in FIG. 10. There are no significant differences between these two spectra and suggest that both reference standards have no significant differences in their tertiary structure.
  • the UV absorbance of aromatic amino acids is influenced by the amino acid's microenvironment.
  • Aromatic amino acids embedded within a protein are in a less polar microenvironment than surface exposed residues. This difference in polarity has a profound effect on the UV absorbance of an aromatic amino acid.
  • Different microenvironments can shift the spectra of aromatic amino acids 4-6 nm in extreme cases.
  • Monitoring these changes for individual proteins is done by calculating the second derivative of the protein's UV absorbance spectrum.
  • the second derivative UV absorbance spectrum of a protein contains a number of minima that correspond to the individual aromatic amino acids. The wavelengths of these minima reflect the microenvironment of the amino acid. Therefore, changes in these minima are indicative of conformational (tertiary) changes in the protein.
  • the second derivative UV absorbance spectrum of the purified protein (from both Process 1 and Process 2, respectively) is characterized by seven minima between 250 and 300 nm. As shown in Table 13 below, and qualitatively in FIG. 11 , the wavelengths of all seven minima for both the DEV10 reference standard and the PX3536G001-H reference standard are not significantly different. These data demonstrate that the microenvironment around the individual aromatic amino acids in both standards are highly similar and suggests significant tertiary differences do not exist between these two reference standards.
  • Differential scanning calorimetric analysis is based upon the detection of changes in the heat content (enthalpy) or the specific heat of a sample with temperature. As thermal energy is supplied to the sample its enthalpy increases and its temperature rises by an amount determined, for a given energy input, by the specific heat of the sample.
  • the specific heat of a protein changes slowly with temperature in a particular physical state, but alters discontinuously at a change of state, e.g. melting or denaturation of the protein.
  • the melting curves for the purified protein are similar and the average Tm (melting temperature) is 62.25 0 C for Process 1 and 62.02 0 C for Process 2. These differences are within the experimental error of the instrument.
  • the sulfhydryl content of the purified protein from Process 1 and Process 2 were measured (Table 14).
  • the purified protein (from Process 1 and Process 2, respectively) was analyzed for total sulfhydryl content spectrophotometrically using 5, 5'-dithio-bis (2-nitrobenzoic acid), commonly referred to as Ellman's reagent.
  • Ellman's reagent 5, 5'-dithio-bis (2-nitrobenzoic acid
  • a new formulation was developed to meet the three requirements for a commercial product: (1) the minimal storage temperature should be 2-8 0 C for ease of distribution; (2) product should be stable at the storage temperature for at least 18 months for a commercial distribution system; and (3) product should be manufactured by commercial scale equipment, and processes should be transferable to various commercial contract manufacturers.
  • the new formulation consists 10 mg/mL of the protein product produced by the process described in Section 6.2 ("Process 2 protein") in 0.5 M arginine as sulfate salt, 0.05 M sodium phosphate monobasic, and 0.01% (w/v) polysorbate 80.
  • the lyophilized product is projected to be stable for at least 18 months at 2-8°C based on accelerated stability data.
  • the previous formulation as described in U.S. Application No. 10/435,087 is not possible to be lyophilized for the following reasons: firstly, the acidic component of the acetate buffer is acetic acid, which sublimes during lyophilization.
  • CG53135-05 was precipitated using the precipitate buffer (50 mM NaPi, 5 mM EDTA, 1 M L-Arginine HCI, 2.5 M (NH4)2SO4).
  • the precipitate was washed with 25 mM sodium phosphate buffer at pH 6.5 to remove the residual arginine and ammonium sulfate.
  • the washed precipitate was then re-dissolved in the following respective buffers listed in the tables. The following are examples of data.
  • All formulation contains 0.2 M arginine.
  • the optimal concentration of the sodium phosphate as a buffering salt was observed (Table 16).
  • the optimal concentration of sodium phosphate is 50 mM with a solubility of at least 1-2 fold increase in comparison with concentrations at 25, 75, and 100 Mm.
  • Table 18 shows a need to add a surfactant during the diafiltration/ultrafiltration step to minimize the formation of aggregates.
  • the experiment was conducted by performing the ultrafiltration/diafiltration at 2.5 mg/mL CG53135-05 in 0.2M arginine and 0.05 M sodium phosphate buffer at pH 7.0. After exchanging with 7 volumes of the final buffer (0.5M arginine and 0.05 M sodium phosphate buffer at pH 7.0), the diafiltrate is concentrated to ⁇ 20 mg/mL. The diafiltrate is then diluted with the final buffer to ⁇ 12.5 mg/mL and lyophilized. Polysorbate 80 is added either before or after the diafiltration to a final concentration of 0.01 %.
  • All formulation contains 0.5 M arginine, 0.05 M sodium phosphate monobasic, and 0.01% polysorbate 80.
  • the new formulation has the following advantages: (1) a lyophilized product with a storage temperature of 2-8 0 C; (2) a lyophilized product with a projected shelf-life of at least 18 months when stored at 2-8°C achieve the solubility of > 30 mg/mL; and (3) The lyophilized product has a collapse temperature of -30°C which can be easily lyophilized by the commercial equipment.
  • the interactions between arginine, sulfate, phosphate, and surfactant and CG53135 were unexpected.
  • a SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA.
  • SNPs occurring within a gene may result in an alteration of the amino acid encoded by the gene at the position of the SNP.
  • Intragenic SNPs may also be silent, when a codon including a SNP encodes the same amino acid as a result of the redundancy of the genetic code.
  • SNPs occurring outside the region of a gene, or in an intron within a gene do not result in changes in any amino acid sequence of a protein but may result in altered regulation of the expression pattern.
  • SeqCallingTM assemblies produced by the exon linking process were selected and extended using the following criteria: genomic clones having regions with 98% identity to all or part of the initial or extended sequence were identified by BLASTN searches using the relevant sequence to query human genomic databases. The genomic clones that resulted were selected for further analysis because this identity indicates that these clones contain the genomic locus for these SeqCallingTM assemblies. These sequences were analyzed for putative coding regions as well as for similarity to the known DNA and protein sequences. Programs used for these analyses include Grail, Genscan, BLAST, HMMER, FASTA, Hybrid and other relevant programs.
  • SeqCallingTM assemblies map to those regions.
  • SeqCallingTM sequences may have overlapped with regions defined by homology or exon prediction. They may also be included because the location of the fragment was in the vicinity of genomic regions identified by similarity or exon prediction that had been included in the original predicted sequence. The sequence so identified was manually assembled and then may have been extended using one or more additional sequences taken from CuraGen Corporation's human SeqCallingTM database. SeqCallingTM fragments suitable for inclusion were identified by the CuraToolsTM program SeqExtend or by identifying SeqCalling fragments mapping to the appropriate regions of the genomic clones analyzed.
  • EXAMPLE 14 STIMULATION OF BROMODEOXYURIDINE INCORPORATION INTO NIH 3T3 CELLS IN RESPONSE TO A TRUNCATED FORM OF FGF-20
  • a vector expressing residues 24-211 of FGF-20 ((d1-23)FGF-20; See Table 1 and SEQ ID NO:32 (CG53135-17) was prepared.
  • the incorporation of BrdU by NIH 3T3 cells treated with conditioned medium obtained using the vector incorporating this truncated form was compared to the incorporation in response to treatment with conditioned medium using a vector encoding full length FGF-20. This experiment was carried out as following:
  • 293-EBNA ceils were transfected using Lipofectamine 2000 according to the manufacturer's protocol (Life Technologies, Gaithersburg, MD). Cells were supplemented with 10% fetal bovine serum (FBS; Life Technologies) 5 hours post-transfection. To generate protein for BrdU and growth assays, cells were washed and fed with Dulbecco's modified Eagle medium (DMEM; Life Technologies) 18 hours post-transfection. After 48 hours, the media was discarded and the cell monolayer was incubated with 100 ⁇ M suramin (Sigma, St. Louis, MO) in 0.5 ml DMEM for 30 min at 4 0 C.
  • DMEM Dulbecco's modified Eagle medium
  • the suramin-extracted conditioned media was then removed, clarified by centrifugation (5 min; 2000 X g), and subjected to TALON metal affinity chromatography according to the manufacturer's instructions (Clontech, Palo Alto, CA) taking advantage of the carboxy-terminal polyhistidine tag. Retained fusion protein was released by washing the column with imidazole.
  • FGF-20 protein concentrations were estimated by Western analysis using a standard curve generated with a V5-tagged protein of known concentration.
  • conditioned media was harvested 48 hours post transfection, and the cell monolayer was then incubated with 0.5 ml DMEM containing 100 ⁇ M suramin for 30 min at 4 0 C. The suramin-containing conditioned media was then harvested.
  • Recombinant FGF-20 and (d1-23)FGF-20 were tested for their ability to induce DNA synthesis in a bromodeoxyuridine (BrdU) incorporation assay.
  • NIH 3T3 cells ATCC number CRL- 1658, American Type Culture Collection, Manassas, VA
  • CCD-1070Sk cells ATCC Number CRL- 2091
  • MG-63 cells ATCC Number CRL-1427
  • Recombinant FGF-20 or (d1-23)FGF-20 was then added to the cells for 18 hours.
  • the BrdU assay was performed according to the manufacturer's specifications (Roche Molecular Biochemicals, Indianapolis, IN) using a 5 hour BrdU incorporation time.
  • CG53135 a full-length tagged variant (CG53135-01), a deletion variant (CG53135-02), and a full-length codon-optimized untagged variant (CG53135-05).
  • CG53135-01 (batch 4A and 6) was used in these experiments. Protein was expressed using Escherichia coli(E. coli), BL21 (Novagen, Madison, Wl), transformed with full-length CG53135-01 in a pETMY-hFGF20X/BL21 expression vector. Cells were harvested and disrupted, and then the soluble protein fraction was clarified by filtration and passed through a metal chelation column. The final protein fraction was dialyzed against phosphate buffered saline (PBS) plus 1 M L-arginine. Protein samples were stored at -70°C.
  • PBS phosphate buffered saline
  • CG53135-02 (batch 1 and 13) was also used in these experiments. Protein was expressed in E. coli, BLR (DE3) (Novagen), transformed with the deletion variant CG53135-02 inserted into a pET24a vector (Novagen). A research cell bank (RCB) was produced and cell paste containing CG53135-02 was produced by fermentation of cells originating from the RCB. Cell membranes were disrupted by high-pressure homogenization, and lysate was clarified by centrifugation. CG53135-02 was purified by ion exchange chromatography. The final protein fraction was dialyzed against the formulation buffer (100 mM citrate, 1 mM ethylenediaminetetraacetic acid (EDTA), and 1 M L-arginine).
  • formulation buffer 100 mM citrate, 1 mM ethylenediaminetetraacetic acid (EDTA), and 1 M L-arginine).
  • CG53135-05, DEV10 which were also used in these experiments, was prepared by Cambrex Biosciences (Hopkinton, MA) according to Process 1 as described in Section 6.18.1 , infra.
  • BrdU Incorporation proliferative activity was measured by treatment of serum-starved cultured cells with a given agent and measurement of BrdU incorporation during DNA synthesis.
  • Cells were cultured in respective manufacturer recommended basal growth medium supplemented with 10% fetal bovine serum or 10% calf serum as per manufacturer recommendations.
  • Cells were grown in 96-well plates to confluence at 37°C in 10% CO 2 /air (to subconfluence at 5% CO 2 for dedifferentiated chondrocytes and NHOst). Cells were then starved in respective basal growth medium for 24-72 hours.
  • coli or pCEP4/Sec or pCEP4/Sec-FGF 2OX enriched conditioned medium was added (10 ⁇ L/100 ⁇ L of culture) for 18 hours.
  • BrdU (10 ⁇ M final concentration) was then added and incubated with the cells for 5 hours. BrdU incorporation was assayed according to the manufacturer's specifications (Roche Molecular Biochemicals, Indianapolis, IN).
  • Growth Assay growth activity was obtained by measuring cell number following treatment of cultured cells with a given agent for a specified period of time. In general, cells grown to ⁇ 20% confluency in 6-well dishes were treated with basal medium supplemented with CG53135 or control, incubated for several days, trypsinized and counted using a Coulter Z1 Particle Counter. Proliferation in Mesenchymal Cells: to determine if recombinant CG53135 could stimulate DNA synthesis in fibroblasts, a BrdU incorporation assay was performed using CG53135-01 treated NIH 3T3 murine embryonic lung fibroblasts.
  • CG53135-01 also induced DNA synthesis in other cells of mesenchymal origin, including CCD-1070Sk normal human foreskin fibroblasts, MG-63 osteosarcoma cell line, and rabbit synoviocyte cell line, HIG-82. In contrast, CG53135-01 did not induce any significant increase in DNA synthesis in primary human osteoblasts (NHOst), human pulmonary artery smooth muscle cells, human coronary artery smooth muscle cells, human aorta smooth muscle cells (HSMC), or in mouse skeletal muscle cells.
  • NHOst primary human osteoblasts
  • HMC human aorta smooth muscle cells
  • CG53135-01 sustained cell growth
  • NIH 3T3 cells were cultured with 1 ⁇ g CG53135-01 or control for 48 hours and then counted (FIG. 14(B)).
  • CG53135 induced an approximately 2-fold increase in cell number relative to control in this assay.
  • CG53135 was found to induce DNA synthesis in the 786-0 human renal carcinoma cell line in a dose-dependent manner (FIG. 14(C)).
  • CG53135-01 induced DNA synthesis in other cells of epithelial origin, including CCD 1106 KERTr human keratinocytes, BaIb MK mouse keratinocytes, and breast epithelial cell line, B5589.
  • CG53135 protein therapeutic agents may inhibit or promote angiogenesis, the process through which endothelial cells differentiate into capillaries. Because CG53135 belongs to the fibroblast growth factor family, some members of which have angiogenic properties, the antiangiogenic or pro-angiogenic effects of CG53135 on endothelial cell lines were evaluated. The following cell lines were chosen because they are cell types used in understanding angiogenesis in cancer: HUVEC (human umbilical vein endothelial cells), BAEC (bovine aortic endothelial cells), HMVEC-d (human endothelial, dermal capillary). These endothelial cell types undergo morphogenic differentiation and are representative of large vessel (HUVEC, BAEC) as well as capillary endothelial cells (HMVEC-d).
  • HUVEC human umbilical vein endothelial cells
  • BAEC bovine aortic endothelial cells
  • HMVEC-d human endothelial, dermal
  • CG53135-01 treatment did not alter cell survival or have stimulatory effects on BrdU incorporation in human umbilical vein endothelial cells, human dermal microvascular endothelial cells or bovine aortic endothelial cells. Furthermore, CG53135-01 treatment did not inhibit tube formation, an important event in formation of new blood vessels, in HUVECS. This result suggests that CG53135 does not have anti-angiogenic properties. Finally, CG53135-01 had no effect on VEGF induced cell migration in HUVECs, suggesting that it does no play a role in metastasis.
  • CG53135-01 which encode the same protein as CG53135-05 induces a proliferative response in mesenchymal and epithelial cells in vitro (i.e., NIH 3T3 mouse fibroblasts, CCD-1070 normal human skin fibroblasts, CCD-1106 human keratinocytes, 786-0 human renal carcinoma cells, MG-63 human osteosarcoma cells and human breast epithelial cells), but not in human smooth muscle, erythroid, or endothelial cells.
  • CG53135-01 and CG53135-05 CG53135-02 also induces proliferation of mesenchymal and epithelial cells.
  • CG53135- 02 induces proliferation of endothelial cells.
  • CG53135 was evaluated for the treatment of chemotherapy-induced oral mucositis in male Golden Syrian hamsters (protein concentrations in this Example were measured by Bradford assay).
  • CG53135-05 used in this study (batch 29-NB849:76) was expressed and purified as described in Section 6.5, with the exception that the final protein fraction was dialyzed against formulation buffer containing 30 mM sodium citrate, 2 mM EDTA, 200 mM sorbitol, 50 mM KCI, 20% glycerol (pH 6.1).
  • Mucositis was induced using 5-fluorouracil, delivered as single bolus (60 mg/kg, IP) on Days -4 and -2.
  • a single submucosatoxic dose of radiation (40 Gy/dose) was locally administered to all animals on Day 0. Animals were treated once daily with 0.1 mL vehicle or 12 mg/kg CG53135-05 IP following mucosa toxic insult according to the schedule shown in Table 11.
  • Mucositis was scored visually as described in Section 6.5 (Table 9) on alternate days beginning on Day 6 and every second day until the conclusion of the experiment on Day 30 (i.e., Days 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30). Each hamster was weighed daily for the period of the study (i.e., Day 0 to Day 30). Weight and survival were monitored as indices for severity of mucositis or possible toxicity resulting from treatment.
  • CG53135 The activity of CG53135 was evaluated in a model of mucositis induced in hamsters treated with 60 mg/kg 5-flourouracil on Days -4 and -2, followed by a single sub-mucosatoxic dose of radiation ( ⁇ 30 Gy) on Day 0. Clinically relevant oral mucositis (mucositis score of ⁇ 3) developed ⁇ Day 15. Intraperitoneal administration of CG53135 for 2, 6, or 18 days significantly reduced severity of mucositis.
  • EXAMPLE 17 EFFECT OF CG53135-05 ADMINISTRATION ON HAMSTER EPITHELIAL PROLIFERATION IN VIVO (N-225 STUDY)
  • BrdU Administration and Immunohistochemistrv all animals received BrdU 50 mg/kg IP two hours before sacrifice, allowing for uptake of the reagent into proliferating tissues. At euthanasia, the following tissues were harvested: cheek pouch mucosa, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum and sternum. All tissue samples were fixed in 10% neutral buffered formalin for 24 hours and then transferred to 70% ethanol. Samples were trimmed, paraffin embedded, sectioned and mounted. Epithelial tissues were stained for incorporation of BrdU by immunohistochemistry using Oncogene Research products BrdU lmmunohistochemistry kit Catalog # HCS24 in accordance with the manufacturer's instructions.
  • CG53135-05 E. coli purified product stimulated the division of the epithelial cells of the cheek pouch, jejunum and rectum as well as the hematopoietic cells of the bone marrow. Peak increases in BrdU incorporation in these tissues were seen at 8 hours after the administration of CG53135-05. All tissues showed the same time response to the administration of CG53135-05 E. coli purified product.
  • EXAMPLE 18 MODULATION OF INTESTINAL CRYPT CELL PROLIFERATION AND APOPTOSIS BY CG53135-05 ADMINISTRATION TO MICE (N-342)
  • a "crypt” is a hierarchical structure with the stem cells towards the crypt base. As cells become more mature, they move progressively from the bottom of the crypt towards the top of the crypt. Therefore, changes that may be affecting stem cells versus their transit amplifying daughter cells can be detected by looking at changes in event frequency at each cell position. The cell positions are marked in FIG. 16. Thus, the effects of CG53135 on the crypt microarchitecture were analyzed in the context of crypt cellularity.
  • mice were sacrificed at various times after a single 12 mg/kg (IP) dose of a CG53135-05 E. coli purified product. Just prior to sacrifice the mice were labeled with a single injection of bromodeoxyuridine to label S-phase cells and determine the effect of the drug on crypt cell proliferation / apoptosis. Mice were weighed and then dosed with a CG53135-05 E. coli purified product (12 mg/kg, single injection, ip). Groups of 6 animals were sacrificed 0, 3, 6, 9, 12, 24, 48 hours post injection with a CG53135-05 E. coli purified product. All received a single injection of bromodeoxyuridine 40 minutes prior to sacrifice (see Table 23).
  • mice An additional two groups of 6 mice were used to assess the effects of CG53135-05 E. coli purified product on stem cell radiosensitivity (groups 8 and 9, see Table 23).
  • One group was treated with a CG53135-05 E. coli purified product (12 mg/kg, single injection, ip) and another group was injected with a placebo control. Twenty-four hours post injection, animals were irradiated with 1Gy X-ray (specifically to induce stem cell apoptosis) followed by routine in vivo BrdU labeling. Animals were sacrificed 4.5 hours later (at time of peak apoptosis).
  • All S-phase dividing cells incorporate the injected bromodeoxyuridine (BrdU) and hence are marked as cycling cells.
  • Animals that were irradiated were placed, unanaesthetised, in a perspex jig and subjected to whole body radiation of 1Gy X-ray at a dose rate of 0.7Gy/min. This low level of radiation induced apoptosis in the small intestinal stem cell population, but not in the more mature cells.
  • the small intestine was removed, fixed in Carnoy's fixative, and processed for histological analysis (paraffin embedded).
  • One set of 3 mm sections were immunolabeled for BrdU and one set of sections were stained with H&E.
  • Longitudinal sections of small intestinal crypts were analyzed for the presence of either BrdU or apoptotic/mitotic nuclei. Fifty half crypts were scored per animal.
  • Groups 1-7 (Group A in the results) were tested to determine the effect of CG53135-05 E. coli purified product over a 48 hour period.
  • Groups 8-9 (Group B in the results) were tested to determine whether CG53135-05 E. coli purified product changes the number of apoptotic cells generated after low dose irradiation, i.e., whether CG53135-05 E coli purified product influences the radiosensitive stem cell population.
  • results generated show a frequency distribution for the crypts in each group of animals that were further analyzed for statistical differences.
  • Tissue samples were harvested at 3, 6, 9, 12, 24, and 48 hours after treatment with CG53135-05 E. coli purified product. Apoptosis, mitotic index, and proliferation were the end points for this study.
  • Table 24 The comparisons shown in Table 24 are between treated groups versus the untreated group.
  • the cell positions shown are the ones that are significantly different from the untreated control (P ⁇ 0.05).
  • mice were weighed and then dosed with a CG53135-05 E. coli purified product (12 mg/kg) or placebo. A single injection was given, intraperitoneally (ip), 24 hours prior to irradiation. Each group of 6 animals was irradiated as per table below. For each radiation dose, the response of a drug treated group and a placebo treated group was compared.
  • the small intestine was removed, fixed in Carnoy's fixative, and processed for histological analysis (paraffin embedded). H&E sections were prepared following conventional protocols. For each animal, ten intestinal circumferences were analyzed, the number of surviving crypts per circumference was scored, and the average per group was determined. Only crypts containing 10 or more strongly H&E stained cells (excluding Paneth cells) and only intact circumferences, not containing Peyers patches, were scored.
  • Corrected number of crypts per circumference Mean number of surviving crypts per circumference in treatment group X (Mean crypt width in untreated control / Mean crypt width in treated animal).
  • EXAMPLE 20 EFFECT OF CG53135 ADMINISTRATION ON CHEMOTHERAPY- RADIATION MODEL OF ORAL MUCOSITIS (N-346 STUDY)
  • Escherichia coli BLR (DE3) cells (Novagen, Madison, Wl) were transformed with full-length, codon-optimized CG53135-05 using pET24a vector (Novagen), and a manufacturing master cell bank (MMCB) of these cells was produced.
  • MMCB manufacturing master cell bank
  • Cell paste containing CG53135-05 produced by fermentation of cells originating from the MMCB was lysed with high-pressure homogenization in lysis buffer and clarified by centrifugation.
  • CG53135-05 was purified from clarified cell lysate by 2 cycles of ion exchange chromatography and ammonium sulfate precipitation.
  • the final protein fraction was dialyzed against the formulation buffer (30 mM citrate, pH 6.0, 2 mM ethylenediaminetetraacetic acid (EDTA), 200 mM sorbitol, 50 mM KCI, 20% glycerol).
  • Vehicle contains 3OmM sodium citrate, pH 6.1 , 2mM EDTA, 20OmM sorbitol, 5OmM KCI, 20% glycerol. Protein concentrations in this example were measured by Bradford assay.
  • Golden Syrian hamsters (Charles River Laboratories or Harlan), of age 5 to 6 weeks, and with an average body weight of 84 g at study commencement, were used in this study. Animals were individually numbered using an ear punch and housed in small groups of up to 7 animals per cage. Animals were acclimated prior to study commencement. During this period, the animals were observed daily in order to reject animals in poor condition.
  • Chemotherapy/Radiation Model of Oral Mucositis the 5-FU/acute irradiation model for oral mucositis in hamsters is an experimental model designed to extend the clinical observations made with the acute radiation model for mucositis (Oral Surg Oral Med Oral Pathol 69(4):437 (1990)).
  • the earlier acute radiation model has proven to be an accurate, efficient and cost-effective technique to provide a preliminary evaluation of anti-mucositis compounds including growth factors and cytokines (see e.g., Oral Oncol 36(4):373-381 (2000), Cytokine 9(8):605-612 (1997); Oral Oncol 33(1 ):47-54 (1997)).
  • Mucositis was induced using 5-fluorouracil, delivered as intraperitoneal (IP) doses (60 mg/kg) on days -4 and -2. .A single dose of radiation (30 Gy/dose) was administered to all animals on day 0. Radiation was generated with a 160 kilovolt potential (18.75-ma) source at a focal distance of 21 cm, hardened with a 3.0 mm Al filtration system. Irradiation targeted the left buccal pouch mucosa at a rate of 1.32 Gy/minute. Prior to irradiation, animals were anesthetized with an IP injection of ketamine (160mg/kg) and xylazine (8mg/kg). The left buccal pouch was everted, fixed and isolated using a lead shield. This resulted in ulcerative oral mucositis that peaked around day 14.
  • IP intraperitoneal
  • Mucositis for the evaluation of mucositis, the animals were anesthetized with an inhalation anesthetic, and the left pouch was everted. Mucositis was scored visually by comparison to a validated photographic scale, ranging from 0 for normal, to 5 for severe ulceration. The scale is described in Table 28. Table 28. Description of Mucositis Score Values
  • a score of 1-2 is considered to represent a mild stage of the disease, whereas a score of 3- 5 is considered to indicate moderate to severe mucositis.
  • a photograph was taken of each animal's mucosa using a standardized technique.
  • All film was developed and the photographs randomly numbered for blinded scoring.
  • Two independent, trained evaluators graded the photographs in blinded fashion using the above- described scale.
  • the final blinded score was the average of the score assigned by the two independent evaluators.
  • the scores from the blinded photographic evaluation were statistically analyzed.
  • Weights and Survival each hamster was weighed daily for the period of the study (i.e., day -4 to day 28). Weight and survival was monitored and recorded in order to assess possible differences amongst treatment groups as an indication for mucositis severity and/or possible toxicity resulting from the treatments. If appropriate, survival was analyzed using a Kaplan Meier log-rank analysis. Differences in weight gain were assessed using a One-Way ANOVA analysis of the area under the curve (AUC) values for the percentage weight gain for individual animals, with a critical value of 0.05.
  • AUC area under the curve
  • Evaluation of Activity the effect of each treatment on mucositis compared to the control group was assessed using a Chi-squared (X2) analysis of the number of animal days with a score of three or higher, and by using the Mann-Whitney Rank Sum test to compare the blinded mucositis scores for each group on each day the evaluations were performed.
  • treatment groups were compared to the control group, with a critical value of 0.05.
  • Mann-Whitney Rank Sum test two days of statistically significant improvement are generally regarded as the minimum improvement necessary for a positive result.
  • Mucositis the mean daily mucositis scores were calculated for each group and are shown in Figure 18. The peak of mucositis in the control group was on day 14 when the mean score for this group reached 3.2. All of the groups treated with CG53135-05 E. coli purified product had their peak scores on day 16, which ranged from a high of 3.0 in the groups treated with CG53135-05 E. coli purified product at 24 mg/kg or 48 mg/kg on day 1 to a low of 2.63 in the group treated with CG53135-05 E. coli purified product at 12 mg/kg on day 1. To evaluate the mucositis scores, an analysis of the number of days with a score of 3 or higher was performed, using the Chi-squared test.
  • Figure 19 depicts the duration of severe mucositis in animals with a mucositis score of >3 as calculated by the chi- square analysis.
  • the groups treated with CG53135- 05 E. coli purified product at 24 mg/kg and 48 mg/kg were essentially the same as controls in this test.
  • Weight Change the mean daily percentage weight change for each group is shown in FIG. 20.
  • the overall increase in weight during the course of this study for animals in the untreated control group was 47.5%, compared with 45.9% in the group treated with CG53135-05 at 6 mg/kg in day 1 , 53.8% in the group treated with CG53135-05 E. coli purified product at 12 mg/kg in day 1 , 41.2% in the group treated with CG53135-05 E. coli purified product at 24 mg/kg in day 1 , 49.7% in the group treated with CG53135-05 E. coli purified product at 48 mg/kg in day 1 , and 46.9% in the group treated with CG53135-05 E.
  • EXAMPLE 21 EFFECT OF CG53135 ON TREATMENT OF ESTABLISHED ORAL MUCOSITIS IN HAMSTER CHEMO/RADIATION MODEL (N-318)
  • Mucositis was evaluated on alternate days beginning on day 6 and every second day until the conclusion of the experiment on day 28 (Ae., days 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, & 28).
  • Mucositis was induced in hamsters. The end points, mucositis, weights and survival, were evaluated. Statistics applied were Chi-squared analysis and Mann-Whitney Rank Sum test. All the three parameters are described in Section 6.20.
  • Mucositis in the untreated control group, the peak of mucositis occurred on day 14 with a mean score of 3. In the vehicle control group the peak of mucositis occurred on day 16 with a mean score of 3.4.
  • the groups receiving CG53135-05 E. coli purified product 12 mg/kg IP on the first and second days after reaching a score of 2 showed similar patterns of mucositis scores to the control groups (FIG. 22(A)).
  • the groups that received CG53135-05 E. coli purified product 12 mg/kg IP on the third and fourth days after reaching a score of 2 showed a reduction in mucositis scores relative to the control groups, predominantly after the peak of mucositis (FlG. 22(B)).
  • EXAMPLE 22 CG53135 CAN BE USED SAFELY AS A SINGLE DOSE THERAPY FOR MUCOSITIS IN HUMAN PATIENTS (STUDIES C-214 AND C-325)
  • a value of the OMAS system is obtained by summing the erythema and ulceration/pseudomembrane scores.
  • UV 0.03 mg/kg
  • UV 0.1 mg/kg
  • CG53135-05 drug substance reached maximum plasma concentration within 1 hour (15 to 35 minutes after completion of infusion).
  • Adverse events that may be related to the study drug include: nausea (2); chills (2); fever (2); vomiting (1); dizziness (1); photopsia (1) (vision-"lights flashing" on day 15) and astigmatism (1) (mild astigmatism on day 28); neuropathy (1) (on soles of the feet on day 15); tachycardia (1); headache (1); and asymptomatic, single premature atrial complex noted on ECG (1). All reported incidences were mild to moderate. No Grade 3 or 4 laboratory toxicity associated with the study drug was noted. Among the 11 patients who have received the drug through September 3, 2004, six serious adverse events determined to be unrelated to study drug were noted from 3 patients.
  • conditioning regimens including melphalan (MeI 200), cyclophosphamide, carmustine and etoposide (CBV), carboplatin and thiotepa (CT), and busulfan/cyclophosphamide (targeted BuCy).
  • the median day of neutrophil engraftment (as determined by ANC>500/uL) occurred on day 13 after stem cell infusion.
  • a larger Phase Il clinical trial will be initiated to evaluate the efficacy of CG53135-05 drug substance in preventing HDCT- induced OM.
  • the CG53135-05 drug substance was generally well tolerated among the 38 patients that were administered in the two phase I trials to date.
  • the doses tested were 0.03, 0.1 , 0.2 and 0.33 mg/kg (UV).
  • No other consistent drug-related or apparent dose-related adverse events or laboratory abnormals have been observed.
  • No study drug-related serious adverse events were observed.
  • Sufficient information on tolerability and preliminary activity is considered to be present to utilize the 0.03, 0.1 and 0.2 mg/kg (UV) doses in Phase Il testing.
  • mice were weighed and then dosed with CG53135-05 E. coli purified product (4 or 16 mg/kg) or untreated. Dosing occurred as described in Tables 34 & 35. Each group of 20 animals was irradiated as per table below. All dosing of CG53135-05 E. coli purified product on day 0 was immediately after irradiation. No anesthesia was administered.
  • mice underwent whole body irradiation at a dose of 14 or 14.5 Gy delivered at a dose rate of 0.7Gy/min. Animals were followed for diarrhea incidence throughout the study period. After 6 days, animals were sacrificed, and the intestinal tract of the mice was harvested for histological analysis.
  • Body Weight Every day for the period of the study, each animal was weighed and its survival recorded, in order to assess possible differences in animal weight among treatment groups as an indication of response to exposure to ionizing radiation.
  • Animals Found Dead or Moribund Animals were assessed 2x/day from Day 3 onwards in order to accurately assess diarrhea onset / progression and detect moribund animals prior to death. Such moribund animals were sacrificed by cervical dislocation. The ileum and mid-colon were removed and fixed in formalin, embedded in paraffin (1 animal per block, two tissues per block) for storage and future analysis/IHC if required. No tissue was removed from animals found dead.
  • Mass specific growth rate was calculated by:
  • Diarrhea score Mice were scored for severity of diarrhea on a scale of 0-3 twice a day for three days beginning at 4 days after irradiation. Average diarrhea score over three days as well as the sum of the diarrhea score over three days was measured and graphed. Significance was obtained by one-way ANOVA and Tukey's Multiple Comparison Test. (FIGs. 25(A) and 25(B))
  • EXAMPLE 24 CG53135 REDUCES CPT-11 INDUCED DIARRHEA IN RATS (STUDY N-392) lrinotecan (CPT-11) is a chemotherapeutic agent which is commonly used against solid tumors which causes gastrointestinal (Gl) mucositis manifest by severe diarrhea.
  • the primary aim of this study was to investigate whether CG53135 reduces CPT-11 -induced Gl mucositis in an in vivo animal model. The secondary aim was to test varying schedules of administration of CG53135.
  • Diarrhea was induced in tumor-bearing rats with a single intraperitoneal dose of CPT-11 (200 mg/kg). Animals were treated with 16 mg/kg CG53135-05 E. coli purified product according to Process 2 described in Section 6.18.2 below (in a vehicle of 0.5M arginine, 0.05M sodium phosphate monobasic, 0.01 % polysorbate 80, and pH adjusted with sulfuric acid to pH 7.0) intraperitoneally either prior to, prior to and during, or post chemotherapy treatment. Rats were monitored closely for the incidence, and severity of diarrhea as well as mortality. Animals were euthanized 168 hours following diarrhea induction. At euthanasia, tissues were harvested for histopathological evaluation of the gastrointestinal tract. .esults:
  • CG53135 pre-treated animals (16 mg/kg) demonstrated an improvement in gastrointestinal iucositis as measured by a reduction in the incidence of diarrhea. A reduction in overall mortality ras also noted in this group. This has important implications for the use of CG53135 in GI iucositis in humans, and should be further studied.
  • Protein concentration was measured by Bradford assay. Mice were exposed to ionizing adiation without anesthesia at a dose range of 484 to 641 cGy on day 0. Animals were dosed with 1 BS (control) or the CG53135-05 E. coli purified product (12 mg/kg, Bradford, daily IP) on day -1 , or ays -2 and -1 before radiation. The schedule is represented in Table 36. The endpoints for the. tudy were survival and weight changes. Survival was followed for 30 days post-irradiation.
  • LD 5O/3 o values were calculated using a probit plot of survivorship with 95% confidence intervals calculated by bootstrapping.
  • the results are indicative of the therapeutic effect of prophylactic administration of 2G53135-05 in radioprotection.
  • one day treatment before radiation (day -1) also protected animals from weight loss in all but the highest radiation level (641 cGy).
  • the invention could be extended to additional dose regimens of the CG53135-05 E. coli purified product, such as prophylactically and/or therapeutically administer the CG53135-05 E. coli purified product prior and/or after the radiation exposure, which could be tested in the same animal model following the same procedures as described herein, in order to define the range of therapeutic efficacy of this compound.
  • the dose regimen for therapeutic treatment may include, but is not limited to, +1 , +1 , and +2 days after radiation exposure.
  • mice were dosed IP with 4 mg/kg (UV) CG53135-05 E. coli purified product 24 hours prior to whole-body irradiation at the indicated doses. The survival of the mice was then followed for 30 days.
  • Figure 28C shows orobit analysis for survival over the range of doses.
  • the LD 50/30 for control and animals treated with the CG53135-05 E coli purified product is 552.4 cGy and 607.4 cGy, respectively, with a dose modification factor (DMF) of 1.10.
  • DMF dose modification factor
  • Example 26 Effects of CG53135 Prophylactic Dose Schedule on Survival of Irradiated Intestinal Crypt Cells (N-375)
  • the objective of this study was to evaluate the ability of CG53135 to protect against 'adiation-induced intestinal crypt cell mortality in vivo when administered once daily for 4 days prior :o irradiation.
  • CG53135-05 E. coli purified product (12 mg/kg) or PBS was administered to BDF1 nice intraperitoneally (IP) once daily for 4 consecutive days prior to exposure to lethal radiation joses from 10-14 Gy on Day 0.
  • IP intraperitoneally
  • the number of surviving regenerating crypt foci was measured 4 jays after irradiation. Protein concentrations in this example were measured by Bradford assay.
  • Table 37 intestinal Crypt Protection Factors Resulting from CG53135-05 E. coli purified product Vlultiple-Dose Administration Prior to Irradiation
  • Fable 38 Intestinal Crypt Protection Factors Resulting from CG53135-05 Administration 6-48 h D rior to Irradiation
  • Example 28 Effects of CG53135 Dose Schedule on Survival of Irradiated Intestinal Crypt Cells (N-416)
  • the objective of this study was to establishing an optimal dosing schedule of CG53135-05 administration to establish the levels of protection against radiation-induced crypt cell mortality. D rotein concentrations in this example were measured by UV absorbance.
  • CG53135-05 E. coli Durified product (4 mg/kg) or phosphate-buffered saline (PBS) was administered to BDF1 male mice ay intraperitoneally (IP) once daily either for 1 , 2, 3, 4 or 5 consecutive days (Days -1 , 0, 1 , 2 and/or 3) prior to, or post-irradiation (13 Gy). The number of surviving regenerating crypt foci was neasured 4 days after irradiation and the dose modification factor (DMF) were calculated.
  • IP intraperitoneally
  • DMF dose modification factor
  • CG53135 has been shown to be a radioprotectant, it is relevant to determine if treatment of cells with CG53135 upregulates any of the genes known to be involved in radioprotection in the interest of "pre-loading" the cells with oxygen radial scavenging pathways. Evaluation of the effect of CG53135 on the intricate pathways involving ROS scavengers and transcription factors will mechanistically describe the observed in vivo radioprotective effects of this agent. Thus, expression studies and survival studies were carried out at the cellular level.
  • NIH3T3 murine fibroblast
  • CCD-1070sk human foreskin fibroblast
  • CCD-18Co human colonic fibroblast
  • HUVEC human umbilical cord vascular endothelial cells
  • MnSOD radioprotective superoxide dismutases
  • Nrf2 transcription factor which is involved in regulation of several antioxidants that were thought to be the "antioxidant response element" (also termed as ARE), was induced by CG53135 in all cell lines studied (Table 40, FIGs. 32(A) - (F)).
  • the ERK and Akt kinases are also activated by CG53135.
  • CCD18Co human colonic fibroblasts were starved for 18 hours in basal media containing 0.1 % BSA or left in complete serum (“Comp”) then stimulated with 100ng/ml FGF-20 and harvested at the time points indicated. Lysates were immunoblotted for human ERK or Akt or their indicated phosphorylated counterparts.
  • CG53135 is active in a tissue that is as equally affected as the hematopoietic stem cells, but no less important to the survival of the animal.
  • Clonogenic assays were performed using CCD-18co cells, FaDu human squamous cell carcinoma cells, IEC6 and IEC18 rat colon crypt cells, and NIH 3T3 mouse fibroblast cells, to assess the effect of CG53135 on radiation protection.
  • NIH 3T3 cells were grown in DMEM + 10% Bovine serum + 50 ⁇ g/ml Pennicilin/Streptomycin; IEC6 and IEC18 cells were grown in DMEM + 10% FBS + 0.1 U/ml Insulin + 50 ⁇ g/ml Pennicilin/Streptomycin; FaDu cells were grown in MEM + 10% FBS + 1 mM Sodium Pyruvate + 50 ⁇ g/ml Pennicilin/Streptomycin + Non-essential amino acids. Cells were plated at a density of 5x10 5 per 10 cm dish (NIH3T3) or 5x10 s per well of a 6-well dish (IEC18, IEC6, FaDu) and allowed to attach.
  • NIH3T3 cells were grown in DMEM + 10% Bovine serum + 50 ⁇ g/ml Pennicilin/Streptomycin
  • IEC6 and IEC18 cells were grown in DMEM + 10% FBS + 0.1 U/ml Insulin + 50 ⁇
  • the number of surviving untreated or CG53135-treated cells was plotted as a function of radiation dose, giving rise to survival curves
  • the slopes of different parts of the survival curves describe different properties of radiation cell killing and can be described as follows
  • DO is the slope of the curve between the final two points, indicating speed of cell killing at the higher doses of radiation
  • the value is interpreted to indicate the amount of radiation required to reduce the fraction of surviving cells by 37% of the previous value on the graph A smaller number indicates a more rapid rate of cell killing
  • D1 is the slope of the curve between the first two points, indicating the speed of cell killing at the lower doses of radiation The value is interpreted as the amount of radiation required to reduce the fraction of surviving cells by 37% of the previous value on the graph A smaller number indicates a more rapid rate of cell killing
  • Dq is the width of the shoulder of the curve before an exponential decrease in cell survival is seen This is essentially the threshold amount of irradiation required before an incidence of cell killing is seen A larger Dq value indicates that the cells are completely protected at the lower doses of radiation
  • the effect of CG53135 treatment on survival of irradiated HUVEC cells is shown in Figure 34
  • the DO value for cells treated with 100 ng/mL CG53135 is higher than that for untreated HUVEC cells or cells treated with 10 ng/ml CG53135, indicating a slower rate of cell death at higher radiation doses
  • the Dq value for cells treated with 100 ng/mL CG53135 suggests that there is a slower rate of cell death at low doses of radiation compared to untreated HUVEC cells or cells treated with 10 ng/ml CG53135 No obvious effects of treatment of CG53135 on D1 values were observed.
  • HUVEC cells treated with 100 ng/ml CG53135-05 E, coli purified product provides a significant decrease in the speed of cell killing at the high dose of radiation.
  • the HUVEC cells treated with 100 ng/ml CG53135-05 £. coli purified product also appeared to be more protected from killing at low doses of radiation compared to untreated cells.
  • the D 1 and D q parameters are indicative of the rate of cell killing at low doses of radiation, whereas he D 0 parameter reflects the rate of killing at high doses of radiation.
  • An increase in these jarameters in CG53135-05-treated cells as compared to untreated indicates a protective effect.
  • Cytokines are important cell signaling proteins mediating a wide range of physiological " esponses. Ionizing radiation can trigger a series of changes in gene expression and cytokine Drofiles. The aim of this study was to evaluate the cytokine profile upon CG53135 treatment in cell ;ulture over a time course.
  • BioPlex cytokine assays which are multiplex bead based assays designed to quantitate nultiple cytokines from tissue culture supernatants, were used for detecting the cytokines.
  • the Drinciple of the assay is similar to a capture sandwich immunoassay.
  • NIH 3T3 cells were plated in a 96 well plate. The cells were washed with DMEM+0.1 % Calf Serum (SFM).
  • SFM Calf Serum
  • Bioplex 18-Plex Cytokine Assay BioRad Laboratories Inc, CA was oerformed following the procedure of the manufacturer.
  • Figure 36 shows the effect of the CG53135-05 E. coli purified product on Mo KC release.
  • WIo KC is also known as the chemokine CXCL1 (which also has been described as Gro1 , Melanoma growth stimulatory activity (MSGA) or neutrophil-activating protein-3 (NAP3)). It functions as a shemoattractant for neutrophils, signalling through the CXCR1 receptor. It has also been implicated in the response to whole body irradiation, raising the possibility that it possesses radioprotective qualities of its own (see Radiat. Res. 160:637-46, 2003).
  • IL-6 and IL-11 expression in response to CG53135 treatment was also examined. Both IL-6 and IL-11 have recently been implicated in the response to total body irradiation. In addition, IL-11 has been used as an agent to combat thrombocytopenia following chemo- or radiotherapy.
  • CCDI8C0 cells were incubated with 100 ng/ml CG53135 in basal media containing 0.1% BSA for the indicated time periods. Conditioned media was removed and analyzed for IL-6 and IL-11 ;oncentration by Luminex or ELISA respectively.
  • Figure 36B shows that IL-6 and IL-11 cytokines are induced upon exposure to the CG53135-05 E. coli purified product in vitro.
  • CG53135-05 in combination with i;XCL1 acts synergistically in radioprotection, both in vitro and in vivo.
  • induction of Dther cytokines e.g., IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, MCP-1 , GM-CSF, RANTES
  • Dther cytokines e.g., IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, MCP-1 , GM-CSF, RANTES
  • CG53135-05 in different cell ines (e.g., HUVEC, CCD-18, NIH3T3).
  • Cells with increased reactive oxygen species upregulate the Superoxide Dismutases - Cu, Zn-SOD, Mn-SOD, and extracellular-SOD to scavenge the superoxide radical to iydrogen peroxide.
  • Activity of these enzymes can be indirectly measured by their byproduct of H 2 O 2 using an acetoxymethyl ester.
  • a derivative of this class 5-(and-6)-chloromethyl-2',7'- Jichlorodihydrofluorescein diacetate, also known as CIvI-H 2 DCFDA, is efficiently retained within the ;ell and fluoresces green when oxidized by H 2 O 2 .
  • IEC18 (rat intestinal epithelial) and CCD-18Co (human colonic fibroblast) cells were plated 0 60 mm dishes at a density of 1x10 5 cells per dish. After attachment, the cells were switched to nedium containing 0.1 % serum and the indicated dose of CG53135. After 18 hours of incubation, he cells were then irradiated at 2 or 4 Gy with X-rays using a Faxitron X-irradiator (Wheeling, IL), ollowed by incubation with 5 mM CM-H 2 DCFDA (Molecular Probes, Eugene, OR) for 15 minutes. The cells were then washed, trypsinized, and analyzed on a Becton Dickinson FACSCalibur (San lose, CA) on the FL1 channel.
  • a Becton Dickinson FACSCalibur Becton Dickinson FACSCalibur (San lose, CA) on the FL1 channel.
  • Results indicate that IEC18 and CCDI 8C0 cells possess increased intracellular H 2 O 2 after reatment with the CG53135-05 E. coli purified product in a dose responsive manner (FIGs. 37 (A)- C)). This is believed to be due to enhanced expression of Superoxide dismutases, predominantly i/lnSOD induced by the CG53135-05 E. coli purified product.
  • Redox Sensor 1 can monitor the redox level of the cytosol upon Dxidation by changing to a red fluorescent agent that can be measured by FACS on the FL2 channel.
  • IEC18 rat intestinal epithelial
  • CCD-18Co human colonic fibroblast cells were plated ;o 60mm dishes at a density of 1x10 5 cells per dish. After attachment, the cells were switched to nedium containing 0.1 % serum and the indicated dose of the CG53135-05 E. coli purified product. Mter 18 hours of incubation, the cells were then irradiated at 2 or 4 Gy with X-rays using a Faxitron K-irradiator, followed by incubation with 5 mM Red CC-1 (Molecular Probes, Eugene, OR) for 15 ninutes. The cells were then washed, trypsinized, and analyzed on a Becton Dickinson -ACSCalibur on the FL2 channel.
  • Red CC-1 Molecular Probes, Eugene, OR
  • IEC18 and CCD18Co cells were found to possess decreased cytosolic redox ootential after treatment with CG53135-05 in a dose responsive manner as shown in FIGs. 38 (A)- [C).
  • the data shown herein is believed to be the result of enhanced expression of superoxide dismutases induced by the CG53135-05 E. coli purified product, which scavenge the more reactive species of superoxide and hydroxyl radicals.
  • the CG53135-05 E. coli purified product is shown :o increase expression of a key antioxidant-controlling transcription factor, Nrf2, which may ;ontribute to this reduction in reactivity in the cytosol in other ways.
  • the radioprotection effect of CG53135 in myeloid cells was also studied by in vitro sxperiment using the myeloid cell line 32D.
  • 32D cells were irradiated at 0, 1 , 2, 3, 4 or 5 Gy then Dlated in methylcellulose-containing growth media including 10 ng/ml IL-3 with or without 100 ng/ml ⁇ G53135-05 E. coli purified product. Cells were allowed to form colonies for 10 days and were then scored.
  • FIG. 39 shows increased survival of 32D cells upon exposure to the CG53135-05 E. coli Durified product. The cell survival is plotted by the natural Log of the surviving fraction, and a linear quadratic equation was used to obtain the curve.
  • CG53135 Long-term effects of CG53135 specifically in the thymus microenvironment on reconstitution of the immune system were also examined. Protein concentrations in this example were measured by UV absorbance.
  • the CG53135 E. coli purified product was tested in a bone marrow ablation and transplantation model and repopulation of the thymus with thymocytes was examined. Mice were irradiated with 9Gy to ablate the bone marrow, and subsequently underwent bone marrow transplantation. Prior to this, one group of mice was dosed with 16 mg/kg (UV) CG53135 (IP), once daily on days -3, -2, -1 , 0 and +1 relative to the day of bone marrow ablation.
  • UV 16 mg/kg
  • mice Thirty days after bone marrow transplantation, the thymi of both untreated and treated mice were harvested and thymocytes collected. Cells were counted (A) as well as stained (B) for the T-cell specific markers CD4 and CD8.
  • EXAMPLE 34 EFFECTS OF CG53135 ON RADIATION INDUCED DIARRHEA (STUDY N439)
  • Test materials The test material used in this study was CG53135-05 E. coli purified product, batch number PT0504A. This was supplied as a frozen stock at 9.9mg/ml and each vial thawed from -80°C and diluted as required in Aminosyn to 1.6 or 0.4mg/ml in a laminar flow hood, and the Aminosyn was sterile filtered prior to the dilution. Animals were then injected ip with 0.1 ml/1 Og body weight. CG53135 was freshly prepared (diluted) each day.
  • Test animals and husbandry 140 male BDF1 mice (Harlan, UK) aged 10-12 weeks at study initiation were individually numbered using an ear punch. Each treatment group consisted of 20 mice, housed in 4 cages of 5 mice. Animals were housed in individually ventilated cages with 27 air changes/hour. Animals were allowed to acclimatize for 14 days prior to study commencement. The rooms were on an automatic timer for a 12 hour light/dark cycle with no twilight. All cages were labeled with the appropriate information necessary to identify the study, dose, animal number, and treatment group. Animals were fed a standard rodent maintenance diet. Food and filtered water were provided ad libitum. Experimental Procedures:
  • mice were divided into 7 treatment groups of 20 animals/ group and ear punched for identification. Animals were then dosed with drug (CG53135) by IP injection or remained untreated according to the schedule in Table xx. Mice were exposed to a single dose of 14GyX ray whole body irradiation using a Pantak PMC 1000, Model HF320 machine with radiation delivered at 0.7Gy/min. All irradiations were performed during 13:00-17:00 hours. Animals were restrained but unanaesthetised for the duration of the irradiation.
  • Body weights and diarrhea Animals were weighed daily to assess possible changes in animal weight among treatment groups. Animals losing more than 20% of their body weight and showing signs of distress were considered moribund and sacrificed. Animals were observed for diarrhea incidence and severity twice a day from Day 3 onwards. In addition to checking incidence, the severity of the diarrhea was recorded on a scale of 0-3 for each treatment group:
  • Histopathology At the time of sacrifice the ileum and mid colon was removed and fixed in formal saline, prior to paraffin embedding. These embedded samples were then cut to produce 3 ⁇ m sections that were stained with H&E and the level of intestinal damage evaluated by eye. The sections from each mouse were evaluated and assigned a score from 1-5:
  • FIG. 41 The relative loss in body weight per group is shown in FIG. 41.
  • the raw data for the diarrhea severity scores are summarized in FIGs. 42 (A) and (B).
  • mice were deemed moribund and were culled if they had lost more than 20% of their body weight and were also displaying signs of distress. If mice had lost more than 20% of their body weight but were active, they were spared and re-evaluated at the next time point. Occasionally mice that had been spared were found dead, and thus tissue was not taken and there are no histology scores.
  • Diarrhea incidence was measured twice daily from Day 3 onwards, although no diarrhea was observed until Day 4. It is immediately obvious that 16mg/kg CG53135-05 reduced diarrhea severity. Dosing using the previously efficacious protocol on Day-1 , 0, +1 with respect to irradiation, again was the most effective protocol, with dosing 0, 6, 12 and 18 hours post irradiation the least effective protocol. A single therapeutic injection immediately following radiation exposure, or dosing immediately following radiation exposure and 24 hours later was also very effective. The former appeared to be have a lesser effect at the earlier time points, but reducing diarrhea severity by the evening of Day 5. During this mid-term phase, there was a correlation between dose frequency and severity, with animals receiving the more daily doses experiencing the more severe diarrhea.
  • mice The histology scores in the small intestine confirm the diarrhea data. Examination of the total and average scores, with moribund mice assigned a score of 4, reveals the best response in mice dosed on day 0 only, and mice dosed -1, 0, +1. Again, CG53135 dosed 0, 6, 12 and 18 hours later was least effective.
  • mice receiving 16mg/kg CG53135 ejaculated immediately on injection This is a new observation, but we have performed most of our previous work using 4mg/kg, so it may be related to the higher dose of drug.
  • HT29 and THP-1 cells were obtained from the American Type Culture Collection (Rockville, MD), HT-29 MTX were provided by Dr. Lesuffler, INSERM, Dillejuis, France. These cell lines (Caco2, HT-29 and HT-29MTX) were grown as described previously. THP-1 cell lines were grown in RPM1-1640 medium (Life Technologies, Gaithersburg, MD) with 10% fetal bovine serum, 100 units /ml of antibiotics/antimycotics (Life Technologies, Gaithersburg, MD).
  • An in vitro healing assay was performed using a modified method. Briefly, reference lines were drawn horizontally across the outer bottom of 24-well plates. HT-29 and Caco-2 cells were seeded and grown to confluence, then incubated with media containing 0.1% FBS for 24 hours.
  • Linear wounds were made with a sterile plastic pipette tip perpendicular to the lines on the bottom of the well. Isolated CG53135-05 E. coli purified product (1 OOng/ml) was then added. The size of the wound was measured at three predetermined locations at various times after wounding (0, 6, 20 and 24 hours). The closure of the wounds was measured microscopically at 2Ox magnification over time, and the mean percentage of wound closure was calculated relative to baseline values (time 0).
  • anti-TGF ⁇ antibody R&Dsystem, Minneapolis, MN
  • polyclonal anti-ITF antibody a gift from D K Podolsky, Harvard Medical School, Boston, MA
  • FlG. 43 shows the effect of FGF-20 in the closure of wounds in various human cell lines. There is a dose dependent increase in the effectiveness of FGF-20 in the closure of wounds in all the cell lines tested, demonstrating the role of FGF-20 in wound repair.
  • RNA from cell lines and the colonic tissue was prepared using TRIzol reagent (Invitrogen) according to the manufacture's instructions.
  • RNA was reverse transcribed using 2 ⁇ g of total RNA, 15U of RNA inhibitors, 1x first strand buffer (Life technologies, Long Island, NY), 5mM dNTP (Pharmacia, Uppsal, Sweden), 125 pmol random hexamer primers (Pharmacia), and 125 LJ of Moloney murine leukemia virus RT (Life Technologies) in a final volume of 25ul.
  • the reaction was carried out for 1 hour at 39 0 C followed by 7 minutes at 93°C and 1 minutes at 24 0 C and then slowly cooled to 4 0 C for 20 minutes.
  • PCR was carried out in a volume of 50 ⁇ l containing 5 ⁇ l of RT mixture, 1X Thermos aquaticus (Taq) buffer, 5 pmol of each primer, 2.5mM dNTP, and 1 U of Taq polymerase.
  • the sequence of primers used were as follows: human COX-2 sense; ⁇ '-AGATCATCTCTGCCTGAGTATCTT-S' (SEQ ID NO: 62), human COX-2 antisense: ⁇ '-TTCAAATGAGATTGTGGGAAAATTGCT-S' (SEQ ID NO: 63), human Intestinal trefoil factor (ITF) sense: 5'-GTGCCAGCCAAGGACAG-3", (SEQ ID NO: 64), human ITF antisense: ⁇ '-CGTTAAGACATCAGCCTCCAG-S', (SEQ ID NO: 65), human PPAR- ⁇ sense: ⁇ '-TCTCTCCGTAATGGAAGACC-S' (SEQ ID NO: 66), human PPAR- ⁇ antisense: ⁇ '-GCATTATGAGACATCCCCAC-S' (SEQ ID NO: 67), human ⁇ -actin sense: ⁇ '-CCAACCGCAAGAAGATGA-S' (SEQ ID NO: 68),
  • PCR was carried out in a Perkin-Elmer 9600 cycler set for 20- 40 cycles to assess linearity of the amplification.
  • the PCR products were electrophoresed on 2% tris-acetate and EDTA agarose gels containing gel star fluorescent dye (FMC Corporation, Philadelphia, PA).
  • a negative from the gels was taken with Alphalmager 2000 (Alpha lnnotech Corporation, CA) and relative abundance of RT-PCR transcript was assessed by Adobe photoshop 3.0.4 software, normalized to the density of ⁇ -actin and G3PDH transcript.
  • COX-2 gene expression in HT29 cell line in the presence of CG53135 was dose dependent, showing highest expression when induced by 10Ong/ml of CG53135-05 E. coli purified product (FIG. 44). At this concentration, the gene expression was higher at 1 hour and 3 hour time periods of incubation and decreased thereafter at 6 hour and 24 hour.
  • COX-2 gene expression in Caco2 cell line was high when stimulated with 100ng/ml of CG53135 as seen in FIG. 45. Increased expression of COX2 was detected at 1 , 3 and 6 hours after incubation with 100ng/ml of CG53135 E. coli purified product.
  • ITF Intestinal Trefoil factor
  • FIG. 49 shows that Epithelial Restitution by XG53135 is mediated in part by TGF- ⁇ pathway (p ⁇ 0.05 vs CG53135 E. coli purified product + anti-TGF- ⁇ ).
  • FIG. 50(C) shows increased expression of phosphorylated STAT3, p-p38MAPK and SOCS-3 genes.
  • FIGs. 50(D)-(E) show increased expression of phosphorylated Elk-1 , ATF-2 and minimal induction of phosphorylated Protein Kinase C in Caco-2 cells in the presence of CG53135.
  • C-Fos and C-Jun were induced, when cultured with CG53135 (FIG. 50(D)-(E)).
  • EXAMPLE 38 DOSE RESPONSIVE EFFECTS OF CG53135 IN FEMALE SWISS WEBSTER MICE WITH DEXTRAN SULFATE-INDUCED COLITIS
  • the CG53135 protein was produced in E. coli as follows: The cDNA for CG53135-01 was identified and cloned into the pRSET vector (Invitrogen) to provide the vector pETMY-CG53135-01.
  • the gene product of this construct provides a polypeptide incorporating (His) 6 -(enterokinase cleavage site)-(multicloning site) at the N-terminal end of the polypeptide; in addition, in this construct, the CG53135 sequence begins with the Ala at position 2 FGF-20 (SEQ ID NO:2). This vector was transformed into Escherichia coli.
  • coli cells were grown up to 10 L scale and infected with CE6 phage to produce the recombinant CG53135.
  • the recombinant protein was purified by disrupting the E. coli cells (resuspended in a 1 M L-arginine solution) in a microfluidizer, followed by multiple metal affinity chromatography steps. The final purified protein was dialyzed into phosphate buffered saline containing 1M L-arginine.
  • Colon content was scored at necropsy according to the following criteria:
  • Pathology Methods Three sections equidistant apart from the distal one third of the colon (area that is most severely affected in this model) were processed for paraffin embedding, sectioned and stained with hematoxylin and eosin for pathologic evaluation.
  • Colon glandular epithelial loss this includes crypt epithelial as well as remaining gland epithelial loss and would equate to crypt damage score; and (2) Colon Erosion - this reflects loss of surface epithelium and generally was associated with mucosal hemorrhage (reflective of the bleeding seen clinically and at necropsy).
  • Colon Erosion this reflects loss of surface epithelium and generally was associated with mucosal hemorrhage (reflective of the bleeding seen clinically and at necropsy).
  • 3 proximal (less severe lesions) and 3 distal (most severe lesion) areas were scored and the mean of the scores for each of the regions was determined. Group means and % inhibition from disease control were determined.
  • mice treated QD with 10 mg/kg AB020858 (CG53135) generally had semi-solid stool and less blood (except animals #5).
  • Clinical benefit was also evident but less impressive in those given doses of 3 or 1 mg/kg QD and absent in those treated with 0.3 mg/kg (FIG. 51(C)).
  • Mice treated BID with 5 mg/kg had the most impressive clinical benefit (68% inhibition) and clinically these mice had the best overall improvement.
  • Absolute colon weights were decreased approximately 26% in mice treated with DSS in vehicle.
  • Treatment with AB020858 (CG53135) at 10 mg/kg QD or 5 mg/kg BID resulted in significant reduction of the DSS-induced changes in colon weights.
  • mice were also treated subcutaneously with CG53135.
  • CG53135 prophylactic administration of CG53135 at doses of 5 or 10 mg/kg ip and 5 or 1 mg/kg sc significantly reduce the extent and severity of mucosal damage induced by dextran sulfate sodium in a murine model of colitis.
  • Rats Female Lewis rats (Harlan, Indianapolis, IN) weighing 175-200 g were acclimated for 8 days (Day -8 through Day -1). Rats were divided into 8 treatment groups: four groups receiving CG53135 (three groups iv and one group sc), two iv controls for normal and the disease model, and two sc controls for normal and the disease model. On Day -1 , treatments with CG53135 or vehicle were initiated and continued through Day 4. CG53135 was injected iv (tail vein) at doses of 5, 1 or 0.2 mg/kg, or 1 mg/kg sc; vehicle controls were injected with BSA (5 mg/mL in PBS + 1 M L-arginine).
  • CG53135 was injected iv (tail vein) at doses of 5, 1 or 0.2 mg/kg, or 1 mg/kg sc; vehicle controls were injected with BSA (5 mg/mL in PBS + 1 M L-arginine).
  • Histopathology Five sections (approximately equally spaced) taken from the weighed 10cm area at risk of small intestine for indomethacin-induced lesions were fixed in 10% neutral buffered formalin, processed for paraffin embedding, sectioned at 5 ⁇ m and stained with hematoxylin and eosin for histopathologic evaluation. Necrosis was scored according to the percent area of the section affected in the same way as described in Example 38 for scoring epithelial cell loss.
  • the means for inflammation and necrosis were determined for each animal, and then the means for each group were calculated.
  • Hematology Administration of 2 doses of indomethacin to rats increased the total white blood cell count as a result of increased neutrophils and lymphocytes. Reductions in red blood cell count, hematocrit, and hemoglobin concentration were also observed. Treatment with CG53135 (5 mg/kg and 0.2 mg/kg iv) resulted in significant reductions in absolute neutrophil counts as compared with disease controls (FIG. 55(B)). Hemoglobin concentration was diminished in the indomethacin controls compared to normal controls, and slightly further diminished in rates treated with CG53135.
  • FIG. 56 Photomicrographs of affected small intestine are shown in FIG. 57 for a normal and disease control, and a rat treated with 0.2 mg/kg CG53135.
  • Panel A shows the small intestine from a normal control animal treated iv with vehicle (BSA). Normal villous architecture and mesentery (arrow) are apparent.
  • Panel B presents a photomicrograph of the small intestine from an indomethacin- treated rat, with vehicle (BSA) iv.
  • BSA vehicle
  • Focal mucosal necrosis extending across most of the area associated with attachment of the mesentery is apparent (see, for example, the asterisks at upper right intestinal wall and lower right intestinal wall). Marked inflammatory cell infiltrate is present in the mesentery (arrow).
  • Panel C shows the image of the small intestine from an indomethacin-treated rat further treated with CG53135, 0.2 mg/kg iv. There is no apparent necrosis, in contrast to the disease control shown in Panel B.
  • BrdU labeling was carried out by injecting 50 mg/kg 1hour prior to necropsy. In the small intestine from a normal control animal, normal pattern of crypt labeling is seen at 100X (FlG. 58, Panel A). BrdU incorporation in the disease model was decreased or absent in epithelial cells in mucosal areas of necrosis, but increased in subajacent inflammatory tissue in which fibroblast labeling was prominent (FIG. 58, Panel B, visualized at 50X). Focal mucosal necrosis (arrow) is delineated by an absence of BrdU immunostaining as well as severe infiltration of inflammatory cells and fibroblast proliferation.
  • Small intestine from a rat treated with indomethacin + CG53135 0.2 mg/kg iv shows an absence of crypt labeling, but relatively intact mucosa (arrow in FIG. 58, Panel C, visualized at 50X).
  • Subadjacent smooth muscle and mesentery is only mildly infiltrated with inflammatory cells, compared with that seen in the disease control (Panel B).
  • CG531335 in which preservation of mucosal integrity occurred, increased crypt labeling was also observed; this is in the direction found in the normal control.
  • EXAMPLE 40 THERAPEUTIC ADMINISTRATION OF CG53135 ENHANCES SURVIVAL IN THE MURINE DSS MODEL.
  • CG53135 was prepared as described in Example 38. Balb/c mice were exposed to DSS for 7 days (day 0 to day 6). The mice were injected daily subcutaneously with various concentrations of CG53135 (5, 1 and 0.2 mg/kg) beginning on the fifth day of DSS exposure (Ae. day 4) and ending 3 days after the termination of DSS exposure (i.e. day 9), or with vehicle only. Animal survival was recorded on a daily basis and the experiment was concluded on day 10.
  • the objective of the study was to assess the ability of CG53135 to therapeutically inhibit the inflammation that occurs in IL-10 deficient mice when transferred from a germ free to a specific pathogen free environment. As inflammatory bowel disease is thought to have an immune component, this study evaluated the efficacy and safety of CG53135 in this immune-mediated model of IBD when dosed therapeutically at the time of significant disease.
  • Test Article CG53135-05 (FGF-20) protein (purity >97%) in 20% glycerol buffer.
  • Glycerol buffer 20% Glycerol, 20OmM Sorbitol, 1 mM EDTA, 10OmM Citrate, 5OmM KCL
  • mice are untreated and maintained in germ free conditions throughout study ' Disease control: vehicle administered ip, once daily using the therapeutic dosing regimen.
  • Table 47 Study Schedule
  • a Mice are dosed orally with a slurry of fecal contents solubilized in PBS from donor SPF documented free of Heliobacter.
  • mice were acclimated for 2 days before bacterial colonization and given autoclaved food and tap water ad libitum during this time. Mice will be treated with CG53135-05 E. coli purified product or buffer for 2 days beginning the day of transfer, then colonized with specific pathogen free bacteria by swabbing their mouth and rectum with solubilized fecal material. Animals were examined prior to initiation of the study to assure adequate health and suitability. Animals that were found to be diseased or unsuitable will not be assigned to the study.
  • mice evenly assigned to all dose groups (e.g., 2 animals per sex per treatment group). If the number of available animals at the time of initiation is not evenly divided between males and females, animals were assigned to groups to balance males and females as best as possible.
  • mice were observed daily for significant clinical signs of toxicity, moribundity and mortality approximately 60 minutes after dosing.
  • Body Weight individual body weights of all mice were recorded pretest (for randomization) and daily through Day 10. Body weights taken on the day of necropsy for animals scheduled for termination was used for determination of organ to body weight ratios. Following are the organs/tissues measured.
  • Histopathology All animals surviving to scheduled termination (Day 10) will be terminated using CO 2 with assessment of gross observations, organ weights and collection of all scheduled tissues into 10% neutral buffered formalin for histopathologic evaluation.
  • Colonic strip cultures were established from the remaining colon fragments, pooled from segments of proximal, middle and distal colon. Colon segments were flushed with phosphate-buffered saline (PBS) to remove fecal contents, opened lengthwise and cut into 0.5 toi .O cm pieces and shaken vigorously in PBS. Approximately 50 to100 mg of tissue was then distributed per well of a 24 well tissue culture plate in duplicate and cultured in 1 mL of complete medium containing antibiotics and an antimycotic agent (Veltkamp et al, 2001 , Gastroenterology, VoI 120(4):900-913).
  • PBS phosphate-buffered saline
  • culture supernatants were collected in aliquots and frozen at -70° C for cytokines and possibly immunoglobulin measurements. lgG2a and IL-12 in the supernatant were measured by ELlSA.
  • MN Mesenteric lymph nodes
  • Cytokine assays IL-12 (Pharmingen, San Diego, CA), TNF- ⁇ and IFN- ⁇ (R&D systems, Minneapolis, MN) was measured in MNL cell and splenocyte culture supernatants by ELISA. Moreover, IL-12 and PGE2 (Assay Design, Ann Arbor, Ml) was measured in supernatants of colon cultures using standard ELISA protocol. Concentrations of these cytokines and PGE2 were measured in duplicate culture supernatants by comparison with standard curves generated using recombinant cytokines.
  • FIG. 60(A) shows weight change when challenged with FGF-20 in IL-10 knock-out (KO).
  • FIG. 60(A) also shows the histopathology of the colon when the mice are challenged with different concentrations of FGF-20 (0.2, 1 , 5 mg/kg). The results indicate that, administration of FGF-20 had a protective effect as compared to the vehicle control.
  • FIG. 60(B) further demonstrates that, upon administration of FGF-20, there is a dose-dependent decrease in the total Cecal Histologic score, as compared to the vehicle (12.2 ⁇ 2.3 vs. 2.5 ⁇ 0.6; p ⁇ 0.001).
  • FIGS. 61(A) (IL-12), 61(B) (IFN ⁇ ) and 61(C) (PGE2) indicate that FGF-20 altered cytokine production in MLN, colonic strip culture, Spleen cell culture, which were prepared from the IL-10KO mice as described above.
  • FIG. 62 also shows FACS analysis of total MLN number (32 ⁇ 3.4 vs. 23 ⁇ 2.5 ; p ⁇ 0.05), CD4+ and CD8+ and CD4+CD69+ cells (3.2 ⁇ 0.3 vs. 1.67 ⁇ 0.1 ; p ⁇ 0.05).
  • Study protocol for the treatment group was established by treatment of established colitis in ex-germ free IL-10 -/- mice colonized with SPF bacteria on day 1. On day 10, treatment was started by intraperitoneally administering either Vehicle or FGF-20 (5mg/kg) and necropsy was performed on day 17.
  • FIG. 63 shows the Weight change in the treatment study, where FGF-20 (5mg/kg) was administered to IL-10 KO mice. Histology of the cecum and rectum are respectively shown in FIGS. 64 and 685, that demonstrates protective effect of FGF-20. Cecal histologic score shows that FGF- 20 decreased as compared to the vehicle control (13.1 ⁇ 1.8 vs. 5.9 ⁇ 1.4; p ⁇ 0.006, FIG. 66).
  • FIG. 67 shows FACS analysis of MLN number, CD4+ and CD8+ and CD69+ cells, all of which were decreased in FGF-20 treated group as compared to the vehicle treatment.
  • EXAMPLE 42 EFFECT OF CG53135-05 IN A CHRONIC 2 WEEK MURINE MODEL OF DSS-INDUCED ULCERATIVE COLITIS (N-404)
  • UC ulcerative colitis
  • IBD inflammatory bowel disease
  • Colitis was induced in female Swiss Webster mice by exposure to 3% DSS in the drinking water (ad libitum) on study Days 1-5 and maintained by exposure to 1 % DSS in the drinking water (ad libitum) on study Days 6-15. Mice were randomly assigned to 4 groups of 15 animals (Table 50). Mice were exposed to 3% DSS in drinking water on test Days 1 through 5 and 1% DSS on test Days 6-15, with concurrent intraperitoneal (IP) treatments of vehicle or CG53135-05 at 0.33, 1.67 and 3.33 mg/kg (UV) Days 6-14 after disease was established. Mice were weighed daily. On Day 15, animals were terminated by cervical dislocation, the colon length was measured, and colon content was scored. Tissues were collected into 10% neutral buffered formalin and processed routinely for histopathology.
  • IP intraperitoneal
  • Histopatholoqy At necropsy, the colon was harvested and divided into 2 approximately equal segments, proximal and distal, to assess regional changes induced in this model. Distal ends were marked to maintain orientation. These colon segments were collected, preserved in 10% neutral buffered formalin, and routinely processed for histopathologic evaluation. During processing for histology, the proximal and distal colon segments were each trimmed to obtain 4 equally spaced segments, and hematoxylin and eosin (H&E) stained slides were prepared for each. Both proximal and distal tissues were examined as these tissues are affected to different degrees of severity in this model (more severe and less variable symptoms predominate in the distal colon).
  • H&E hematoxylin and eosin
  • submucosal edema was quantified by measuring the distance (mm) from the muscularis mucosa to the internal border of the outer muscle layer.
  • the parameters reflecting epithelial cell loss/damage were scored individually using a percent area involved scoring method. Parameters that were scored using the percent involvement scale included colon glandular loss and colon erosion.
  • Mucosal epithelial hyperplasia (basophilia, mitotic figures, multilayered on basement membranes, absence of goblet cells) was scored 0-5 based on the following criteria
  • Mucosal thickness (an indicator of proliferative changes or edematous inflammatory mucosal expansion) was measured by placing an ocular micrometer at the base of the glands and the overlying surface epithelium in a non-tangential area of section representative of the thickest areas of mucosa.
  • the scores for proximal tissues and distal colon were averaged to determine a score for the entire colon for each parameter evaluated.
  • the 3 scored parameters ⁇ i.e., inflammation, glandular loss, and surface epithelial erosion) were combined to arrive at a sum of overall histopathology scores for proximal or distal sections of the colon, and then proximal and distal overall scores were averaged to arrive at an overall histopathology score for the entire colon. These summations indicate the overall damage in the distal, proximal, or entire colon and would have a maximum severity score of 15.
  • Additional tissues were collected and divided into two equal sections. One section was snap frozen in liquid nitrogen, the other was placed in 10% neutral buffered formalin for paraffin embedding and immunohistochemical staining with Ki67 antibody. The formalin preserved tissues were trimmed into approximately 0.5 cm sections (1 section/tissue), processed through graded alcohols and a clearing agent, infiltrated, embedded in paraffin and sectioned. Slides were then re-hydrated and stained with mouse Ki67 antibody (Dako) at 1 :70 dilution followed by streptavidin/HRP detection system with a DAB peroxidase indicator and counterstained with Gill's Hematoxylin. The slides were exposed to diaminobenzadine (DAB) for four minutes to provide optimal specific staining with minimal background nonspecific staining.
  • DAB diaminobenzadine
  • the mean and maximal scores were analyzed with a Kruskal-Wallis test followed by a Dunn's multiple comparison test between each day and normal control animals. In all other cases, multiple comparison test results were adjusted for the number of comparisons being performed using a Bonferonni correction. In the case of pathology scores, the comparisons were effectively doubled, by analyzing the mean and the maximum values.
  • mice Fifty-nine of the sixty mice survived the duration of the study and were included in the analysis of body weight loss, terminal colon lengths, and colon content. Decreased body weight gain was seen in all groups with the most severe effects occurring on Day 9 and recovery of gain beginning on Day 10. A dose-dependent trend in inhibition of DSS-induced body weight loss was observed, corresponding to 19, 35 and 67% reduction in weight loss for groups receiving IP injections of 0.33, 1.67 or 3.33 mg/kg (UV) CG53135-05, respectively. Gross pathology evaluation indicated that colon length decreases and colon content score increases were greatest in the vehicle-treated disease control group.
  • mice that received 3.33 mg/kg (UV) CG53135-05 showed significant improvements in on (35%) and total (34%) inflammation, distal (38%) and total (37%) gland loss as well as distal (38%) and total (37%) summed histopathologic scores.
  • Protective effects (46-62% inhibition, non-significant) of CG53135-05 were also seen on proximal, distal and total colon erosion in the mid and high dose groups.
  • Beneficial effects (dose responsive, non-significant) on inhibition of mucosal thickness changes and hyperplasia scores occurred with 39% inhibition of the total mucosal thickness in mice treated with 3.33 mg/kg (UV) and 31% improvement in the hyperplasia score.
  • MCA Middle cerebral artery
  • coli purified product or vehicle (4OmM acetate, 200 mM mannitol (pH 5.3)) by percutaneous injection into the cisterna magna, once at 1 day, (approximately 24 hours) and once at 3 days, (approximately 72 hours) after MCA. Animals were given test article (2 dose groups) or vehicle treatment according to the study design.
  • Body Weight Animals were weighed on Days 1 , 3, 7, 14 and 21.
  • Limb Placing Test limb placing tests were carried out on all animals on Day -1 (pre- operation), Day 1 (just prior to injection), Day 3 and then every 7 days thereafter (Days 7, 14, 21).
  • the forelimb placing test measures sensorimotor function in each forelimb as the animal places the limb on a table top in response to visual, tactile, and proprioceptive stimuli.
  • the forelimb placing test consists of the following evaluations and scoring, where the combined total score for the forelimb placing test reflects a range from 0 (no impairment) to 10 (maximal impairment): visual placing (forward, sideways): 0 - 4 tactile placing (dorsal, lateral): 0 - 4 proprioceptive placing: 0 - 2 Total score for all forelimb tests: 0 - 10
  • Hindlimb Placing Test Assessment Score Similarly, the hindlimb placing test measures sensorimotor function of the hindlimb as the animal places it on a tabletop in response to tactile and proprioceptive stimuli.
  • the hindlimb placing test consists of the following evaluations and scoring, where the combined total score for the hindlimb placing test reflects a range from 0 (no impairment) to 6 (maximal impairment): tactile placing (dorsal, lateral): 0 - 4 proprioceptive placing: 0 - 2
  • Body Swing Test The body swing test was carried out on all animals on Day -1 (pre- operation), Day 1 (just prior to injection), Day 3 and then every 7 days thereafter (Days 7, 14, 21).
  • Cylinder Test the cylinder test was carried out on all animals on Day -1 (pre-operation) and 7 days thereafter (Days 7, 14, 21). The cylinder test measures spontaneous motor activity of the forelimbs. Animals are placed in a narrow glass cylinder (16.5 x 25 cm) and videotaped for 5 min on the day before stroke surgery and at weekly intervals thereafter. Videotapes are then scored independently by one experienced observer and up to 50 spontaneous movements will be counted ( ⁇ 5 min per rat per day). Spontaneous movements include those made by each forelimb to initiate rearing, to land on or to move laterally along the wall of the cylinder, or to land on the floor after rearing.
  • the area of cerebral infarcts on each of seven slices (+4.7, +2.7, +0.7, - 1.3, -3.3, -5.3, and -7.3 mm compared with Bregma) was determined using a computer interface imaging system using the indirect method (area of the intact contralateral hemisphere - area of the intact ipsilateral hemisphere) to correct for brain shrinkage during processing. Infarct volume was then expressed as a percentage of the intact contralateral hemispheric volume. Volumes of the infarction in the cortex and striatum were also determined separately using these same methods. H&E stained section was examined for histological changes such as hemorrhage, abscess or tumor formation.
  • Visual placing (scored 0-4), tactile placing (scored 0-4), and proprioceptive placing (scored 0-2) were summed to generate a range of potential total scores from 0 to 12, with 12 representing maximal impairment (FIG. 68 (A)).
  • Hindlimb Placing on days -1 , 1 , 3, 7, 14, and 21 relative to MCA occlusion, animals were examined by using a limb placing test to assess sensorimotor function in the hindlimb in response to tactile and proprioceptive stimuli (Kawamata, T., Dietrich, W. D., Schallert, T., Gotts, E., Cocke, R. R., Benowitz, L. I. & Finklestein, S. P. (1997) Proc. Natl. Acad. Sci. USA 94, 8179-8184; De Ryck, M., Van Reempts, J., Duytschaever, H., Van Deuren, B. & Clincke, G. (1992) Brain Res.
  • Tactile placing (scored 0-4), and proprioceptive placing (scored 0-2) were summed to generate a range of potential total scores from 0 to 6, with 6 representing maximal impairment (FIG. 68(B)).
  • Body Weight animals were weighed on days -1 , 1 , 3, 7, 14, and 21 relative to MCA occlusion and the results indicate no significant difference between the vehicle and CG53135-05 treatment (FIG. 68(E)).
  • CG53135 administration will be useful in the treatment of pathological conditions including ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning and neurodegenerative diseases (such as Alzheimer's, Parkinson's Disease, Amyotrophic Lateral Sclerosis, Huntington's Disease).
  • pathological conditions including ischemic stroke, hemorrhagic stroke, trauma, spinal cord damage, heavy metal or toxin poisoning and neurodegenerative diseases (such as Alzheimer's, Parkinson's Disease, Amyotrophic Lateral Sclerosis, Huntington's Disease).
  • MMPs matrix metalloproteinases
  • OA Osteoarthritis
  • RA Rheumatoid Arthritis
  • SW1353 cells were plated in a 24-well plate at 1 x105 cells/ml (1 ml) in DMEM medium-10 % FBS. Following overnight incubation, the medium was replaced with DMEM + 0.2 % Lactabulmin serum.
  • CG53135-05 E. coli purified product was added to the wells at doses ranging from 10 to 5000 ng/ml, in the absence or presence of IL-1 beta (0.1 to 1 ng/ml, R&D systems Minneapolis, MN), TNF-alpha (10 ng/ml, R&D systems) or vehicle control to a final volume of 0.5 ml.
  • IL-1 beta and TNF-alpha are both potent stimulators of MMP activity. All treatments were done in triplicate wells.
  • TIMP-1 tissue inhibitor of matrix metalloproteinase
  • ELISA tissue inhibitor of matrix metalloproteinase
  • CG53135 significantly decreased MMP-13 production in the presence of either IL-1 beta or TNF-alpha as demonstrated in FIG. 69(A) and FIG. 69(B), respectively.
  • IL-1 beta and TNF-alpha are both potent stimulators of MMP activity.
  • MMP-13 affinity for type Il collagen, the main collagen that is degraded in OA is ten times higher that of MMP-1. Since MMP-13 expression increases in OA and RA, the decrease of MMP-13 observed with addition of CG53135 indicates that the protein can be used as an OA and RA therapeutic.
  • CG53135 up-regulated the production of TIMP-1, a natural inhibitor of MMP activity (FIG. 69(C)).
  • CG53135 This enhancement of TIMP-1 production by CG53135 is beneficial in reducing the matrix breakdown by MMP-1 and -13 observed in OA and RA. In addition, CG53135 had no effect on MMP-3 production constitutively or after IL-1 induction. Similarly, CG53135-05 E. coli purified product showed increase in basal expression of MMP-1 in SW1353 cells.
  • EXAMPLE 45 EFFECT OF CG53135 ON NORMAL RATS: PROOF OF PRINCIPLE TO THE MENISCAL TEAR MODEL
  • CG53135 The effect of CG53135 on the normal rats was studied as a proof of principle to drive further studies in disease model (ex: meniscal tear model of osteoarthritis in rats). The effect of CG53135 on synovium and cartilage was assessed by injecting the protein into normal male Lewis rats.
  • the rats were injected intra-articularly three times per week for 2 weeks with vehicle solution (8mM acetate, 40 mM arginine, and 0.6% glycerol (pH 5.3) in approximately 1% hyaluronic acid), 10 ⁇ g CG 53135-05 E. coli purified product or 100 ⁇ g CG 53135-05 E. coli purified product.
  • vehicle solution 8mM acetate, 40 mM arginine, and 0.6% glycerol (pH 5.3) in approximately 1% hyaluronic acid
  • 10 ⁇ g CG 53135-05 E. coli purified product or 100 ⁇ g CG 53135-05 E. coli purified product.
  • CG53135 Male Lewis rats weighing 293-325 grams on day 0 were obtained from Harlan Sprague Dawley (Indianapolis, Indiana) and acclimated for 8 days. The rats were divided into three treatment groups with three animals in each group: two groups received CG53135 and one received only the vehicle control. The rats were anesthetized with isoflurane and injected through the patellar tendon into the area of the cruciate attachments of both knees. CG53135 was injected at doses of 0.1 mg/ml (0.01 mg/joint) or 1.0 mg/ml (0.1 mg/joint). Controls were injected with the vehicle solution as described above. Injections were done Monday, Wednesday and Friday for 2 weeks. The animals were terminated on day 15 at which time they were injected ip with BRDU (100 mg/kg) in order to pulse label proliferating cells.
  • BRDU 100 mg/kg
  • Histopathology Preserved and decalcified (5% formic acid) knees were trimmed into 2 approximately equal longitudinal (ankles) or frontal (knees) halves, processed through graded alcohols and a clearing agent, infiltrated and embedded in paraffin, sectioned, and stained with toluidine blue (knees). Multiple sections (3 levels) of right knee were analyzed microscopically with attention to the parameters of interest listed below. Each parameter was graded as normal, minimal, mild, moderate, marked or severe. Evaluation of the cartilage was done using descriptive parameters rather than the scoring criteria generally used in the osteoarthritis model because of the type of alterations generated by the repetitive injection of the protein. Although animals were injected with BRDU prior to termination, the proliferative changes were readily observed in toluidine blue stained sections.
  • Knees injected with 100 ⁇ g CG 53135-05 E. coli purified product had moderate to marked synovial hyperplasia, inflammation and fibroplasia with moderate matrix deposition in fibrotic synovium.
  • Articular cartilage had none to minimal proteoglycan loss or fibrillation.
  • the central area of the joint where the cruciates attach and in which the intra-articular injections are made had minimal to marked fibroplasia and cartilage/bone damage. All knees had mild to moderate marginal zone chondrogenesis.
  • One animal had chondrogenesis in areas associated with articular cartilage.
  • inflammation in the joint can induce bone resorption and marginal zone chondrogenesis so these results need to be interpreted in light of the possibility that the inflammatory response to the protein injection contributed to the proliferative response.
  • the morphologic appearance of the proliferative changes and chondrogenesis clearly indicates that the biological activity of this protein (CG53135) is important in generating the response.
  • Example 45 utilized CG53135 administration into the joints of normal rats to identify effects on relevant cell populations by histomorphometric analysis.
  • CG53135 induced significant marginal zone chondrogenesis similar to that seen with other growth factors such as TGF- beta, suggesting an effect on pluripotent stem cells within the marginal zone.
  • TGF- beta growth factor
  • the potentially adverse effects of observed synovial fibroplasia and bone resorption could have been due to either FGF-20 activity or endotoxin levels within the non-clinical grade hyaluronic acid used to formulate the protein.
  • Prophylactic Dosing intra-articular dosing (CG53135-05 E. coli purified product) of the right knee joint was initiated on the day of surgery and is continued for 2 weeks post-surgery with intra- articular injections given Thursday, Saturday, and Monday (day 0, 2, 4, 7, 9, and 11) with rats under Isoflurane anesthesia. Indomethacin, a nonsteroidal anti-inflammatory drug, was dosed (1 mg/kg/day) daily by the oral route starting on the day of surgery to reduce any potential inflammation due to the injection. Body weights were recorded on days 0, 7 and 14. After animal termination on day 14 post- surgery, both knees were collected for histopathologic evaluation. The study design is shown in Table 53.
  • intra-articular dosing CG53135-05 E. coli purified product
  • intra-articular injections given Friday, Sunday, and Tuesday (day 22, 25, 27, 29, 32, and 34) with rats under isoflurane anesthesia
  • lndomethacin is dosed daily by the oral route starting on the day of surgery.
  • Body weights are recorded on days 0, 7, 14, 21 , 28, and 35.
  • both knees are collected for histopathologic evaluation.
  • Table 54 The study design is shown in Table 54.

Abstract

La présente invention concerne des formulations améliorées comprenant du FGF-20, certains de ses fragments, dérivés, variantes, homologues, analogues ou certaines de leurs combinaison, et des procédés améliorés de production ainsi que des procédés d'utilisation des compositions de l'invention.
EP05851340A 2004-11-03 2005-11-03 Formulations, procedes de production et utilisations du fgf-20 Withdrawn EP1812460A1 (fr)

Applications Claiming Priority (10)

Application Number Priority Date Filing Date Title
US10/980,659 US20050208514A1 (en) 1999-07-27 2004-11-03 Novel fibroblast growth factor and nucleic acids encoding dame
US10/980,695 US20050215473A1 (en) 2003-05-09 2004-11-03 Prophylactic and therapeutic uses of FGF-20 in radiation protection
US10/980,459 US20050256042A1 (en) 2003-05-09 2004-11-03 Methods of preventing and treating alimentary mucositis
US10/980,458 US20080287349A1 (en) 2003-05-09 2004-11-03 Fibroblast growth factor 20 and methods of use thereof
US10/980,370 US20060094651A1 (en) 2004-11-03 2004-11-03 Formulations and methods of production of FGF-20
US10/980,764 US20050164929A1 (en) 2000-11-06 2004-11-03 Methods of preventing and treating inflammatory bowel disease
PCT/US2005/010731 WO2005113809A2 (fr) 2004-05-10 2005-03-29 Nouveau facteur de croissance des fibroblastes et acides nucleiques codant pour ce facteur
PCT/US2005/010732 WO2006073417A2 (fr) 2004-05-10 2005-03-29 Utilisations prophylactiques et therapeutiques de fgf-20 en protection antirayonnement
PCT/US2005/010545 WO2005112979A2 (fr) 2004-05-10 2005-03-29 Procédés de prévention et de traitement de la mucosite alimentaire
PCT/US2005/039833 WO2006055264A1 (fr) 2004-11-03 2005-11-03 Formulations, procedes de production et utilisations du fgf-20

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EP2350271B1 (fr) 2008-11-20 2016-01-27 Biogen MA Inc. Inactivation de virus enveloppés par l'arginine
WO2014035695A1 (fr) 2012-08-30 2014-03-06 The Board Of Trustees Of The Leland Stanford Junior University Immunité antitumorale due aux lymphocytes t induite par radiation à haute dose
TW201713772A (zh) * 2015-05-15 2017-04-16 葛蘭素集團有限公司 製造重組蛋白質之方法
EP3302494A4 (fr) * 2016-01-08 2019-01-23 Gufic Biosciences Limited Composition parentérale lyophilisée de tigécycline et procédé de préparation associé
CA3054534A1 (fr) * 2017-03-02 2018-09-07 Glytech, Inc. Procede de production d'un polymere d'acide amine
JP2021536241A (ja) * 2018-08-30 2021-12-27 グラクソスミスクライン、インテレクチュアル、プロパティー、ディベロップメント、リミテッドGlaxosmithkline Intellectual Property Development Limited 核酸を検出する方法
CN111638370B (zh) * 2020-04-30 2023-03-03 吉林省格瑞斯特生物技术有限公司 胃功能及胃癌发生风险检测装置及其制备方法

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US6180096B1 (en) * 1998-03-26 2001-01-30 Schering Corporation Formulations for protection of peg-interferon alpha conjugates
US20020037557A1 (en) * 2000-03-13 2002-03-28 Amgen, Inc. Fibroblast growth factor-like molecules and uses thereof
WO2003099201A2 (fr) * 2002-05-09 2003-12-04 Curagen Corporation Compositions et procedes d'utilisation d'un facteur de croissance de fibroblastes
EP1773372A2 (fr) * 2004-05-10 2007-04-18 Curagen Corporation Procédés de prévention et de traitement de la mucosite alimentaire

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