WO2006073417A2 - Utilisations prophylactiques et therapeutiques de fgf-20 en protection antirayonnement - Google Patents

Utilisations prophylactiques et therapeutiques de fgf-20 en protection antirayonnement Download PDF

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WO2006073417A2
WO2006073417A2 PCT/US2005/010732 US2005010732W WO2006073417A2 WO 2006073417 A2 WO2006073417 A2 WO 2006073417A2 US 2005010732 W US2005010732 W US 2005010732W WO 2006073417 A2 WO2006073417 A2 WO 2006073417A2
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protein
amino acid
seq
subject
composition
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PCT/US2005/010732
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English (en)
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WO2006073417A3 (fr
Inventor
Enrique Alvarez
William Hahne
Timothy K. Maclachlan
Bisni Narayanan
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Curagen Corporation
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Priority claimed from US10/842,206 external-priority patent/US20050256039A1/en
Priority claimed from US10/980,459 external-priority patent/US20050256042A1/en
Application filed by Curagen Corporation filed Critical Curagen Corporation
Priority to JP2007513141A priority Critical patent/JP2007536382A/ja
Priority to EP05856585A priority patent/EP1789072A2/fr
Priority to CA002566507A priority patent/CA2566507A1/fr
Priority to JP2007540036A priority patent/JP2008519032A/ja
Priority to AU2005306898A priority patent/AU2005306898A1/en
Priority to PCT/US2005/039833 priority patent/WO2006055264A1/fr
Priority to CA002586213A priority patent/CA2586213A1/fr
Priority to EP05851340A priority patent/EP1812460A1/fr
Publication of WO2006073417A2 publication Critical patent/WO2006073417A2/fr
Publication of WO2006073417A3 publication Critical patent/WO2006073417A3/fr

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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]

Definitions

  • the present invention relates to methods of stimulating stem cell proliferation and engraftment, and methods of preventing and/or treating disorders associated with radiation exposure, chemotherapy, exposure to chemical/biological warfare agents, and/or any other insults affecting rapidly proliferating tissues in a body, or one or more symptoms thereof.
  • the present invention provides methods of stimulating stem cell proliferation and/or engraftment and methods of preventing and/or treating disorders associated with one or more insults affecting rapidly proliferating tissues in a body (e.g., radiation exposure, chemical and/or biological insults) or one or more symptoms thereof by administering to a subject a composition comprising a Fibroblast Growth Factor- 20 (FGF-20) protein, or its fragments, derivatives, variants, homologs, analogs, or a combination thereof.
  • FGF-20 Fibroblast Growth Factor- 20
  • the FGF family consists of more than 20 members, each containing a conserved amino acid core (see, e.g., Powers etal., 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 etal., Int. Rev. Cytol., 185:45-106 (1999)).
  • FGF family are involved in various physiological and pathological processes during embryogenesis and adult life, including morphogenesis, limb development, tissue repair, inflammation, angiogenesis, and tumor growth and invasion (see, e.g., Powers etal., Endocr. Relat. Cancer, 7(3):165-197 (2000); or Szebenyi etal., Int. Rev. Cytol. 185:45-106 (1999)).
  • morphogenesis limb development, tissue repair, inflammation, angiogenesis, and tumor growth and invasion
  • FGF-20 Through a homology-based genomic mining process, a new member of the FGF family, FGF-20, has been identified (see U.S. Patent Application Nos. 09/494,585, filed January 13, 2000, and 09/609,543, filed July 3, 2000, the disclosures of which are incorporated herein by reference in their entireties).
  • a single exposure to ionizing radiation can produce immediate effects on tissue through free radical generation and often results in radiation sickness.
  • Radiation-induced lethality depends on dose exposure.
  • Three main syndromes are associated with single-dose exposure to radiation: cardiovascular/central nerve system (CNS) syndrome, gastrointestinal (Gl) syndrome, and hematopoietic (bone marrow) syndrome (see, e.g., Coleman etal., Radiat. Res., 159(6):812-834 (2003)).
  • the time of death, depending on the dose of exposure is within hours for cardiovascular/central nerve system (CNS) syndrome, within 3-10 days for Gl syndrome, and 30-60 days for hematopoietic syndrome.
  • Other symptoms associated with radiation sickness include, but are not limited to, nausea, vomiting, diarrhea, skin burns and sores, fatigue, dehydration, inflammation, hair loss, neutropenia, ulceration of the oral mucosa and Gl system, xerostomia, and bleeding (e.g., bleeding from the nose, mouth and rectum).
  • a common underlying cause for the symptoms associated with radiation sickness is the direct effect of ionizing radiation on stem cell precursor cells.
  • Mitotically active hematopoietic progenitors are unable to divide after a whole body exposure of 2-3 Gy, resulting in lymphopenia, thrombocytopenia, anemia, bone marrow aplastic, followed by infection, bleeding and poor wound healing, which contribute to lethality of the hematopoietic syndrome.
  • Radiation at doses of less than 2 Gy induces mild cytopenias without significant bone marrow damage (see e.g., Geiselhart etai, J. Immunol. 166(5):3019-3027 (2001)).
  • Peripheral blood lymphopenia may develop within the first 6-24 hrs after a moderate to high dose exposure (see e.g., Mackall et al., Blood 97(5): 1491 -1497 (2001)).
  • Neutropenia a decrease in the number of neutrophils resulting in increased risk of infection
  • gastrointestinal mucositis represent two of the most significant causes of mortality resulting from ionizing radiation.
  • Vesicant agents such as mustard gas, like radiation, cause similar effects at the cellular level; their use in combination will have a geometric effect on morbidity.
  • Radioprotectants include, e.g., the protection of personnel involved in accidental exposure to ionizing radiation in, e.g., an industrial, medical, or military setting, and protection of personnel exposed to radiation in the event of nuclear accident or terrorist attack.
  • Acidic FGF increases the survival of C3H/HeCNR mice receiving 840 centi-Grey (cGy) of ionizing radiation to the whole body (see Cytokine, 9(1):59-65 (1997)).
  • Ionizing radiation is named such because of the ability of the radiation particles to generate reactive ions when bombarded against another molecule within the cell or elsewhere.
  • H 2 O As one of the most common molecules within a cell is water, H 2 O, very often the radicals generated are a form of this molecule's breakdown (see, e.g., Int. J. Radiat. Biol, 65:27-33 (1994)).
  • the reactive oxygen species (ROS) include, but are not limited to, superoxide (O 2 " ), hydroxyl (OH) and hydrogen peroxide (H 2 O 2 ) (see Proc. Natl. Acad. Sci. USA, 78:1001-1003 (1981)).
  • the present invention provides methods of preventing or treating one or more disorders associated with radiation exposure(s), chemotherapy, exposure(s) to chemical and/or biological warfare agents with radiomimetic properties, and/or exposure(s) to any other insults affecting rapidly proliferating tissues in a body, or one or more symptoms thereof by administering to a subject in need thereof a composition comprising one or more CG53135 proteins.
  • the disorder to be prevented and/or treated is a disorder of hematopoiesis, including but is not limited to, anemia, leukopenia (e.g., netropenia), thrombocytopenia, pancytopenia, and a clotting disorder.
  • the disorder to be prevented and/or treated is bone marrow failure.
  • the disorder to be prevented and/or treated is graft-versus-host disease.
  • the disorder to be prevented or treated is alimentary mucositis, including but is not limited to, oral mucositis, esophagitis, stomatitis, enteritis, and proctitis.
  • the disorder to be prevented and/or treated is radiation induced prostatitis, vaginitis, and/or urethritis.
  • the disorder to be prevented or treated is a cardiovascular and/or central nervous system syndrome. In some embodiments, more than one disorders described above may be prevented and/or treated by the methods of the invention.
  • 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).
  • CG53135 protects rapidly proliferating tissues, such as hematopoietic and gastrointestinal tissues, from insults such as radiation exposure(s), chemotherapy, and exposure(s) to one or more chemical and/or biological warfare agents, and CG53135 stimulates proliferation and engraftment of stem cells such as hematopoietic stem cells and/or gastrointestinal stem cells. While not limited by any theory, CG53135 is believed to protect stem cells associated with the regenerative capacities of the proliferating tissues from the adverse effects of cytotoxic agents. This general protection ultimately leads to the amelioration of symptoms and/or improvement of morbidity and mortality associated with insults affecting rapidly proliferating tissues.
  • the present invention also provides methods of upregulating oxygen scavenging pathways in a subject, where the methods comprises administering to the subject a composition comprising one or more CG53135 proteins.
  • the oxygen scavenging pathways comprise one or more superoxide dismutases ("SOD”).
  • the oxygen scavenging pathways comprise genes selected from the group consisting of an extracelluar signal regulated kinase ("ERK”), an adhesion related kinase (“AKT'), a superoxide dismutase, cyclooxygenase-2 (“COX-2”), and NF-E2-related factor 2 (“Nrf-2").
  • the present invention further provides methods of stimulating secretion of one or more endogenous cytokines or endogenous chemokines from cells of a subject, where the method comprises administering to the subject a composition comprising one or more CG53135 proteins.
  • the present invention provides methods of stimulating proliferation of hematopoietic stem cells and/or gastrointestinal stem cells of a subject, where the method comprises administering to the subject a composition comprising one or more CG53135 proteins.
  • the present invention also provides methods of optimizing engraftment of hematopoietic stem cells in a subject, where the method comprises administering to the subject a composition comprising one or more CG53135 proteins.
  • the present invention provides a method of protecting and/or regenerating hematopoietic tissues of a subject exposed to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents with radiomimetic properties) comprising administering to the subject a therapeutically effective amount of a composition comprising one or more CG53135 proteins.
  • the present invention provides a method of treating leukopenia (e.g., neutropenia) of a subject exposed to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents with radiomimetic properties) comprising administering to the subject a therapeutically effective amount of a composition comprising one or more CG53135 proteins.
  • leukopenia e.g., neutropenia
  • rapidly proliferating tissues such as radiation, chemotherapy, and chemical/biological warfare agents with radiomimetic properties
  • the present invention provides a method of protecting and/or regenerating gastrointestinal tissues of a subject exposed to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents with radiomimetic properties) comprising administering to the subject a therapeutically effective amount of a composition comprising one or more CG53135 proteins.
  • the present invention provides a method of treating gastrointestinal mucositis of a subject exposed to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents with radiomimetic properties) comprising administering to the subject a therapeutically effective amount of a composition comprising one or more CG53135 proteins.
  • the present invention provides a method of preventing and/or treating a disorder (e.g., alimentary mucositis, bone marrow failure, radiation induced prostatitis, vaginitis and/or urethritis, a disorder of hematopoiesis, or a cardiovascular/central nervous system syndrome) or ameliorating a symptom (e.g., diarrhea, skin burn, sores, fatigue, dehydration, inflammation, hair loss, ulceration of oral mucosa, xerostomia, and bleeding) associated with an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents with radiomimetic properties) comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a composition comprising one or more CG53135 proteins.
  • a disorder e.g., alimentary mucositis, bone marrow failure, radiation induced prostatitis, vaginitis and/or urethritis, a disorder of
  • a composition comprising one or more CG53135 proteins is administered to a subject prior to the subject's exposure to the insult. In another embodiment, a composition comprising one or more CG53135 proteins is administered to a subject after the subject's exposure to the insult, but prior to any disorder associated with the insult, or a symptom thereof developed in the subject. In another embodiment, a composition comprising one or more CG53135 proteins is administered to a subject after one or more disorders associated with the insult or symptoms thereof developed in the subject.
  • a composition comprising one or more CG53135 proteins is administered a subject in need thereof both prior to the development of any radiation associated disorder and/or symptom (e.g., prior to the occurrence of the insult, and/or after the occurrence of the insult but prior to the development of any disorder and/or symptom) and after the development of a radiation associated disorder and/or symptom.
  • a composition comprising one or more CG53135 proteins is administered to a subject who is at risk of exposing to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents with radiomimetic properties).
  • a composition comprising one or more CG53135 proteins is administered to a subject in need thereof no more than 24 hours, 20 hours, 15 hours, 10 hours, or 5 hours prior to the subject's exposure to an insult affecting rapidly proliferating tissues (e.g., radiation, chemotherapy, and chemical/biological warfare agents with radiomimetic properties).
  • a composition comprising one or more CG53135 proteins is administered to a subject in need thereof 3 days, 2 days, 1 day prior to exposure to radiation (day -3, -2, and -1 ), the day exposed to radiation (day 0), and the day after exposure to the radiation (day 1), respectively.
  • composition comprising one or more CG53135 proteins is administered to a subject in need thereof on day -1 , 0, and 1 , respectively.
  • dosing schedules can be used, and such schedules are encompassed by the present invention.
  • the present invention provides a method of improving survival of subjects exposed to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents with radiomimetic properties) comprising administering to the subjects a prophylactically or therapeutically effective amount of a composition comprising one or more CG53135 proteins.
  • the therapeutically effective dose may be a single dose, two doses or more than two doses of a composition comprising one or more CG53135 proteins.
  • a single prophylactic dose of a composition comprising one or more CG53135 proteins is administered to a subject followed by an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents with radiomimetic properties), where such prophylactic dose causes a defined, short acting proliferative effect on various compartments in the proliferating tissues (e.g., intestinal villi).
  • an insult affecting rapidly proliferating tissues such as radiation, chemotherapy, and chemical/biological warfare agents with radiomimetic properties
  • more than a single prophylactic dose which may be two or more than two doses of a composition comprising one or more CG53135 proteins, is administered to a subject exposed to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents with radiomimetic properties) to preventing, treating or ameliorating a symptom associated with the insult.
  • an insult affecting rapidly proliferating tissues such as radiation, chemotherapy, and chemical/biological warfare agents with radiomimetic properties
  • an insult affecting rapidly proliferating tissues is radiation exposure.
  • an insult affecting rapidly proliferating tissues is one or more alkylating agents, one or more vesicant agents (e.g., mustard agents), or one or more other chemotherapeutic agents, or a combination thereof.
  • an insult affecting rapidly proliferating tissues is a radiation exposure in combination with one or more alkylating agents, one or more chemical warfare agents (e.g., mustard agents), or one or more other chemotherapeutic agents.
  • a composition comprising one or more CG53135 proteins is used in combination with one or more other therapies known in the art to prevent, treat, or ameliorate one or more symptoms associated with an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents with radiomimetic properties).
  • therapies known in the art to prevent, treat, or ameliorate one or more symptoms associated with an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents with radiomimetic properties).
  • compositions, formulations, and kits are also encompassed by the present invention.
  • CG53135" 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 (SEQ ID NO: 2).
  • 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 (i.e., 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, ⁇ 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. Table 1 A. Summary of some of the CG53135 family members
  • the term "effective amount” refers to the amount of a therapy (e.g., a composition comprising one or more CG53135 proteins) which is sufficient to reduce and/or ameliorate the severity and/or duration of a disease or disorder associated with an insult affecting a rapidly proliferating tissue (such as radiation, chemotherapy, and chemical/biological warfare agents) or one or more symptoms thereof, prevent the advancement of said disease or disorder, cause regression of said disease or disorder, prevent the recurrence, development, or onset of one or more symptoms associated with the insult, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., prophylactic or therapeutic agent).
  • the term “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 and/or a complimentary strand thereof.
  • hybridizes under stringent conditions describes conditions for hybridization and washing under which nucleotide sequences at least 30% (preferably, 35%, 40%,
  • 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, and at least one wash in 0.2 X SSC, 0.01% BSA.
  • stringent hybridization conditions are hybridization at 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.1 XSSC, 0.2% SDS at about 68°C.
  • stringent hybridization conditions are hybridization in 6XSSC at about 45 0 C, followed by one or more washes in 0.2 X SSC, 0.1 % SDS at 50-65 0 C ⁇ i.e., one or more washes at 50 0 C, 55°C, 60 0 C or 65°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 that 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.
  • in combination refers to the use of more than one therapy. The use of the term “in combination” does not restrict the order in which therapies are administered to a subject in need thereof.
  • a first therapy can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy to a subject in need thereof.
  • the terms "prevent,” “preventing,” and “prevention” refer to the prevention of the recurrence, onset, or development of a disorder associated with an insult affecting a rapidly proliferating tissue 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 therapy e.g., a composition comprising a CG53135 protein
  • prophylactically effective amount refers to the amount of a therapy (e.g., a composition comprising one or more CG53135 proteins) which is sufficient to result in the prevention of the development, recurrence, or onset of a disease or disorder associated with an insult to rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents) 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 one or more CG53135 proteins
  • 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.
  • the subject is a mammal, preferably a human, who has been exposed to or is going to be exposed to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, or chemical/biological warfare agents).
  • the subject is a farm animal (e.g., a horse, pig, or cow) or a pet (e.g., a dog or cat) which has been exposed to or is going to be exposed to a similar insult.
  • farm animal e.g., a horse, pig, or cow
  • pet e.g., a dog or cat
  • subject' is used interchangeably with "patient” in the present invention.
  • the terms “treat,” “treatment,” and “treating” refer to the reduction of the progression, severity, and/or duration of a disorder associated with an insult affecting a rapidly proliferating tissue 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).
  • the term "therapeutically effective amount” refers to the amount of a therapy (e.g., a composition comprising one or more 53135 proteins) that is sufficient to reduce the severity of a disease or disorder characterized by an insult to rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents), reduce the duration of such a disease or disorder, prevent the advancement of such a disease or disorder, cause regression of such a disease or disorder, ameliorate one or more symptoms associated with an insult to rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents), or enhance or improve the therapeutic effect(s) of another therapy.
  • a therapy e.g., a composition comprising one or more 53135 proteins
  • Figure 1 shows Liquid Chromatography and Mass Spectrometry analysis of CG53135-05 E. Co// purified product.
  • Figure 2 depicts tryptic map of CG53135-05 E. coli purified product.
  • Figure 3 shows the effect of CG53135 on mouse survival after single exposure to acute radiation dose of 600 cGy.
  • Figure 4 shows the mean weight change in mice after single exposure to acute radiation of 600 cGy.
  • Figure 5 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
  • Figure 6 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.
  • Figure 7 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.
  • Figure 8 shows the cell positions in the crypt.
  • the bottom of the crypt is cell position 1 , the crypt base.
  • the stem cells are located around cell position 4 and the proliferative cells occupy roughly half of the crypt.
  • the cells are constantly maturing such that the cells are fully differentiated and not cycling at the top of the crypt. Changes that may affect stem cells versus their transit amplifying daughter cells can be detected by examining changes in event (labeling, apoptosis, mitosis, etc.) frequency at each cell position.
  • Figure 9 shows a survival curve of intestinal crypt cells from mice prophylactically administered CG53135 or PBS, following different radiation dosages.
  • Figure 10 shows the effect of prophylactic administration of CG53135 on mice intestinal crypt survival after radiation insult.
  • Figure 11 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.
  • Figure 12 shows effect of CG53135 multiple-dose administration on crypt survival curves.
  • IP intraperitoneal
  • the plot represents the level of protection of crypt cells in response to treatment schedule. Protection factor value indicates the number of surviving crypts per circumference in the CG53135-05-treated animals compared to PBS, expressed as a ratio.
  • Figure 13A and 13B show CG53135 induced expression of scavengers, cycloxegenase, trefoil factor, and transcription factors in NIH 3T3 cells.
  • Figure 14 shows CG53135 induced expression of scavengers, cycloxegenase, trefoil factor, and transcription factors in CCD1070sk cells.
  • Figure 15 shows CG53135 induced expression of scavengers, cycloxegenase, trefoil factor, and transcription factors in CCD18Co cells.
  • B shows activation of ERK and AKT kinases by CG53135.
  • Figure 16 shows CG53135 induced expression of scavengers, cycloxegenase, trefoil factor, and transcription factors in human umbilical vein endothelial cells (HUVEC).
  • Figure 17 shows the effect of CG53135 on the survival of IEC 18 cells irradiated with different X-ray doses.
  • Figure 18 shows the effect of CG53135 on the survival of NIH 3T3 cells irradiated with different X-ray doses.
  • Figure 19 shows the effect of CG53135 on the survival of HUVEC irradiated with different X- ray doses.
  • Figure 20 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
  • 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.
  • Figure 21 (A) shows the effect of CG53135 on the release of cytokine in NIH 3T3 cells.
  • (B) shows IL-6 and IL-11 expression in response to CG53135.
  • Figure 22 shows dose response of CM-HaDCFDA fluorescence from IEC18 cells treated with CG53135 after 4Gy irradiation.
  • Figure 23 shows response of CM-H 2 DCFDA fluorescence from IEC18 cells treated with CG53135 after 2Gy and 4Gy irradiation.
  • Figure 24 shows dose response of CM-H 2 DCFDA fluorescence from CCD-18Co cells treated with CG53135 after 4Gy irradiation.
  • Figure 25 shows dose response of Red CC-1 fluorescence from IEC18 cells treated with CG53135 after 4Gy irradiation.
  • Figure 26 shows response of Red CC-1 fluorescence from IEC18 cells treated with CG53135 after 4Gy and 6Gy irradiation.
  • Figure 27 shows response of Red CC-1 fluorescence from CCD-I8C0 cells treated with CG53135 before and after 10Gy irradiation.
  • Figure 28 shows in vitro radioprotection of the myeloid cell line 32D by CG53135.
  • Figure 29 shows effect of CG53135 on repopulation of thymus following bone marrow ablation and subsequent bone marrow transplant.
  • Figure 30 (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.
  • Figure 31 (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.
  • Figure 32 shows analysis of diarrhea score for each day of observation.
  • Figure 33 shows the relative loss in body weight per group for study 439.
  • Figure 34 (A) shows the average diarrhea score over 3 days.
  • Figure 34 (B) shows the mean diarrhea severity. 5.
  • the present invention provides methods of stimulating stem cell proliferation and/or engraftment and methods of preventing and/or treating disorders associated with one or more insults affecting rapidly proliferating tissues in a body (e.g., radiation exposure, chemical and/or biological insults) or one or more symptoms thereof by administering to a subject a composition comprising one or more CG53135 proteins.
  • CG53135" 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 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
  • Additional examples of FGF-20 SNPs can be found in Example 2 of U.S. Patent Application No. 10/435,087, filed May 9, 2003, the content of which is incorporated by reference by its entirety.
  • 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 a CG53135 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 a CG53135 protein fragment includes, but is not limited to, residues 2-211 , 3-211 , 9-211 , 12-211 , 15-211 , 24-211 , 52-211 , 54-211 , 55-211 , 63-196, 63-211 , or 63-194 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 , 52-211 , 54-211 , 55-211 , 63-196, 63-211 , or 63-194 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. In one embodiment, such alteration of an FGF-20 sequence is done in a region that is not conserved in the FGF protein family.
  • nucleotide coding 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 that 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 that 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.
  • a derivative or an analog of FGF-20 can also be made according to the methods described in, e.g., PCT Publication No. WO 2004/018499 A2, and US Publication Nos. US 2004/0087505 A1 , and US 2004/0038348 A1 (the content of each is incorporated herein by reference in its entirety).
  • the FGF-20 derivatives and analogs of the invention can be produced by various methods known in the art.
  • the manipulations that 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. era/., 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.
  • 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
  • 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 an extended half-life in vivo using any methods known in the art.
  • 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), CG53135-15 (SEQ ID NOs:27 and 28), CG53135-16 (SEQ ID NOs:29 and 30), CG53135-17 (SEQ ID NOs:31 and 32), IFC 250059629 (SEQ ID NOs:33 and 34), IFC 20059669 (SEQ ID NOs:35 and 36), IFC 317459553 (SEQ ID NOs:37 and 38), IFC 317459571 (SEQ ID NOs:39 and 40), IFC 250059596 (SEQ ID NOs:41 and 10), IFC316351224 (SEQ ID NOs:41 and 10), or a combination thereof.
  • any techniques known in the art can be used in purifying a CG53135 protein, including but 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., gel
  • CG53135 proteins employed in a composition of the invention can be in the range of 80 to 100 percent of the total mg protein, or at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of the total mg protein.
  • one or more CG53135 proteins employed in a composition of the invention is at least 99% of the total protein.
  • CG53135 is purified to apparent homogeneity, as assayed, e.g., by sodium dodecyl sulfate polyacrylamide gel electrophoresis.
  • 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 that 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 (ATG) 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.
  • 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 et al., 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 B.
  • 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 cytomegalocirus for effective expression of a CG53135 sequence (Foecking etal., 1986, Gene 45:101 ; and Cockett etal., 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 that direct the expression of high levels of readily purified fusion protein products 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.
  • These 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 that 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.
  • mammalian host cells include, but are not limited to, PC12, CHO, VERY, BHK, HeIa, COS, MDCK,
  • CG531335 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. For long-term, high-yield production of properly processed CG53135, stable expression in cells is preferred.
  • 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 arid media may also be used to enhance production of CG53135.
  • CG531335 Any techniques known in the art may be applied to establish the optimal conditions for producing CG53135.
  • An alternative to producing CG53135 or a fragment thereof by recombinant techniques 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. During synthesis, 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.
  • 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.
  • Non-limiting examples of methods for preparing CG53135 can be found in Section 6, infra. 5.3. Prophylactic and Therapeutic Uses of CG53135
  • 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 ail 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. Messenchymal 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 mmarrow-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
  • 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, 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 proliferating 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 proliferating 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.
  • 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.
  • 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.
  • Toxicity and therapeutic efficacy of a composition of the 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 of LD50/ED50.
  • Compositions that exhibit large therapeutic indices are preferred. While compositions that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such composition 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 for use in humans.
  • the dosage of complexes 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, the route of administration utilized, the severity of the disease, age and weight of the subject, and other factors normally considered by a medical professional (e.g., a physician).
  • 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 cultures. Such information can be used to more accurately determine useful doses in humans. 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 CG53135 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, at least 10 mg/kg, at least 25 mg/kg, at least 50 mg/kg, at least 75 mg/kg, or at least 100 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 0.001-100 mg/kg, between 0.001 -50 mg/kg, between 0.001-25 mg/kg, between 0.001-10 mg/kg, between 0.005-5 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.
  • a composition comprising one or more CG53135 proteins is administered to a subject in a single dose to prevent a disorder associate with an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents) or ameliorate one or more symptoms thereof.
  • a disorder associate with an insult affecting rapidly proliferating tissues such as radiation, chemotherapy, and chemical/biological warfare agents
  • such composition is administered no more than 24 hours, no more than 20 hours, no more than 15 hours, no more than 10 hours, or no more than 5 hours prior to the exposure to the insult.
  • a composition comprising one or more CG53135 proteins is administered to a subject in two or more doses to prevent and/or treat a disorder associate with an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical/biological warfare agents) or ameliorate one or more symptoms thereof.
  • a composition is preferably administered to a subject both before exposure to an insult (such as radiation, chemotherapy, and chemical/biological warfare agents) and after exposure to an insult (such as radiation, chemotherapy, and chemical/biological warfare agents).
  • such composition is administered 3 days prior, 2 days prior, 1 day prior to the insult, and on the day of insult, and 1 day after the insult, respectively.
  • chemotherapeutic agents such as chemotherapeutic agents, radiation therapy and biological/immunotherapeutic agents such as cytokines
  • cytokines are known in the art and described in such literature as the Physician's Desk Reference (58th ed., 2004).
  • compositions Various delivery systems are known and can be used to administer a composition used in
  • 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, transmucosal, rectal, and oral routes.
  • compositions used in accordance to the methods 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., eye mucosa, oral mucosa, vaginal 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.
  • administration can be by direct injection at the site (or former site) of rapidly proliferating tissues that are most sensitive to an insult, such as radiation, chemotherapy, or chemical/biological warfare agent.
  • the nucleic acid can be administered in vivo to promote expression of their encoded proteins (e.g., CG53135 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 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 nucleic acid of the invention 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 various salt forms, 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.
  • a composition comprising CG53135 is to be administered topically, 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, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.
  • a topical delivery agent such as but not limited to, lipids, liposomes, 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.
  • 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. 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); nonaqueous 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-lysine, 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.
  • 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.
  • 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, as an emulsion in an acceptable oil
  • 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.
  • the ingredients of the compositions used in accordance to the methods of the invention are derived from a subject that is the same species origin or species reactivity as recipient of such compositions.
  • a formulation used in accordance to the methods of the invention comprises 0.02 M - 0.2 M acetate, 0.5-5% glycerol, 0.2-0.5 M arginine-HCI, and one ore more CG53135 proteins, preferably 0.5-5 mg/ml (UV).
  • a formulation used in accordance to the methods of the invention comprises 0.04M sodium acetate, 3% glycerol (volume/volume), 0.2 M arginine-HCI at pH 5.3, and one or more isolated CG53135 proteins, preferably 0.8 mg/ml (UV).
  • a formulation used in accordance to the methods of the invention comprises 0.01-1 M of a stabilizer, such as arginine in various salt forms, 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 CG53135 proteins, preferably 0.005-50 mg/ml (UV).
  • a stabilizer such as arginine in various salt forms, sulfobutyl ether Beta-cyclodextrin sodium, or sucrose
  • 0.01 %-0.1% weight/volume (“w/v”) polysorbate 80 or polysorbate 20 0.01 %-0.1% weight/volume
  • CG53135 proteins preferably 0.005-50 mg/ml (UV).
  • a formulation used in accordance to the methods of the invention comprises 3OmM sodium citrate, pH 6.1 , 2mM EDTA, 20OmM sorbitol, 5OmM KCI, 20% glycerol, and one or more isolated CG53135 proteins.
  • 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 (e.g., a composition comprising one or more CG53135 proteins) 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 kit of the invention further comprises a needle or syringe, preferably packaged in sterile form, for injecting the formulation, and/or a packaged alcohol pad. 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 (e.g., a composition comprising one or more CG53135 proteins) sufficient for a single administration.
  • a pharmaceutical formulation or composition comprising a dose of the composition of the invention (e.g., a composition comprising one or more CG53135 proteins) sufficient for a single administration.
  • compositions of the invention are stored in containers with biocompatible detergents, including but not limited to, lecithin, taurocholic acid, and cholesterol; or with other proteins, including but not limited to, gamma globulins and serum albumins.
  • the products of the invention include instructions for use or other informational material that advise the physician, technician, or patient on how to appropriately prevent or treat the disease or disorder in question.
  • Recombinant human CG53135 was purified from Escherichia coli BLR (DE3) cells (Novagen, Madison, Wl) as follows: the DE3 cells were transformed with full-length, codon-optimized CG53135- 05 cloned in a 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 two 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, and 20% glycerol).
  • Vehicle (without CG53135 protein) contains 3OmM sodium citrate, pH 6.1 , 2 mM EDTA, 20OmM sorbitol, 5OmM KCI, 20% glycerol.
  • the final purified protein product includes some truncated form of FGF-20 (e.g., 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)) in addition to the full length FGF-20, and a protein consisting of amino acid 3-211 (CG53135-13, SEQ ID NO:24) of FGF-20 constitutes the majority of the final purified protein product. All the variants/fragments in the final purified product have high activity in the proliferation assays.
  • FGF-20 e.g., 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)
  • CG53135-05 E. coli purified product is used herein to refer to a purified protein product from E. coli expressing a CG53135-05 construct.
  • a CG53135-05 E. 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).
  • a CG53135 E. coli purified product may also contain one or more carbamylated CG53135 proteins.
  • Purified drug substance (by both Process 1 and Process 2, respectively, see Section 6.19, infra) 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 ( Figure 1 ).
  • 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 3 below.
  • the predominant sequence for each reference standard was identical and corresponded to residues 3-20 in the theoretical N-terminal sequence of CG53135-05.
  • 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 0 C in 100 ⁇ L of 6 N HCI, 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 4 below. Note that Cys and trp are destroyed during acid hydrolysis of the protein. Asn and gin are converted to asp and glu, respectively, during acid hydrolysis and thus their respective totals are reported as asx and glx. Met and his were both unresolved in this procedure.
  • 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 L ) V and electrospray mass spectrometric detection.
  • 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 absorption at 214 nm (Figure.
  • 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 5).
  • the PI 20O 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 Pl 200 of 0.7 - 11 ng/mL (Table 5).
  • mice Irradiation of the animals was performed at the Brigham and Women's Hospital in Boston, MA. The studies were conducted at The Massachusetts College of Pharmacy, Boston, MA. Animals were randomly divided into 4 groups with a control group of 15 mice and 3 test groups of 15 mice each
  • mice were exposed to a single dose of 600 cGy of total body ionizing radiation.
  • CG53135- 05 (12 mg/kg) was administered intraperitoneally (IP) daily on Days 2-3 after radiation, Days 4-7 after radiation, or Days 2 and 1 (-2 and -1 ) before radiation, respectively.
  • Mortality (animal number, day of death) was recorded in each group.
  • mice with CG53135-05 on Days -1 and -2 prior to total body irradiation are effective in radioprotection and also supports the radioprotective effect on gastrointestinal or hematopoietic tissues from the damaging effects of total body irradiation.
  • 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.
  • Figure 6C shows probit analysis for survival over the range of doses.
  • the LD 50Z30 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
  • 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 Figure 8. 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 (Bradford, IP) dose of the 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. Two further groups of mice were used to assess effects on stem cell radiosensitivity. One group was treated with the CG53135-05 E. coli purified product (12 mg/kg, Bradford single injection, IP) and another group was injected with a placebo control.
  • mice Twenty-four hours post injection, animals were irradiated with 1 Gy 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).
  • mice were weighed and then dosed with the CG53135-05 £. coli purified product (12 mg/kg, Bradford, single injection, IP). Groups of 6 animals were sacrificed 0, 3, 6, 9, 12, 24, 48 hours post injection with CG53135-05. All received a single injection of bromodeoxyuridine 40 minutes prior to sacrifice (see Table 8).
  • mice An additional two groups of 6 mice were used to assess the effects of CG53135-05 on stem cell radiosensitivity (groups 8 and 9, see Table 8).
  • One group was treated with CG53135-05 (12 mg/kg Bradford, single injection, ip) and one group was injected with a placebo control. 24 hours post injection, animals were irradiated with 1 Gy X-ray and sacrificed 4.5 hours later.
  • 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 the CG53135-05 E. coli purified product over a 48 hour period.
  • Groups 8-9 (Group B in the results) were tested to determine whether the CG53135-05 E. coli purified product changes the number of apoptotic cells generated after low dose irradiation, i.e., whether the 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 the CG53135-05 E. coli purified product. Apoptosis, mitotic index, and proliferation were the end points for this study.
  • mice were weighed and then dosed with the CG53135-05 E. coli purified product (12 mg/kg) or placebo. A single injection was given, intraperitoneal ⁇ (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.
  • the average crypt width (measured at its widest point) was also measured in order to correct for scoring errors due to crypt size difference. The correction was applied as follows:
  • 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).
  • the crypt survival following prophylactic administration of the CG53135-05 E. coli purified product showed inverse correlation to the irradiation dose, that is, the smaller the radiation dose, the higher the crypt survival ( Figures 9 and 10).
  • Prophylactic administration of the CG53135-05 E. coli purified product significantly increased the number of crypts (P ⁇ 0.001).
  • Table 11 shows the protection factor achieved for the radiation doses following prophylactic administration of the protein (the CG53135-05 E. coli purified product). Protection factor (Table 11) represents the ratio of surviving crypt cells between treated and untreated cells. On average, 1.55 times as many cells survived irradiation dose of 12 Gy, when animals were administered with the CG53135-05 E. coli purified product prior to the radiation insult.
  • the objective of this study was to evaluate the ability of CG53135 to protect against radiation- induced intestinal crypt cell mortality in vivo when administered once daily for 4 days prior to irradiation.
  • CG53135-05 E. coli purified product (12 mg/kg) or PBS was administered to BDF1 mice intraperitoneal ⁇ (IP) once daily for 4 consecutive days prior to exposure to lethal radiation doses from 10-14 Gy on Day 0.
  • IP intraperitoneal ⁇
  • the number of surviving regenerating crypt foci was measured 4 days after • irradiation. Protein concentrations in this example were measured by Bradford assay.
  • Example 7 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. Protein concentrations in this example were measured by UV absorbance.
  • CG53135-05 E. coli purified product (4 mg/kg) or phosphate-buffered saline (PBS) was administered to BDF1 male mice by intraperitoneal ⁇ (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 measured 4 days after irradiation and the dose modification factor (DMF) were calculated. Single dose administration of CG53135-05 on Day -1 resulted in a DMF of 2.3 ( Figure 12).
  • CG53135 is a Gl crypt cell radioprotectant with prophylactic and intervention (treatment) properties.
  • Example 8 Radioprotective Mechanisms of CG53135 Among the many changes that occur in a cell upon an attack of ionizing radiation, an increase of reactive oxygen species occurs via the ionization of H 2 O. As this process produces the most reactive molecules within the cell, in order to reduce cellular damage, the nucleus increases transcription of enzymes that scavenge these radicals to less reactive intermediates. As 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.
  • NIH3T3 murine fibroblast
  • CCD-1070sk human foreskin fibroblast
  • CCD-18Co human colonic finbroblast
  • HUVEC human umbilical cord vascular endothelial cells
  • MnSOD radioprotective superoxide dismutases
  • CCD18C0 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. Both ERK and Akt kinases were active by 2 minutes of treatment with the CG53135-05 E. coli purified product ( Figure 15). The activation of these kinases, particularly Akt, has been associated with radioprotective events.
  • 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 + 0.1U/ml Insulin + 50 ⁇ g/ml Pennicilin/Streptomycin; FaDu cells were grown in MEM + 10% FBS + 1mM Sodium Pyruvate + 50 ⁇ g/ml Pennicilin/Streptomycin + Nonessential amino acids. Cells were plated at a density of 5x10 5 per 10 cm dish (NIH3T3) or 5x10 5 per well of a 6-well dish (IEC18, IEC6, FaDu) and allowed to attach. Cells were then treated with the
  • CG53135-05 E. coli purified product at doses of 10 or 100 ng/ml (IEC18, IEC6, FaDu) or at 50 and 200 ng/ml (NIH 3T3) in basal media containing 0.1% serum (IEC18, IEC6, FaDu) or 1% serum (NIH 3T3) and incubated for 16 hours (IEC18, IEC6, FaDu) or 1 hour (NIH 3T3).
  • Cells were then irradiated using a Faxitron X-ray irradiator (Wheeling, IL) fitted with a 0.5 mm aluminum filter at 2.5, 5, 7.5, 10, 12.5 and 15 Gy at 13OkVp, resulting in a radiation rate of 50 cGy/min.
  • 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 IEC18 cells is shown in Figure 17. While the DO and D1 values showed no obvious treatment-related trends, the Dq value indicates that IEC18 cells treated with CG53135 are more protected from killing at the lower doses of radiation compared to untreated cells (shoulder of survival curves of cells treated with 10ng/ml and 100ng/ml CG53135 is broader).
  • treatment of IEC18 cells with the CG53135-05 E. coli purified product results in cell killing at a higher dose of radiation compared to untreated cells, indicating that CG53135 acts as a radioprotectant in these cells.
  • CG53135 The effect of CG53135 treatment on survival of irradiated NIH 3T3 cells is shown in Figure 18.
  • the DO values for NIH 3T3 cells treated with 50 ng/ml or 200 ng/ml CG53135 appear larger than the DO value for untreated cells, and the difference approaches significance for the 50 ng/ml dose.
  • CG53135 may act as a radioprotectant, promoting survival of NIH 3T3 cells at the higher doses of radiation.
  • the D1 value for cells treated with 100 ng/mL CG53135 is smaller than for untreated cells, indicating a slower rate of the cell death at lower doses of radiation. No trend in Dq values of the survival curves could be determined, largely due to the variation of survival in untreated cells.
  • the effect of CG53135 treatment on survival of irradiated HUVEC cells is shown in Figure 19.
  • 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.
  • treatment of HUVEC cells with 100 ng/ml CG53135-05 E 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 E. 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 the D 0 parameter reflects the rate of killing at high doses of radiation.
  • An increase in these parameters 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 responses. Ionizing radiation can trigger a series of changes in gene expression and cytokine profiles. The aim of this study was to evaluate the cytokine profile upon CG53135 treatment in cell culture over a time course.
  • BioPlex cytokine assays which are multiplex bead based assays designed to quantitate multiple cytokines from tissue culture supernatants, were used for detecting the cytokines. The principle 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). The CG53135-05 E.
  • coli purified product at 10 ng/ml or 100 ng/ml, was added to the cells. The cell supernatant was collected after 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, and 24 hours. Fifty ng of TNF was used as a positive control. Bioplex 18-Plex Cytokine Assay (BioRad Laboratories Inc, CA) was performed following the procedure of the manufacturer.
  • Figure 21 shows the effect of the CG53135-05 E. coli purified product on Mo KC release.
  • 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.
  • CCD18Co 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 concentration by Luminex or ELISA respectively.
  • Figure 21 B 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 CXCL1 acts synergistically in radioprotection, both in vitro and in vivo.
  • induction of other cytokines e.g., IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, MCP-1 , GM-CSF, RANTES
  • cytokines e.g., IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, MCP-1 , GM-CSF, RANTES
  • Cells with increased reactive oxygen species upregulate the Superoxide Dismutases - Cu, Zn-SOD, Mn-SOD, and Extracellular-SOD to scavenge the superoxide radical to hydrogen 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'- dichlorodihydrofluorescein diacetate, also known as CM-H 2 DCFDA, is efficiently retained within the cell and fluoresces green when oxidized by H 2 O 2 .
  • Cu, ZnSOD 5-(and-6)-chloromethyl-2',7'- dichlorodihydrofluorescein diacetate
  • IEC18 rat intestinal epithelial
  • CCD-I8C0 human colonic fibroblast cells
  • the cells were switched to medium containing 0.1% serum and the indicated dose of CG53135.
  • the cells were then irradiated at 2 or 4 Gy with X-rays using a Faxitron X-irradiator (Wheeling, IL), followed 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 Jose, CA) on the FL1 channel.
  • Results indicate that IEC18 and CCDI8C0 cells possess increased intracellular H 2 O 2 after treatment with the CG53135-05 E. coli purified product in a dose responsive manner ( Figures 22, 23 and 24). This is believed to be due to enhanced expression of Superoxide dismutases, predominantly MnSOD induced by the CG53135-05 E. coli purified product. Production of intracellular H 2 O 2 by the CG53135-05 E. coli purified product in IEC18 cells is enhanced with increasing dose of ionizing radiation ( Figure 23). This result reflects the increased production of more reactive oxygen species such as superoxide and hydroxyl by radiation, thus increasing substrate for the Superoxide Dismutases induced by the CG53135-05 E. coli purified product. Red CC-1 method
  • Redox Sensor 1 can monitor the redox level of the cytosol upon oxidation by changing to a red fluorescent agent that can be measured by FACS on the FL2 channel.
  • IEC18 rat intestinal epithelial
  • CCD-I8C0 human colonic fibroblast cells
  • the cells were switched to medium containing 0.1% serum and the indicated dose of the CG53135-05 E. coli purified product.
  • the cells were then irradiated at 2 or 4 Gy with X-rays using a Faxitron X- irradiator, followed by incubation with 5 mM Red CC-1 (Molecular Probes, Eugene, OR) for 15 minutes.
  • the cells were then washed, trypsinized, and analyzed on a Becton Dickinson FACSCalibur on the FL2 channel.
  • IEC18 and CCD18Co cells were found to possess decreased cytosolic redox potential after treatment with CG53135-05 in a dose responsive manner as shown in Figures 25, 26, and 27.
  • 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 to increase expression of a key antioxidant-controlling transcription factor, Nrf2, which may contribute 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 experiment using the myeloid cell line 32D.
  • 32D cells were irradiated at 0, 1 , 2, 3, 4 or 5 Gy then plated in methylcellulose-containing growth media including 10 ng/ml IL-3 with or without 100 ng/ml CG53135- 05 E. coli purified product. Cells were allowed to form colonies for 10 days and were then scored.
  • Figure 28 shows increased survival of 32D cells upon exposure to the CG53135-05 E. coli purified 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.
  • the qualities of the curve i.e., D1 , Dq and DO, indicating at what points radioprotection are observed, were derived using the methods described in Hall et al.,
  • 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 are dosed with 0, 16 or 24 mg/kg IV (UV) of the CG53135-05 E. coli purified product on -3, -2, -1 , 0 and +1 days revolving around the day of irradiation with 60 mice per group.
  • the day -3, -2, -1 , 0 and +1 CG53135 dosing schedule has provided the most consistent results in both the survival study and thymus reconstitution models described in previous sections, supra. Twenty mice from each group are irradiated on day 0 with 3, 4, or 5 Gy X-ray at a dose rate of 1Gy/minute.
  • the optimal dose and schedule for IV administration of CG53135 can also be tested in a nonhuman primate radiation model for hematopoietic injury. Historically, studies involving radiation protection have utilized the male rhesus monkey, Macaca mulatta. Five groups of five animals are used. Based on activity in the rodent model, a dose of the CG53135-05 E. coli purified product is chosen and the equivalent dose in monkey is identified. This optimal dose is bracketed with one higher and one lower dose.
  • the schedule of CG53135 dosing (day -3, -2, -1 , 0, +1) is identical to the schedule used in the rodent study.
  • Mononuclear cells are isolated by density centrifugation and placed in methylcellulose-based media containing cytokines (GM-CSF, erythropoietin, IL-3, c-kit, etc.) to identify the presence and health of myeloid progenitors.
  • cytokines GM-CSF, erythropoietin, IL-3, c-kit, etc.
  • Clinical support including antibiotics, fresh irradiated whole blood and fluids, is provided to the animals throughout the experiment as needed.
  • Antibiotics i.e., Gentamicin, Baytril
  • Gentamicin i.e., Gentamicin, Baytril
  • an absolute neutrophil count i.e., 500/ ⁇ l.
  • fresh irradiated whole blood is administered at approximately 30 ml/transfusion.
  • At the end of the study animals are euthanized, and bone marrow, spleen, liver and jejunum are harvested for histopathological examination. This study should determine the optimal dose for this schedule and route of administration for nonhuman primates, with the objectives of demonstrating clinical benefit of CG53135 treatment in a second animal species and predicting the optimal dose for humans.
  • Example 15 Direct Effect of CG53135 on Hematopoietic and Mesenchymal Progenitors From Irradiated Human Bone Marrow
  • Fresh bone marrow obtained from normal human donors is purchased from a commercial vendor. Mononuclear cells are isolated by density centrifugation, then transferred to tissue culture dishes containing Iscove's modified Dulbecco's medium with or without CG53135-05 E. coli purified product at concentrations of 10, 100 and 500 ng/ml and incubated for one hour. Cells will then be irradiated with 0, 1 , 2 or 4 Gy using an X-ray radiation source.
  • Irradiated cells are then transferred at the appropriate densities to dishes containing methylcellulose-based media containing hematopoietic stem cell factors c-kit and IL-3 as well as the factors GM-CSF and erythropoietin to allow differentiation along the granulocytic and erythrocytic lineages, respectively.
  • CG53135-05 E. coli purified product is added to the media at a concentration of 100 ng/ml. This dosing schedule will be used to determine whether CG53135 is active in pre-treatment, post treatment, or both with respect to irradiation as follows:
  • Cells are grown for 10 days and are scored for colonies representative of erythroid lineage (BFU-E, CFU-E), granulocytic/macrophage lineage (CFU-GM), or a progenitor of both lineages (CFU- GEMM).
  • a subset of irradiated cells are placed in media consisting of Iscove's modified Dulbecco's media, 25% equine serum and hydrocortisone to allow the growth of stromal cells, and are subjected to a similar treatment protocol as described above. After 2 weeks, colonies are stained and scored for morphologies resembling fibroblasts or mixed fibroblast/adipocyte colonies. 6.16.
  • Example 16 Indirect Effect of CG53135 on Bone Marrow Repopulation Through Stimulation of Bone Marrow Stromal Response
  • CG53135 imparts a protective or proliferative effect on the bone marrow stem cells by, among other mechanisms, stimulating fibroblasts within the marrow stroma to secrete factors that facilitate the health and proliferative capacity of hematopoietic stem cells.
  • human marrow stromal cells are purchased and plated out in 96-well dishes.
  • the CG53135-05 E. coli purified product is added to the medium at concentrations of 10, 100 and 500 ng/ml for time periods of 0, 1 , 4, 8, 24, and 48 hours.
  • cytokine release After incubation, the conditioned media is removed and analyzed for cytokine release using a Bio-Rad Bio-Plex to screen for 17 human cytokines, as well as ELISA to test for additional known radioprotectants such as IL-1 ⁇ and IL-11 , and known stimulants of hematopoietic expansion such as IL-3 for stem cells and IL-7 for T- cells.
  • CG53135 has been demonstrated to have a proliferative effect on mouse and human fibroblasts (Jeffers et.al., 2001). Bone marrow stromal cells are tested for their ability to replicate in response to CG53135 treatment.
  • CG53135 may be able to create conditions wherein surviving marrow stem cells can proliferate and expand. This can be tested in vitro by measuring the expansion of pluripotent CD34 + cells on stroma that has been treated with CG53135. Stromal cells are plated at subconfluent densities and either treated with CG53135 or left untreated for 24 hours.
  • CD34 + cells are then plated on top of the fibroblast layer and allowed to grow for 2, 4 or 6 days. At each time point, floating cells are collected and counted to measure proliferation, as well as stained for CD34 and Lin expression to ascertain that the cells have not differentiated.
  • EXAMPLE 17 CG53135 REDUCES THE INCIDENCE. LENGTH AND SEVERITY OF RADIATION-INDUCED DIARRHEA (N-438)
  • 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.
  • 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. ( Figure 31 (A) and (B)) An analysis of for each day of observation was also made to determine differences at days of peak diarrhea. Significance was determined as described above ( * - P ⁇ 0.05, ** - P ⁇ 0.01 , *** - P ⁇ 0.001). ( Figure 32)
  • EXAMPLE 18 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 -8O 0 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.
  • mice 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 acclimatise 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.
  • 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 14Gy X 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:
  • 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.
  • 16mg/kg CG53135-05 consistently reduced the diarrhea severity in mice exposed to 14Gy X irradiation. Dosing day 0 only or days -1 , 0, +1 yielded the best protection when examined by a variety of parameters. Further dosing (Days 0, 1 ; Day 0, +1 , +2; and Day 0, +1 , +2, +3) provided no extra benefit. Dosing 0, 6, 12 and 18 hours post irradiation also failed to reduce the total diarrhea severity.
  • untagged molecules were generated in a phage-free bacterial host.
  • the codon-optimized, full-length, untagged molecule (CG53135-05) has the most favorable pharmacology profile and was used to prepare product for the safety studies and clinical trials.
  • PRODUCTION PROCESS AND PHARMACEUTICAL FORMULATIONS PROCESS 1
  • 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 ⁇ M 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. Formulation and Fill/Finish
  • vial vial
  • 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 24).
  • 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.
  • PI 20O concentration of CG53135-05 that results in incorporation of BrdU at 2 times the background
  • 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°C with samples tested at intervals of 3, 6, 9, 12, and 24 months.
  • 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 0 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. This loss of acetic acid to lyophilization increases the pH to > 7.5, which is far from the target pH of 5.3. Secondly, the glycerol has a collapse temperature of ⁇ -45°C, which renders this formulation not be able to be lyophilized commercially. Most of the commercial lyophilizers have a shelf temperature ranged from -45 0 C to - 5O 0 C with temperature variation of ⁇ 3 0 C.
  • 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.
  • the solubility was lower as there was not sufficient protein in the experiment to be dissolved Table 28.
  • the use of the sulfate salt of arginine increases the solubility by at least 2 to 6-fold.
  • All formulation contains 0.2 M Arginine.
  • the optimal concentration of the sodium phosphate as a buffering salt was observed (Table 29).
  • 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, or 100 mM.
  • Table 30 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 E. coli purified product in 0.2 M Arginine and 0.05 M sodium phosphate buffer at pH 7.0. After exchanging with 7 volumes of the final buffer (0.5 M 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 buffer 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; with a projected shelf-life of at least 18 months (2) the solubility of CG53135 is increased, allowing concentrations of > 30 mg/mL to be obtained; and (3) the lyophilized product has a collapse temperature of -3O 0 C which can be easily lyophilized by the commercial equipment.
  • the interactions between arginine, sulfate, phosphate, and surfactant and CG53135 were unexpected.
  • MMCB Manufacturing Master Cell Bank
  • MWCB Manufacturing Working Cell Bank
  • Inoculum Preparation the initial cell expansion occurs in shake flasks. Seed preparation is done by thawing and pooling 2 -3 vials of the MWCB in chemically defined medium and inoculating 3 - 4 shake flasks each containing 500 ml. of chemically defined seed medium. 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 OD600 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 0 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 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 1 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.
  • 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.
  • 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 transported to Formatech Inc, MA from Diosynth - RTP, NC 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.
  • the CG53135-05 reference standard was prepared at Diosynth RTP Inc, using a 140L scale manufacturing process that was representative of the bulk drug substance manufacturing process (as described in the General Method of Manufacture).
  • the reference standard was stored as 1 mL aliquots in 2 mL cryovials at -80 0 C ⁇ 15 0 C.
  • Such alterations and changes may include, for example, different pharmaceutical compositions for the administration of the proteins according to the present invention to a mammal; different amounts of protein in the compositions to be administered; different times and means of administering the proteins according to the present invention; and different materials contained in the administration dose including, for example, combinations of different proteins, or combinations of the proteins according to the present invention together with other biologically active compounds for the same, similar or differing purposes than the desired utility of those proteins specifically disclosed herein.
  • Such changes and alterations also are intended to include modifications in the amino acid sequence of the specific desired proteins described herein in which such changes alter the sequence in a manner as not to change the desired potential of the protein, but as to change solubility of the protein in the pharmaceutical composition to be administered or in the body, absorption of the protein by the body, protection of the protein for either shelf life or within the body until such time as the biological action of the protein is able to bring about the desired effect, and such similar modifications. Accordingly, such changes and alterations are properly intended to be within the full range of equivalents, and therefore within the purview of the following claims.

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Abstract

L'invention concerne des procédés permettant de stimuler la prolifération et la prise de greffe de cellules souches, et des méthodes permettant de prévenir et/ou de traiter des troubles associés à une exposition à des rayonnements, à la chimiothérapie, à une exposition à des agents de conduite de guerre chimiques/biologiques et/ou à d'autres agressions quelconques affectant des tissus corporels à prolifération rapide, ou à un ou plusieurs symptôme(s) de celles-ci. L'invention concerne, en particulier, des méthodes permettant de stimuler la prolifération et la prise de greffe de cellules souches, et des méthodes permettant de prévenir et/ou de traiter des troubles associés à des agressions affectant des tissus corporels à prolifération rapide (par exemple, exposition à des rayonnements, agressions chimiques/biologiques) ou à un ou plusieurs symptôme(s) de celles-ci par administration à un sujet d'une composition comprenant une protéine de facteur de croissance fibroblaste 20 (FGF-20) ou ses fragments, ses dérivés, ses variantes, ses homologues, ses analogues ou une combinaison de ceux-ci.
PCT/US2005/010732 2004-05-10 2005-03-29 Utilisations prophylactiques et therapeutiques de fgf-20 en protection antirayonnement WO2006073417A2 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
JP2007513141A JP2007536382A (ja) 2004-05-10 2005-03-29 放射線保護におけるｆｇｆ−２０の予防的使用および治療的使用
EP05856585A EP1789072A2 (fr) 2004-05-10 2005-03-29 Utilisations prophylactiques et therapeutiques de fgf-20 en protection antirayonnement
CA002566507A CA2566507A1 (fr) 2004-05-10 2005-03-29 Utilisations prophylactiques et therapeutiques de fgf-20 en protection antirayonnement
EP05851340A EP1812460A1 (fr) 2004-11-03 2005-11-03 Formulations, procedes de production et utilisations du fgf-20
CA002586213A CA2586213A1 (fr) 2004-11-03 2005-11-03 Formulations, procedes de production et utilisations du fgf-20
JP2007540036A JP2008519032A (ja) 2004-11-03 2005-11-03 Ｆｇｆ−２０の調合物、生成方法および使用
AU2005306898A AU2005306898A1 (en) 2004-11-03 2005-11-03 Formulations, methods of production and uses of FGF-20
PCT/US2005/039833 WO2006055264A1 (fr) 2004-11-03 2005-11-03 Formulations, procedes de production et utilisations du fgf-20

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US84217904A 2004-05-10 2004-05-10
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US10/842,206 US20050256039A1 (en) 2003-05-09 2004-05-10 Novel fibroblast growth factors and methods of use thereof
US61301304P 2004-09-22 2004-09-22
US60/613,013 2004-09-22
US10/980,459 US20050256042A1 (en) 2003-05-09 2004-11-03 Methods of preventing and treating alimentary mucositis
US10/980,459 2004-11-03

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WO2009100265A2 (fr) 2008-02-08 2009-08-13 Colgate-Palmolive Company Compositions et méthodes pour le traitement de la xérostomie
US10385113B2 (en) 2016-03-30 2019-08-20 The Board Of Trustees Of The University Of Arkansas Engineered FGF compositions and methods of use thereof
WO2019199476A1 (fr) * 2018-04-12 2019-10-17 Amgen Inc. Procédés de préparation de compositions protéiques stables
RU2709195C1 (ru) * 2017-08-08 2019-12-17 ЭсСиЭм ЛАЙФСАЙЕНС КО., ЛТД. Фармацевтическая композиция для предотвращения или лечения выпадения волос, содержащая белок-хемокинового лиганда 1, содержащего с-х-с мотив (схсl1), и миноксидил в качестве активных ингредиентов
RU2711298C2 (ru) * 2016-08-08 2020-01-16 ЭсСиЭм ЛАЙФСАЙЕНС КО., ЛТД. Композиция для предотвращения или лечения выпадения волос, содержащая белок хемокинового лиганда 1, содержащего с-х-с мотив (cxcli)
US11267855B2 (en) 2018-03-16 2022-03-08 The Board Of Trustees Of The University Of Arkansas Engineered FGF1 and FGF2 compositions and methods of use thereof

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WO2001007595A2 (fr) * 1999-07-27 2001-02-01 Curagen Corporation Nouveau facteur de croissance des fibroblastes et acides nucleiques codant ce dernier
WO2001072957A2 (fr) * 2000-03-31 2001-10-04 Nobuyuki Itoh Molecules de type facteur de croissance des fibroblastes et leurs utilisations

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WO2001007595A2 (fr) * 1999-07-27 2001-02-01 Curagen Corporation Nouveau facteur de croissance des fibroblastes et acides nucleiques codant ce dernier
WO2001072957A2 (fr) * 2000-03-31 2001-10-04 Nobuyuki Itoh Molecules de type facteur de croissance des fibroblastes et leurs utilisations

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WO2009100265A2 (fr) 2008-02-08 2009-08-13 Colgate-Palmolive Company Compositions et méthodes pour le traitement de la xérostomie
EP2249789A2 (fr) * 2008-02-08 2010-11-17 Colgate-Palmolive Company Compositions et méthodes pour le traitement de la xérostomie
EP2249789A4 (fr) * 2008-02-08 2014-01-15 Colgate Palmolive Co Compositions et méthodes pour le traitement de la xérostomie
US10471283B2 (en) 2008-02-08 2019-11-12 Colgate-Palmolive Company Compositions and methods for the treatment of xerostomia
US10385113B2 (en) 2016-03-30 2019-08-20 The Board Of Trustees Of The University Of Arkansas Engineered FGF compositions and methods of use thereof
RU2711298C2 (ru) * 2016-08-08 2020-01-16 ЭсСиЭм ЛАЙФСАЙЕНС КО., ЛТД. Композиция для предотвращения или лечения выпадения волос, содержащая белок хемокинового лиганда 1, содержащего с-х-с мотив (cxcli)
RU2709195C1 (ru) * 2017-08-08 2019-12-17 ЭсСиЭм ЛАЙФСАЙЕНС КО., ЛТД. Фармацевтическая композиция для предотвращения или лечения выпадения волос, содержащая белок-хемокинового лиганда 1, содержащего с-х-с мотив (схсl1), и миноксидил в качестве активных ингредиентов
US11267855B2 (en) 2018-03-16 2022-03-08 The Board Of Trustees Of The University Of Arkansas Engineered FGF1 and FGF2 compositions and methods of use thereof
WO2019199476A1 (fr) * 2018-04-12 2019-10-17 Amgen Inc. Procédés de préparation de compositions protéiques stables
CN111971302A (zh) * 2018-04-12 2020-11-20 美国安进公司 制备稳定的蛋白质组合物的方法

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EP1789072A2 (fr) 2007-05-30
WO2006073417A3 (fr) 2009-05-07

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