JP2007531702A - Therapeutic use of G53135-05 (FGF-20) in radiation protection - Google Patents

Abstract

The present invention relates to a method for the prevention or treatment of one or more symptoms associated with radiation exposure. Specifically, the present invention is a method of preventing or treating one or more symptoms associated with radiation exposure (eg, gastrointestinal (GI) syndrome, hematopoietic (bone marrow) syndrome and cerebrovascular (CNS) syndrome). Providing a composition comprising CG53135-05, fibroblast growth factor-20 (FGF-20) protein to a subject in need of such treatment.

Description

Detailed Description of the Invention

  This application is identified by US Provisional Application Nos. 60 / 469,353 (filed May 9, 2003) and 60 / 544,308 (filed February 26, 2004) and Attorney Docket Number Cura 57UN US Provisional Application (titled “Therapeutic Use of G53135-05 (FGF-20) in Radiation Protection” filed April 26, 2004), which is hereby incorporated by reference in its entirety. And

(Technical field)
The present invention relates to a method for preventing or treating one or more symptoms associated with radiation exposure. In particular, the present invention is a method for preventing or treating one or more symptoms associated with radiation exposure (eg, gastrointestinal (GI) syndrome, hematopoietic (bone marrow) syndrome and cerebrovascular (CNS) syndrome) comprising CG53135 -05, providing a method comprising administering a composition comprising fibroblast growth factor-20 (FGF-20) protein to a subject in need of the treatment.

(Conventional technology)
The FGF family consists of more than 20 members, each containing a conserved amino acid core (see, eg, Powerset et al., Endocr. Relat. Cancer, 7 (3): 65-197 (2000)). FGF regulates diverse cellular functions such as growth, survival, apoptosis, motility and differentiation (see, eg, Szebenyet et al., Int. Rev. Cytol., 185: 45-106 (1999)). Members of the FGF family are involved in various physiological and pathological processes in embryogenesis and adulthood, including morphogenesis, limb development, tissue repair, inflammation, angiogenesis and tumor growth and wetting. (See, eg, Powerset et al., Endocr. Relat. Cancer, 7 (3): 165-197 (2000); or Szebenyi et al., Int. Rev. Cytol. 185: 45-106 (1999)). Homology-based genomic mining has identified a new member of the FGF family, FGF-20 (also known as CG53135) (eg Jeffers et al., Cancer Res., 61 (7): 3131-8 (2001)). checking).

  A single exposure to a large amount of ionizing radiation can produce immediate effects on the tissue due to the generation of free radicals and can often cause radiation sickness. Radiation-induced lethality depends on exposure dose. Three major syndromes are associated with a single dose of radiation exposure: cerebrovascular (CNS) syndrome, gastrointestinal (GI) syndrome and hematopoietic (bone marrow) syndrome (eg Coleman et al. Radiat. Res., 159 (6 ): 812-834 (2003)). The death time depends on the dose of exposure, but is within a few hours for CNS syndrome, within 3 to 10 days for GI syndrome, and within 30 to 60 days for hematopoietic syndrome. Other signs associated with radiation sickness include nausea, vomiting, diarrhea, skin burns and wounds, fatigue, dehydration, inflammation, hair loss, neutropenia, oral mucosa and GI ulcers, and from the nose, mouth and rectum Including, but not limited to, bleeding. A common and root cause of the signs associated with radiation sickness is that ionizing radiation acts directly on the stem cell progenitor cells. Stem cells that are particularly affected in the gastrointestinal and / or hematopoietic system are cellular targets for pharmacological intervention. Neutropenia (reducing the number of neutrophils and increasing the risk of infection) and gastrointestinal mucositis are two of the most serious causes of death resulting from ionizing radiation. Like radiation, mustards cause similar effects at the cellular level; these combinations will have a geometrical effect on morbidity.

  Symptomatic medical interventions such as blood cell replacement, antibiotics, cytokines, and transplantation of hematopoietic stem cells in the case of high doses could increase overall survival. Biological modification of radiation response is an area that has been actively studied for many years. The development of both effective radiosensitizers and protective agents could provide therapeutic benefits to patients undergoing radiotherapy (see, for example, Cancer Chemotherapeutic Agents, 1995: 501-527). Further indications of radiation protection agents include protection of individuals involved in unforeseen ionizing radiation exposures in industrial or military environments. Treatment with members of the FGF family improved survival and hematopoietic recovery in mice following total body exposure to ionizing radiation (eg, Cytokine, 9 (1): 59-65 (1997); ActaOncol., 36 (3 ): 337-340 (1997); Acta Oncol., 34 (3): 435-438 (1995); Radiat. Res., 150 (2): 204-11 (1998); and Am. J. Clin. Oncol. 24 (5): 491-5 (2001)). Acidic FGF (aFGF or FGF-1, 1-24 mg) increased the survival of C3H / HeCNR mice receiving 840 centimeter-gray (cGy) ionizing radiation systemically (Cytokine, 9 (1): 59- 65 (1997)). Radioprotective effects of the other three members of the FGF family, basic FGF (bFGF or FGF-2), keratinocyte growth factor 1 and 2 (KGF-1 or FGF-7, and KFG-2 or FGF-10) Were tested in both C3H / He and BalbC mice (see Am. J. Clin. Oncol., 24 (5): 491-5 (2001)). These studies showed that the LD50 of ionizing radiation increased by about 100 cGy at a single dose (6 μg) of growth factor.

Ionizing radiation is so called because of the ability of radiation particles to produce reactive ions when irradiated against another molecule in a cell or elsewhere. Since one of the most common molecules in the cell is water, H ₂ O, the radicals that occur very often are a degraded form of this molecule (eg Int. J. Radiat. Biol, 65:27 -33 (1994)). Reactive oxygen species (ROS) include superoxide (O ₂ ), hydroxyl (HO) and hydrogen peroxide (H ₂ 0 ₂ ) (Proc. Natl. Acad. Sci. USA, 78: 1001-1003 (1981). )checking). These molecular intermediates, such as producing energy in the mitochondria by an electron transport system, are useful for cells, but excessive amounts can cause tissue destruction due to high reactivity. Genomic DNA breaks, mitochondrial membrane depolarization, and protein denaturation are damages that can be caused by such radicals. The body's first line of defense against these factors is an enzyme that scavenges radicals and makes them less reactive species. These enzymes include genes induced by superoxide dismutase (MnSOD, Cu, ZnSOD, extracellular-SOD), glutathione peroxidase and the transcription factor Nrf2 (Free Radic. Biol. Med., 17: 389-395). 1994); FASEB J., 7: 361-368 (1993); Proc. Natl. Acad. Sci. USA 94: 5361-5366). In fact, many of these proteins have been shown to be radioprotective to cells when overexpressed (Free Radic. Biol. Med., 17: 389-395 (1994), and FASEB J., 7: 361-368 (1993)). In addition, other members of the fibroblast growth factor family such as KGF have been shown to affect Nrf2 expression (see Mol. Cell Biol., 22: 5492-5505 (2002)). Therefore, understanding the effects of FGF-20 on the pathway mediated by ROS is some of the mechanisms of radioprotective effects on all animals, as ROS plays a major role in cell damage after exposure to ionizing radiation. Will draw.

(Summary of Invention)
The present invention is a method of preventing or treating one or more symptoms associated with radiation exposure, chemotherapy, chemical warfare and / or any other injury affecting rapidly proliferating body tissue, comprising CG53135-05. A method of administering a composition containing FGF-20 protein to a subject in need of such treatment is provided. In part, the present invention is based on the findings of the present invention that CG5135-05 protects rapidly proliferating tissues such as hematopoietic and gastrointestinal tissues from radiation exposure, chemotherapy and chemical weapons. Without being limited to any theory, it is believed that CG53135-05 protects stem cells associated with proliferative tissue regeneration potential from cytotoxic adverse effects. Ultimately, this general protection leads to an improvement in symptoms and / or an improvement in morbidity and mortality associated with injury affecting rapidly proliferating tissues.

  The present invention is a method of treating a subject exposed to injuries (eg, radiation, chemotherapy and chemical weapons) that affect rapidly proliferating tissue, comprising the therapeutic efficacy of a composition comprising a CG53135-05 polypeptide. A method comprising administering an amount to a subject is provided.

  The present invention is a method of protecting and / or regenerating a hematopoietic tissue of a subject exposed to injuries (eg, radiation, chemotherapy and chemical weapons) that affect rapidly proliferating tissue, comprising a CG53135-05 polypeptide. Also provided is a method comprising administering to a subject a therapeutically effective amount of the composition. In one embodiment, the present invention is a method of treating neutropenia in a subject exposed to injuries (eg, radiation, chemotherapy and chemical weapons) that affect rapidly proliferating tissue, comprising CG53135-05. There is provided a method comprising administering to a subject a therapeutically effective amount of a composition containing the polypeptide.

  The present invention is a method of protecting and / or regenerating the gastrointestinal tissue of a subject exposed to injuries (eg, radiation, chemotherapy and chemical weapons) that affect rapidly proliferating tissue, comprising a CG53135-05 polypeptide. Also provided is a method comprising administering to a subject a therapeutically effective amount of the composition. In one embodiment, the invention provides a method of treating gastrointestinal mucositis in a subject exposed to injuries (eg, radiation, chemotherapy, and chemical weapons) that affect rapidly proliferating tissue, comprising: A method comprising administering to a subject a therapeutically effective amount of a composition comprising

  In addition, the present invention provides signs (eg, diarrhea, skin burns, wounds, fatigue, dehydration, inflammation, hair loss, oral mucosal ulcers associated with injuries (eg, radiation, chemotherapy and chemical weapons) that affect rapidly proliferating tissues. , And nasal, oral or rectal bleeding, but not limited thereto), a prophylactically or therapeutically effective amount of a composition comprising a CG53135-05 polypeptide Is provided to a subject in need of such treatment. In one embodiment, the composition containing CG53135-05 is administered before the subject is exposed to the injury. In another embodiment, the composition containing CG53135-05 is administered after the subject has been exposed to injury. In yet another embodiment, the CG53135-05 polypeptide is administered to a subject at risk of exposure to injuries (eg, radiation, chemotherapy and chemical weapons) that affect rapidly proliferating tissue.

  The present invention administers to a subject a prophylactically effective amount of a composition containing a CG53135-05 polypeptide before the subject is exposed to injury that affects rapidly proliferating tissues such as radiation, chemotherapy and chemical weapons. A method of protecting and / or regenerating hematopoietic tissue, comprising: In one embodiment, the invention provides for the prevention of a composition comprising a CG53135-05 polypeptide before the subject is exposed to injuries (eg, radiation, chemotherapy and chemical weapons) that affect rapidly proliferating tissue. A method of preventing neutropenia in a subject, comprising administering to the subject an effective amount.

  The present invention provides a prophylactically effective amount of a composition containing a CG53135-05 polypeptide before the subject is exposed to injuries (eg, radiation, chemotherapy and chemical weapons) that affect rapidly proliferating tissue. Also provided is a method of protecting and / or regenerating a subject's gastrointestinal tissue comprising administering. In one embodiment, the present invention provides for the prophylactic of a composition containing a CG53135-05 polypeptide before the subject is exposed to injuries (eg, radiation, chemotherapy and chemical weapons) that affect rapidly proliferating tissue. Provided is a method of preventing gastrointestinal mucositis in a subject comprising administering to the subject an effective amount.

  In one embodiment, the present invention provides a rapidly acting tissue affecting injury (eg, comprising administering to a subject a prophylactically or therapeutically effective amount of a composition comprising a CG53135-05 polypeptide (eg, Provide a method for improving the survival of subjects exposed to radiation, chemotherapy and chemical weapons. The therapeutically effective amount may be a single dose of CG53135-05, or two or more doses.

  In another embodiment, a single and prophylactic dose of CG53135-05 will later be exposed to injuries (eg, radiation, chemotherapy and chemical weapons) that affect rapidly proliferating tissue. Administered to the subject. Here, such prophylactic doses produce a limited, short-acting proliferative effect on various compartments (eg, intestinal villi) within the growing tissue. In another embodiment, one or more prophylactic doses (CG53135-05 polypeptide may be two or more doses) are injuries that affect rapidly proliferating tissue (eg, radiation , Chemotherapy and chemical weapons) may be administered to prevent, treat or ameliorate symptoms associated with the injury.

  In some embodiments, the injury that affects rapidly proliferating tissue is radiation exposure. In some embodiments, the injury acting on rapidly proliferating tissue is one or more alkylating agents, one or more mustard agents, or one or more other chemotherapeutic agents, or combinations thereof It is. In some embodiments, the disorder that affects rapidly proliferating tissue is radiation exposure, one or more alkylating agents, one or more mustard agents, or one or more other chemotherapeutic agents, or It is a combination of them.

  In some embodiments, the CG53135-05 polypeptide is used to prevent, treat or ameliorate one or more symptoms associated with injury (eg, radiation, chemotherapy and chemical weapons) that affect rapidly proliferating tissue. Used in combination with one or more other therapies known to those skilled in the art.

  Pharmaceutical compositions, formulations and kits are also provided by the present invention.

3.1. The present invention is based on the identification of novel FGF-20 nucleic acids and amino acids, and variants thereof described herein and elsewhere with respect to CG53135 (US Provisional Application Nos. 09 / 494,585 and 10/174). , No. 393, “FGF-CX”, both of which are incorporated herein by reference). As used herein, the term “CG53135-05 polypeptide” refers to an isolated polypeptide comprising an amino acid sequence consisting of 211 amino acids. Furthermore, the present invention also includes fragments, derivatives, variants, homologs or analogs of CG53135-05. In some embodiments, the invention encompasses variants of the CG53135-05 polypeptide, where several amino acid residues of the amino acid sequence of CG53135-05 (eg, only 1%, 2%, 3%, 5% %, 10% or 15%). In some embodiments, the isolated CG53135-05 polypeptide comprises the amino acid sequence of the mature form of the amino acid sequence provided by CG53135, or a modified form of the mature form of the amino acid sequence provided by CG53135. Preferably, only 1%, 2%, 3%, 5%, 10% or 15% of the amino acid sequence of CG53135-05 is altered in a variant of the mature form of the amino acid sequence.

  As used herein, the term “effective amount” refers to the severity of a disease or injury characterized by rapidly growing tissue damage (eg, radiation, chemotherapy and chemical weapons), or one or more symptoms thereof. And / or reduce and / or improve the duration, prevent progression of the disease or injury, cause remission of the disease or injury, and relapse, development or onset of one or more symptoms associated with the injury Of a therapy (eg, a composition containing a CG53135-05 polypeptide) sufficient to prevent or enhance the prophylactic or therapeutic effect of another therapy (eg, a prophylactic or therapeutic agent) or Say dose.

  As used herein, the term “in combination” refers to the use of one or more therapies. The use of the term “in combination” does not limit the order in which therapies are administered to a subject in need of the therapy. The first therapy is performed before subjecting the second therapy to the subject in need of the therapy (for example, 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), simultaneously or after (eg, 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 later).

  As used herein, the term “prophylactically effective amount” refers to the development of a disease or disorder associated with rapidly growing tissue damage (eg, radiation, chemotherapy and chemical weapons), or one or more symptoms thereof. Refers to a dose of a therapy (eg, CG53135-05 polypeptide) sufficient to result in prevention of recurrence or onset, or to enhance or improve the prophylactic effect of another therapy.

  As used herein, the term “subject” refers to an animal, preferably a mammal comprising a non-primate (eg, cow, pig, horse, cat, dog, rat or mouse) and primate (eg, monkey, chimpanzee or human), Even more preferably humans. In one embodiment, the subject is a mammal, preferably a human, that has been or will be exposed to injuries (eg, radiation, chemotherapy and chemical weapons) that affect rapidly proliferating tissue. In another embodiment, the subject has been exposed to or will be exposed to injuries (eg, radiation, chemotherapy and chemical weapons) that affect rapidly proliferating tissue (eg, horses, pigs or cattle). ) Or pet (eg dog or cat). In another embodiment, the subject is a mammal, preferably a human, that may be exposed to injuries (eg, radiation, chemotherapy and chemical weapons) that affect rapidly proliferating tissue. The term “subject” is used interchangeably with “patient” herein.

  As used herein, the term “therapeutically effective amount” reduces the severity of a disease or disorder characterized by injuries to rapidly proliferating tissues (eg, radiation, chemotherapy and chemical weapons), and the disease Or reduce the duration of the disorder, prevent progression of the disease or disorder, cause remission of the disease or disorder, improve one or more symptoms associated with injury to rapidly proliferating tissue, Alternatively, it refers to a dose of a therapy (eg, 53135-05 polypeptide) sufficient to enhance or improve the therapeutic effect of another therapy.

  The terms “therapy” and “therapeutic agent” prevent, treat or ameliorate a disease or disorder characterized by rapidly injuring tissues (eg, radiation, chemotherapy and chemical weapons), or one or more symptoms thereof. Any protocol, method and / or agent that may be used in In certain embodiments, the terms “therapy” and “therapeutic agent” refer to a disease or disorder characterized by rapidly growing tissue damage (eg, radiation, chemotherapy and chemical weapons), or one or more signs thereof. Refers to antiviral therapy, antibacterial therapy, antibacterial therapy, biological therapy, supportive therapy and / or other therapies that are useful for treatment or improvement, and these are known to physicians.

(Detailed description of the invention)
The present invention relates to a method for preventing or treating one or more symptoms associated with radiation exposure, chemotherapy, chemical warfare and / or any other disorder acting on rapidly proliferating body tissues, comprising FGF-20 protein A composition comprising a CG53135-05 polypeptide is provided to a subject in need of such treatment.
CG53135-05 polypeptides and methods for making such polypeptides are described in US application Ser. Nos. 09 / 494,585 and 10 / 174,394, both of which are hereby incorporated by reference. Part.

  The present invention relates to a method for preventing, treating or ameliorating a disease or one or more symptoms associated with injuries (eg radiation, chemotherapy and chemical weapons) acting on rapidly proliferating tissue, comprising CG53135-05 polypeptide A method comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a composition comprising

  In some embodiments, the injury that affects rapidly proliferating tissue is radiation exposure. In certain embodiments, the injury is ionizing radiation. In another embodiment, the injury may be one or more chemotherapy, or one or more chemical weapons, or a combination thereof. Examples of chemotherapy and chemical weapons are, but are not limited to, alkylating agents and mustard agents. In some embodiments, the injury acting on rapidly proliferating tissue is one or more radiation exposure, one or more chemotherapy, one or more chemical weapons, or combinations thereof.

  Organs and body systems that are most susceptible to injury such as ionizing radiation include the skin, hematopoietic and lymphatic systems, gonads, lungs, nervous tissue and GI tract. In one embodiment, the injury is in particular hematopoiesis and / or damage to gastrointestinal tissue of the subject. In another particular embodiment, the disease to be prevented or treated is mucositis. In some embodiments, symptoms associated with injuries (eg, radiation, chemotherapy and chemical weapons) that affect rapidly proliferating tissue are diarrhea, skin burns, wounds, fatigue, dehydration, inflammation, hair loss, oral mucosal Includes but is not limited to ulcers and bleeding from the nose, mouth or rectum.

  A group of patients that may be targeted using the methods of the present invention includes subjects exposed to rapidly acting tissues (eg, radiation, chemotherapy and chemical weapons), injuries acting on rapidly growing tissues (eg, Subjects suspected of being exposed to radiation, chemotherapy and chemical weapons), subjects who will later be exposed to injuries (eg radiation, chemotherapy and chemical weapons) that affect rapidly proliferating tissues, and rapidly proliferating Including, but not limited to, subjects at risk of being exposed to injuries (eg, radiation, chemotherapy and chemical weapons) that affect tissue.

  In one embodiment, the composition containing CG53135-05 is administered before the subject is exposed to the injury. In another embodiment, the composition containing CG53135-05 is administered after the subject has been exposed to the injury. In yet another embodiment, the CG53135-05 polypeptide is administered to a subject at risk for exposure to injuries (eg, radiation, chemotherapy and chemical weapons) that affect rapidly proliferating tissue.

  Compositions containing CG53135-05 are used to prevent, treat or ameliorate diseases or one or more symptoms associated with injuries (eg, radiation, chemotherapy and chemical weapons) that affect rapidly proliferating tissues. Alternatively, it can be administered in combination with other therapies. In one preferred embodiment, a composition containing CG53135-05 is used to prevent disease or one or more symptoms associated with injuries (eg, radiation, chemotherapy and chemical weapons) that affect rapidly proliferating tissues, It is administered in combination with one or more therapies known to be used in treatment or amelioration. Examples of such other therapies are mesna (sodium 2-mercaptoethanesulfonate) and other analogs having a free thiol moiety, dimesna (disodium 2,2′-dithiobisethane sulfonate) and other disulfides and US application numbers. Including, but not limited to, compounds as described in US20030092681.

  In one embodiment, in combination therapy, the CG53135-05 polypeptide and / or another therapeutic agent is administered in sub-optimal dosages that exhibit a detectable therapeutic effect when administered, for example, alone. No dose (measurement is by methods known to those skilled in the art). In such methods, co-administration of CG53135-05 polypeptide and other therapeutic agents improves the overall treatment effect.

  In one embodiment, the CG53135-05 polypeptide and one or more therapeutic agents are administered in a single patient visit. In another embodiment, the CG53135-05 polypeptide is administered prior to administration of one or more other therapeutic agents. In yet another embodiment, the CG53135-05 polypeptide is administered after administration of one or more therapeutic agents. In certain embodiments, the CG53135-05 polypeptide and one or more other therapeutic agents are periodically administered to the subject. Periodic administration includes administration of CG53135-05 polypeptide over a period of time, followed by administration of one or more other therapeutic agents over a period of time, and repetition of this series of administrations. Periodic administration may reduce the development of resistance to one or more therapeutic agents, avoid or reduce side effects from one therapeutic agent, and / or improve the effectiveness of the treatment.

The toxic and therapeutic effects of the compositions of the invention (eg CG53135-05 polypeptide) are therapeutically effective, for example, LD ₅₀ (dose that kills 50% of the population) and ED ₅₀ (50% of the population). Can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as determining doses). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD ₅₀ / ED ₅₀ . Compositions that exhibit high therapeutic indices are preferred. Compositions that exhibit toxic side effects may be used, but delivery systems that allow such compositions to reach diseased tissue sites to minimize potential damage to uninfected cells and thereby reduce side effects Care should be taken when designing.

In one embodiment, data obtained from cell culture assays and animal studies can be utilized in devising dose ranges for use in humans. Preferably, the conjugate dose is within a range of circulating concentrations, including ED ₅₀ with little or no toxicity. The dose will fall within this range depending on the mode of administration used, the route of administration utilized, the severity of the disease, the age and weight of the subject, and other factors normally considered by a medical professional (eg, a physician). It may change. For any composition used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. As determined in cell culture, the dose may be formulated in animal models to reach a range of circulating plasma concentrations, including IC50 (ie, the concentration of the test compound that inhibits symptoms halfway). 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.

  In some embodiments, the dose of CG53135-05 polypeptide provided by the present invention for administration to a human patient is at least 0.1 mg / kg, at least 0.5 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 15 mg / kg, at least 20 mg / Kg, at least 25 mg / kg, at least 30 mg / kg, at least 35 mg / kg, at least 40 mg / kg, at least 45 mg / kg, at least 50 mg / kg, at least 60 mg / kg, at least 70 mg / kg g, at least 80 mg / kg, at least 90 mg / kg, at least 100 mg / kg, at least 150 mg / kg, or at least 200 mg / kg. In some embodiments, the dose of CG53135-05 polypeptide provided by the present invention for administration to a human patient is 0.1-300 mg / kg, 0.5-250 mg / kg, 1-200 mg / kg. 1-150 mg / kg, 1-125 mg / kg, 1-100 mg / kg, 1-90 mg / kg, 1-80 mg / kg, 1-70 mg / kg, 1-60 mg / kg, 1-50 mg / kg, 1 -40 mg / kg, 1-35 mg / kg, 1-30 mg / kg, 1-25 mg / kg, 1-20 mg / kg, 1-15 mg / kg, 1-10 mg / kg or 1-5 mg / kg.

  Appropriate and recommended doses, formulations and routes of administration for treatment modalities such as chemotherapeutic agents, radiotherapy and biological / immunological therapies such as cytokines are known to those skilled in the art and Physician's It is described in documents such as Desk Reference (58th ed., 2004).

  Various delivery systems are known and can be used to administer the compositions of the present invention. Such delivery systems include the construction of nucleic acids of the invention as part of liposomes, microparticles, microcapsules, expression by recombinant cells, receptor-mediated endocytosis, retroviruses or other vectors. However, it is not limited to these. Introduction methods include intradermal, intramuscular, intraperitoneal, intrathecal, intraventricular, epidural, intravenous, subcutaneous, nasal, intratumoral, transdermal, enteral and oral routes. It is not limited to. The compositions of the present invention may be administered by any useful route such as, for example, infusion or bolus injection, absorption from epithelial or mucocutaneous linings (eg, oral, vaginal, rectal and intestinal mucosa). Furthermore, it may be administered in combination with other biologically active agents. Administration can be systemic or local.

  In some embodiments, it may be desirable to administer the pharmaceutical composition of the present invention locally to the site in need of treatment. This may be, for example, by local injection during surgery or by topical application, such as in combination with a wound dressing after surgery, such as injections, catheters, suppositories or implants (such as a sealable membranes) Or a porous, non-porous or gelatinous material, including membranes or fibers. In one embodiment, administration can be by direct injection into a rapidly proliferating tissue site (or anterior) that is most susceptible to injury, such as radiation, chemotherapy or chemical weapons.

  In some embodiments where the composition of the invention is a nucleic acid encoding a prophylactic or therapeutic agent, the nucleic acid is in vivo to promote expression of the encoded protein (eg, CG53135-05 polypeptide). It can be administered by constructing the nucleic acid as part of an appropriate nucleic acid expression vector, and the nucleic acid is transferred into the cell by using a retroviral vector, direct injection, or using microparticle irradiation (eg, a gene gun). Or coated with lipids or cell surface receptors or transfection agents, or in combination with homeobox-like peptides known to enter nucleic acids, etc. . Alternatively, the nucleic acids of the invention can be introduced into cells and incorporated into host cell DNA by homologous recombination for expression.

  The compositions of the present invention include bulk drug compositions that are useful in the manufacture of pharmaceutical compositions that can be used in unit dosage form preparations. In a preferred embodiment, the composition of the present invention is a pharmaceutical composition. Such compositions contain a prophylactically or therapeutically effective amount of one or more compositions of the invention (eg, CG53135-05 polypeptide) and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition is formulated to be stable to the route of administration to the subject.

  In one embodiment, the term “pharmaceutically acceptable” is approved by a federal or state government supervisory authority, S. Means described in pharmacopoeia or other pharmacopoeia generally recognized for use in animals, more preferably humans. The term “carrier” refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient or vehicle that is administered in combination with a prophylactic or therapeutic agent. Such pharmaceutical carriers may be sterile liquids such as water and oils (eg, synthetic oils such as petroleum, animal oils, vegetable or peanut oil, mineral oil, sesame oil) or diluents, flavorings, solubilizers, lubricants, suspending agents, binders. It can be a solid carrier such as one or more substances that may act as a tablet disintegrant or an encapsulating substance. 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 are starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol , Water, ethanol, or a combination thereof. If desired, the composition may also contain minor amounts of wetting agents, emulsifying agents or pH buffering agents.

  The compositions of the present invention can be in any number of possible dosage forms such as, but not limited to, liquid syrups, tablets, capsules, gel capsules, soft gels, pills, powders, enemas, sustained release formulations and the like. It may be prescribed. The compositions of the invention may be formulated as suspensions in aqueous, non-aqueous or mixed media thereof. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may contain a stabilizer. The composition can also be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations may include standard amounts of pharmaceutical carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carboxylate.

  In some embodiments, the compositions of the present invention may be formulated and used as a foam. Pharmaceutical foams include but are not limited to emulsions, microemulsions, creams, jellies and liposomes. Although essentially similar in nature, these formulations depend on the components and consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the art of pharmaceutical formulation and may be further applied to the formulation of the compositions of the present invention.

  A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. In certain embodiments, the composition is formulated according to conventional methods as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, nasal, intratumoral or topical administration to humans. Conventionally, compositions for intravenous administration are solutions in sterile aqueous isotonic buffer. Where necessary, the composition may include a solubilizing agent and a local anesthetic such as lidocaine to reduce pain at the site of the injection.

  When the composition of the present invention is administered topically, the composition can be formulated in the form of skin 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 essential or desired. Coated condoms, gloves and the like are also useful. Preferred topical formulations include those in which the molecular complexes of the invention are mixed with agents for local delivery, such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Although there is, it is not limited to these. Preferred lipids and liposomes are neutral (eg dioleoylphosphatidyl DOPE ethanolamine, dimyristoyl phosphatidylcholine DMPC, distearoyl phosphatidylcholine), anionic (eg dimyristoyl phosphatidylglycerol DMPG) and cationic (eg dioleoyltetramethylamino). Propyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The molecular complex of the present invention may be encapsulated in liposomes or may form complexes with them, particularly with cationic liposomes. Alternatively, the molecular complex may be complexed with lipids, particularly cationic lipids. Preferred fatty acids and esters are arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, 1- Including glyceryl monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine or C1-10 alkyl esters (eg, isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof However, it is not limited to these. For topical dosage forms that are not sprayable, a viscous to semi-solid or solid form that is adapted for topical application and preferably comprises one carrier or one or more excipients having a kinematic viscosity greater than water Are typically used. Another suitable topical dosage form is a sprayable aerosol preparation, wherein the active ingredient is preferably combined with a solid or liquid inert carrier to form a pressurized volatile substance (such as freon). Gas spray) or packaged in a compression bottle. Humidifying agents or wetting agents can also be added to pharmaceutical compositions and dosage forms as desired. Examples of such additive ingredients are well known to those skilled in the art.

  Where the method of the invention comprises nasal administration of the composition, the composition may be formulated in aerosol form, spray, mist or drop form. In particular, prophylactic or therapeutic agents for use in accordance with the present invention are by using a suitable high pressure gas (eg dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas), It can be suitably delivered in the form of an aerosol spray from a pressurized pack or nebulizer. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a certain amount. For use by inhalation or powder inhalation, capsules and cartridges (eg consisting of gelatin) containing a powder mix of the compound and a suitable powder base such as lactose or starch may be formulated.

  When the method of the invention comprises oral administration, the composition can be a powder, granule, microparticle, nanoparticle, suspension or solution in water or non-aqueous medium, capsule, gel capsule, starch, It can be formulated in the form of tablets or minitablets. Thickeners, fragrances, diluents, emulsifiers, dispersion aids or binders may be desired. Tablets or capsules include a binder (eg pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); a filler (eg lactose, microcrystalline cellulose or calcium hydrogen phosphate); a lubricant (eg magnesium stearate, Talc or silica); disintegrants (eg potato starch or sodium starch glycolate); or can be prepared by conventional means together with pharmaceutically acceptable excipients such as wetting agents (eg sodium lauryl sulfate). The tablets may be coated by methods known to those skilled in the art. Liquid preparations for oral administration include suspensions (eg, sorbitol syrup, cellulose derivatives or hydrogenated edible fat); emulsifiers (eg, lecithin or acacia); non-aqueous vehicles (eg, almond oil, oily esters, ethyl alcohol) Or fractionated vegetable oils); and preservatives (eg, methyl or propyl p-hydroxybenzoate or sorbic acid) and may be prepared by conventional means together with pharmaceutically acceptable additives. The preparation may contain buffer salts, flavors, colorants and sweeteners as required. Preparations for oral administration may be suitably formulated to release a prophylactic or therapeutic agent in a delayed, controlled or sustained form.

  In one embodiment, the molecular complex of the invention is orally administered with one or more penetration enhancers such as, for example, surfactants and chelating agents. Preferred surfactants include, but are not limited to, fatty acids and esters or salts thereof, bile acids and salts thereof. Preferred bile acids / salts are chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, This includes but is not limited to sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate. Preferred fatty acids are arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicouple, tricaprate, monoolein, dilaurin, 1-monocaprin Examples include, but are not limited to, glyceryl acid, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine or monoglyceride, diglyceride or a pharmaceutically acceptable salt thereof (eg, sodium). In some embodiments, penetration enhancers are used in combination, such as fatty acids in combination with bile acids / salts. In one particular embodiment, lauric acid, the sodium salt of 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. The molecular conjugates of the present invention may be delivered orally in granular form, including but not limited to those that are conjugated to form dry particles or micro- or nanoparticles to be sprayed. Complexing agents that can be used to complex with the molecular complex of the present invention are poly-amino acids, polyimines, polyacrylates, polyalkyl acrylates, polyoxetanes, polyalkyl cyanoacrylates, cationized gelatin, albumin, acrylate , Polyethylene glycol (PEG), polyalkyl cyanoacrylates, polyimines derived from DEAE, porlan, cellulose and starch. Particularly preferred complexing agents are chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P (TDAE), polyaminostyrene (eg p- Amino), poly (methyl cyanoacrylate), poly (ethyl cyanoacrylate), poly (butyl cyanoacrylate), poly (isobutyl cyanoacrylate), poly (isohexyl cyanoacrylate), DEAE-methacrylate, DEAE -Hexyl acrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethyl acrylate, polyhexyl acrylate, poly (D, L-lactic acid), poly (DL-lactic acid) / glycolic acid Coalescence (PLGA), encompasses alginate and polyethylene glycol (PEG), but are not limited to.

  The methods of the invention may include pulmonary administration of a composition formulated with an aerosolizing agent, for example, by use of an inhaler or nebulizer.

  The methods of the invention may include administration of a composition formulated for parenteral administration by injection (eg, bolus injection or continuous infusion). Injectable formulations may be provided in unit dosage forms (eg, ampoules or multiple dose containers) with preservatives added. The composition may take forms such as suspensions, solutions or emulsions in oily or aqueous vehicles, and may further contain formulation agents such as suspending, stabilizing and / or dispersing agents. But you can. Alternatively, the active ingredient may be in powder form comprised with a suitable vehicle (eg, sterile pyrogen-free water) prior to use.

  In one preferred embodiment, the composition is formulated according to conventional procedures, as are pharmaceutical compositions adapted for intravenous administration to humans. In general, compositions for intravenous administration are solutions in sterile aqueous isotonic buffer. If desired, the composition may contain a solubilizing agent and a local anesthetic such as lignocaine to reduce pain at the site of the injection. Generally, the ingredients are supplied separately or mixed as a unit dosage form, such as a lyophilized dry powder or a water-free concentrate, in a sealed container indicating the amount of active ingredient such as an ampoule or sachet. Where the composition is to be administered by inhalation, it can be dispensed with an inhalation bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

  The compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts are those formed with free amino groups, such as those derived from hydrogen chloride, phosphoric acid, acetic acid, succinic acid, artaric acid, and sodium, potassium, ammonium, calcium, ferric hydroxide , A salt formed with a free carboxyl group such as, but not limited to, derived from isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine and the like. Examples of pharmaceutically acceptable salts are acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium acetate, cansylate, carbonate, chloride, citrate, dihydrochloride, edetate, edicylate, estrate, esylate, fumarate, glucaptate , Gluconate, glutamate, glycolylarsanilate, hexyl resorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl Nitrate, Methylsulfate, Mukat, Napsilate, Nitrate, Pamoart (Embonato), Panto tenor , Phosphaterdiphosphate, polygalactronate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, theocrate, triethiodide, benzathine, chloroprocaine, choline, diethanolamine, ethyl enineamine, meglumine, procaine, Aluminum, calcium, lithium, magnesium, potassium, sodium and zinc are not limited to these.

  In addition to the formulations described above, the composition may be formulated as a sustained release preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the composition may be a suitable polymeric or hydrophobic material (eg, as an emulsion in an acceptable oil) or ion exchange resin, or a poorly soluble derivative such as, for example, a poorly soluble salt. May be prescribed as Liposomes and emulsions are well known examples of vehicles or carriers for hydrophilic drugs.

  In one embodiment, the components of the composition of the invention originate from the same species as the recipient of such composition or from a species having the same reactivity.

  The invention also provides kits for carrying out the therapeutic regimen of the invention. Such kits contain a composition of the invention (eg, CG53135-05 polypeptide) in a pharmaceutically acceptable form in a prophylactically or therapeutically effective amount in one or more containers. The composition in the vial of the kit of the invention may be in the form of a pharmaceutically acceptable solution, such as with sterile saline, dextrose solution or buffer, or other pharmaceutically acceptable sterile fluid. It may be a combination. Alternatively, the composition may be lyophilized or dried; in this case, the kit is optionally pharmaceutically acceptable in a container to reconstitute the composition and form a solution for injection purposes. It further comprises a solution (eg, saline, dextrose solution, etc.), preferably sterilized.

  In another embodiment, the 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. . If desired, instructions for administering the formulations of the present invention by a physician or patient are included.

  In some embodiments, the invention comprises a plurality of containers each containing a pharmaceutical formulation or composition containing a dose of a composition of the invention (eg, CG53135-05 polypeptide) sufficient for a single administration. Provide kit.

As with any pharmaceutical product, the packaging materials and containers are designed to protect the stability of the product during storage and shipping. In one embodiment, the compositions of the present invention include biocompatible surfactants including but not limited to lecithin, taurocholic acid and cholesterol; or gamma globulin and serum albumin, Stored in a container with other proteins not limited to. In addition, the products of the present invention include instructions or other informational material that advises a physician, technician or patient on how to appropriately prevent or treat the disease or disorder in question.
The invention is further illustrated by the following examples.

5.1. Example 1: Protein Expression and Purification Recombinant human CG53135-05 was purified from Escherichia coli BLR (DE3) cells (Novagen, Madison, WI) as follows. DE3 cells were transformed with full length codon optimized CG53135-05 cloned into the pET24a vector (Novagen) to create a master cell bank (MMCB) for the production of these cells. A cell paste containing CG53135-05 obtained by fermentation of cells derived from MMCB was lysed by high-pressure homogenization in a lysis buffer and clarified by centrifugation. CG53135-05 was purified from the clarified cell lysate by two cycles of ammonium sulfate precipitation and ion exchange chromatography. The final protein fraction was dialyzed against preparation buffer (30 mM citrate, pH 6.0, 2 mM ethylenediaminetetraacetic acid (EDTA), 200 mM sorbitol, 50 mM KCl and 20% glycerol). The vehicle (without CG53135-05 protein) contains 30 mM sodium citrate, pH 6.1, 2 mM EDTA, 200 mM sorbitol, 50 mM KCl, 20% glycerol.

5.2. Example 2: Preventive protective effect of CG53135-05 from radiation exposure 5.2.1. Effect of CG53135-05 on survival after a single exposure to acute radiation (N-272)
This study was conducted to investigate the effects of CG53135-05 administered under different schedules as radioprotective agents in mice after lethal whole body ionizing radiation irradiation.
Male C3H / He mice (total number “n” = 60) with an average weight of 22.1 grams at the start of the study were used as treatment groups. Animals were fed a standard commercial mouse diet. Food and water were given as appropriate.

Study design Irradiation of the animals was performed at the Brigham and Women's Hospital (Boston, Mass.). The study was conducted at The Massachusetts College of Pharmacy (Boston, Mass.). Mice were randomly divided into 4 groups: a control group consisting of 15 mice and 3 test groups each consisting of 15 mice. Mice were exposed to a single dose of whole body ionizing radiation of 600 cGy. CG53135-05 (12 mg / kg) was administered intraperitoneally (IP) daily 2 days and 3 days after irradiation, 4 to 7 days later, or 2 days before and 1 day before irradiation (-2 and -1), respectively. The number of deaths (number of animals, date of death) was recorded in each group.

全動物に１日１回ＣＧ５３１３５−０５またはビヒクルを与えた（０．１ｍＬ／体重１０ｇ）。 Table 1: Research plan

All animals received CG53135-05 or vehicle once daily (0.1 mL / 10 g body weight).

Survival of statistical treatment groups was compared using Kaplan-Meier log rank analysis. Body weight changes were analyzed by determining the area under the curve (AUC) using a trapezoidal variant. The area under the curve, the equation:
i = {yi (x + 1−xi) + (1/2) (yi + 1−yi) (x + 1−xi)}
[Where x = time (days), y = rate of weight change and i = time point, so xi is the x value at time point i, yi is the y value at that time point, and xi + 1 And yi + 1 are the x and y values at the next time point. ]
It calculated using. Both t-test and Mann-Whitney rank sum test were used to compare AUCs for body weight change between different treatment groups.

Results Treatment of mice with CG53135-05 (12 mg / kg) two days before and one day before irradiation significantly increased survival compared to untreated controls (P = 0.04). On the other hand, treatment with CG53135-05 after 2-3 days or 4-7 days after irradiation had no significant effect on survival (P> 0.05) (FIG. 1).
Treatment plans with CG53135-05 after 2-3 days and after 4-7 days had no significant effect on survival. Furthermore, prophylactic CG53135-05 administration (on days -2 and -1) also prevented post-radiation weight loss (FIG. 2). Statistical analysis of the area under the curve (AUC) for body weight change by one-way analysis of variance (one-way ANOVA) showed no significant difference between groups (P = 0.051), but two days before and one day before ( 13.9%) as well as after 4-7 days (14.8%), treatment with CG53135-05 also increased the overall average rate of weight gain compared to the untreated control (9.7%) (FIG. 2). ). The results of this study show that treatment of mice with CG53135-05 one day before and two days before whole body irradiation is effective in radioprotection, and also on gastrointestinal or hematopoietic tissues from the damage effects of whole body irradiation Supports protective effects.

5.2.2. Prophylactic effect of CG53135-05 on mice after exposure to acute ionizing radiation (N-308)
This study was conducted to investigate the effect of CG53135-05 prophylactically administered to mice later exposed to various doses of systemic ionizing radiation. The subsequent procedure was the same as described in section 5.2.1. On day 0, mice were exposed to ionizing radiation in a dose range of 484 to 641 cGy without anesthesia. Animals were administered PBS (control) or CG53135-05 (12 mg / kg, daily, IP) one day before irradiation or two and one day before. The schedule is shown in Table 2. The end points of this study were survival and weight change. Survival was followed for 30 days after irradiation.

Table 2. Research plan

result:
Survival decreased as radiation dose increased in all treatment groups. In animals dosed with PBS, the 30-day survival rate at the lowest radiation dose (484 cGy) was 93.75%, 50.0% for 534 cGy, 31.25% for 570 cGy, 12.5% for 606 cGy, and 641 cGy Then, it decreased to 6.25% (FIG. 3). In animals that received GC53135 (12 mg / kg IP) only on day −1, the 30-day survival rate at the lowest radiation dose (484 cGy) was 87.5%, compared to 87.5% for 534 cGy and 570 cGy 81.25%, 43.75% for 606cGy and 31.25% for 641cGy (FIG. 4). In animals that received GC53135 (12 mg / kg IP) on days -2 and -1, the 30-day survival rate at the lowest radiation dose (484 cGy) was 87.5% compared to 75.0 for 534 cGy %, 570 cGy, 37.5%, 606 cGy, 31.25% and 641 cGy, 0 (FIG. 5).

  A combined comparative study showed a 4.8-fold increase in survival of animals treated on day -1 relative to control animals (p = 0.00016). LD50 / 30 values were calculated using survival probit plots with 95% confidence intervals calculated by the bootstrap method. However, the survival rate of animals treated on days -2 and -1 relative to control animals was not significant (p = 0.4162). The results show that prophylactic administration of CG53135-05 has a therapeutic effect in radioprotection. Furthermore, treatment one day before irradiation (day -1) also protected animals from weight loss in all but the maximum radiation level (641 cGy, data not shown). In this particular system, especially at higher radiation levels, a single dose (Day-1 only) was significantly more effective than a double dose schedule (Day-2 and Day-1 respectively).

  In addition to the above results, the present invention may extend to further regimens of CG53135-05, such as prophylactic and / or therapeutic administration of CG53135-05 before and / or after exposure to radiation. This can be tested in the same animal model according to the same procedure as described herein to define the therapeutic efficacy range of this compound. Dosage regimens for therapeutic treatment include, but are not limited to, 1 day, 1 day and 2 days after radiation exposure.

5.3. Example 3: Regulation of intestinal crypt cell proliferation and apoptosis by administration of CG53135-05 to mice (N-342)
The present study aims to distinguish between effects on stem cells and daughter cells and to gain insight into the mechanism of action of CG53135-05 in syndromes associated with gastrointestinal stem cell damage (eg mucositis). Assess the effect of CG53135-05 on cell turnover. Furthermore, the effect of CG53135-05 on the radiosensitivity of stem cells was also evaluated.

  The “crypt” has a hierarchical structure with stem cells toward the bottom of the crypt. As cells mature, they gradually move from the bottom of the crypt to the top of the crypt. Thus, by observing changes in the frequency of events at each cell location, changes that may be an effect on stem cells and daughter cells that undergo translocation amplification can be detected. The cell position is shown in FIG. Therefore, the effect of CG53135-05 on the crypt microstructure was analyzed in light of crypt cellularity.

Experimental design After a single dose of 12 mg / kg (IP) CG53135-05, the animals were sacrificed at various times. Immediately prior to sacrifice, mice were labeled with a single injection of bromodeoxyuridine to label S-phase cells and to determine the effect of the drug on crypt cell proliferation / apoptosis. Two more groups of mice were used to assess the effect of stem cells on radiosensitivity. One group was treated with CG53135-05 (12 mg / kg, single injection, ip) and another group was injected with a placebo control. The animals were irradiated with X-ray 1 Gy 24 hours after injection (especially to induce apoptosis of stem cells) and then subjected to normal in vivo BrdU labeling. The animals were sacrificed 4.5 hours later (at the peak of apoptosis).

  Mice were weighed and then CG53135-05 (12 mg / kg, single injection, ip) was administered. Groups of 6 animals were sacrificed at 0, 3, 6, 9, 12, 24, 48 hours after injection of CG53135-05. All were injected once with bromodeoxyuridine 40 minutes before sacrifice (see Table 3).

  To evaluate the effect of CG53135-05 on stem cell radiosensitivity, two groups of 6 mice were used (see Groups 8 and 9, see Table 3). One group was treated with CG53135-05 (12 mg / kg, single injection, ip) and another group was injected with a placebo control. The animals were irradiated with 1 Gy X-ray 24 hours after injection and sacrificed 4.5 hours later.

Table 3. Research plan

Modulation of small intestinal crypt cell proliferation and apoptosis: Procedure All S-phase dividing cells take up injected bromodeoxyuridine (BrdU) and are therefore marked as cells in cycle. Irradiated animals were set on perspec jigs under non-anaesthesia and exposed to X-ray 1 Gy whole body radiation at a dose rate of 0.7 Gy / min. This low level of radiation induced apoptosis in the small intestinal stem cell population, but not in more mature cells.

  The small intestine was removed, fixed with Carnoy's fixative, and processed for histological analysis (embedded in paraffin). One set of 3 mm sections was immunolabeled for BrdU and another set of sections was stained with H & E. Longitudinal sections of small intestine crypts were analyzed for the presence of BrdU or apoptotic / mitotic nuclei. 25 crypts (Fifty half cryptos) were recorded per animal.

  Groups 1-7 (result group: A) were tested to determine the effect of CG53135-05 over 48 hours. To determine whether CG53135-05 alters the number of apoptotic cells that occur after low dose irradiation, ie, whether CG53135-05 affects the radiosensitive stem cell population (Group of Results: B ) Was tested.

  The results obtained showed a distribution of frequencies for crypts in each group of animals (further analyzed for statistical differences). Tissue samples were taken at 3, 6, 9, 12, 24 and 48 hours after treatment with CG53135-05. Apoptosis, mitotic index and proliferation were the endpoints of this study.

result:
Group A
In groups 1-7 (Table 3), CG53135-05 had no significant effect on spontaneous apoptosis. Similar results were obtained for the split index (Table 4). However, as shown in Table 4, BrdU incorporation results showed the following:
a) At 3 hours there was an extension / expansion of the growth area (positions 12-22).
b) Great proliferative effects were seen at many positions up to 9 hours.
c) Up to 12 hours, only positions 4-8 (stem cells) showed an increase in uptake.
d) There was significant inhibition of proliferation up to 24 hours.
e) Up to 48 hours the uptake was comparable to the control level.

表４に示す比較は処置群対無処置群のものである。示した細胞位置は無処置対照と有意な差があるものである（Ｐ＜０．０５）。 Table 4. Outline of significant cell location in crypts after assessing apoptosis, mitosis and proliferation

The comparison shown in Table 4 is for the treated group versus the untreated group. The cell locations shown are significantly different from untreated controls (P <0.05).

Group B:
In groups 8 and 9, the radiosensitivity of stem cells was evaluated (Table 3). As shown in Table 3, CG53135-05 or PBS was administered one day before irradiation with 1 Gy of radiation. Tissues were collected 4.5 hours after irradiation. There was no significant effect on either radiation-induced apoptosis or mitotic index (data not shown). However, increased uptake and significant inhibition of proliferation at positions 4-8 up to 12 hours was seen in mice pretreated with CG53135-05 and irradiated, which is consistent with the results of Group A (Table 4).

5.4. Example 4: Effect of prophylactic administration of CG53135-05 on intestinal crypt survival in mice following radiation injury (N-343)
The purpose of this study was to evaluate the efficacy of CG53135-05 on radiation-induced crypt cell mortality in vivo using the Clonquant® assay.

  Mice were weighed and then administered CG53135-05 (12 mg / kg) or placebo. An intraperitoneal (ip) injection was made once 24 hours prior to irradiation. As shown in the table below, each group of 6 animals was irradiated. For each radiation dose, the response of the drug treated group and the placebo treated group were compared.

  The small intestine was removed, fixed with Carnoy's fixative, and further processed for histological analysis (embedded in paraffin). H & E sections were prepared according to routine protocols. Ten tentestine circumferences were analyzed for each animal, the number of surviving crypts per margin was evaluated, and the average per group was determined. The crypts comprising 10 or more cells strongly stained with H & E (excluding Paneth cells) and intact margins without Peyer's patches were evaluated.

The average of the crypt width (measured at the widest position) was also measured to correct the evaluation error due to the crypt size difference. The correction was made as follows:
Corrected number of crypts per rim = average number of crypts surviving per rim in group X treatment (average crypt width in untreated controls / crypts in treated animals) Width average).

Table 5: Research plan

result:
The survival of crypts after prophylactic administration of CG53135-05 showed an inverse correlation to the irradiation dose, and the lower the irradiation dose, the higher the survival of the crypts (FIGS. 7 and 8). ). Prophylactic administration of CG53135-05 significantly increased the number of crypts (P <0.001). Table 6 shows the protection factors obtained for radiation dose after prophylactic administration of protein (CG53135-05). The protection factor represents the ratio between treated and untreated cells (Table 6). When CG53135-05 was administered to animals before radiation injury, 1.55 times as many cells survived at a radiation dose of 12 Gy on average.
Table 6:

5.5. Example 5: CG53135-05 upon mechanisms ionizing radiation attacks radiation protection in a number of changes that occur in cells, increased reactive oxygen species occurs via the ionization of H _{2 O.} This process results in the most reactive molecules in the cell to reduce cell damage, so that the cell nucleus increases transcription of enzymes that scavenge these radicals into less reactive intermediates. Since CG53135-05 has been shown to be a radioprotectant, treatment of cells with CG53135-05 is involved in radioprotection to “preload” cells into the oxygen radical scavenging pathway. It is appropriate to find out whether up-regulates any of the known genes. Assessing the effects of CG53135-05 on complex pathways involving ROS scavengers and transcription factors will mechanistically explain the observed in vivo radioprotective effects of this substance. Therefore, expression studies and survival studies were performed at the cellular level.

Expression studies:
To delineate the mechanism of radioprotection by CG53135-05, the expression characteristics of free oxygen radical scavengers and transcription factors in fibroblasts and endothelial cells were studied. NIH3T3 (mouse fibroblasts), CCD-1070sk (human foreskin fibroblasts), CCD-18Co (human colon fibroblasts) and HUVEC (human umbilical cord vascular endothelial cells) cells with 0.1% FBS and a given concentration Transferred to basal medium containing CG53135. After 18 hours of incubation, total RNA was recovered from the cells using RNEasy (Qiagen, Valencia, CA). RNA was reverse transcribed using SuperScript First Strand Synthesis System (for RT-PCR) (Invitrogen, Carlsbad, CA), and the gene of interest was amplified using the primers and the number of cycles shown below.

Table 7: RT-PCR primers (human)

Table 8: RT-PCR primers (mouse)

Results The results are summarized in Table 9 and further shown in FIGS.
Table 9

Among the radioprotective superoxide dismutases, MnSOD, which is the most radioprotective, was found to be consistently induced by CG53135-05 among cell lines (Table 9, FIGS. 9-12). .
The Nrf2 transcription factor involved in the regulation of several antioxidants, thought to be an “antioxidant response element” (also referred to as ARE), was induced by CG53135 in all cell lines studied (Table 9, FIGS. 9 to 12).

  It is well established that one mechanism by which cells are protected from ionizing radiation is through the introduction of oxygen radical scavenging pathways. The gene expression studies described herein indicate that this may be one of the pathways where CG53135-05 may modulate radioprotection. In addition, one of the major target organs for whole body irradiation (TBI) is the gastrointestinal tract. The data disclosed herein are (1) the most reactive of all the cell lines studied is gastrointestinal fibroblasts (CCD18CO); and (2) well-characterized gastrointestinal radiation. The protective agent Tff3 is shown to be strongly upregulated by CG53135-05. Tissues in which CG53135-05 is affected in the same way as hematopoietic stem cells, considering that other radioprotectants well known in the art strictly affect bone marrow survival and not any other compartment While it is important to notice that it is active in, it is equally important for animal survival.

Survival studies:
Cells exposed to a specific dose of radiation prepare for the onslaught of ionizing radicals, repair the damage caused by the radicals, or delay the onset of apoptosis in the face of irreparable damage or possibly a combination of all of these mechanisms. Would have to. Thus, each of these pathways is important for the final survival of the cell and its ability to replicate. This was assessed by the ability of the cells to form colonies in vitro after irradiation.

  To evaluate the effect of CG53135-05 on radioprotection, clonogenic assays were performed using CCD-18co cells, FaDu human squamous cell carcinoma cells, IEC6 and IEC18 rat colon crypt cells and NIH3T3 cells. Cell culture conditions were as follows: NIH3T3 cells were grown in DMEM + 10% bovine serum + 50 μg / ml penicillin / streptomycin; IEC6 and IEC18 cells in DMEM + 10% FBS + 0.1 U / ml insulin + 50 μg / ml penicillin / streptomycin. FaDu cells were grown in MEM + 10% FBS + 1 mM sodium pyruvate + 50 pg / ml penicillin / streptomycin + non-essential amino acids.

Cells were seeded and allowed to attach at a density of 5 × 10 ⁵ (NIH3T3) per 10 cm dish or 5 × 10 ⁵ (IEC18, IEC6, FaDu) per 6-well dish. Then, doses of 10 or 100 ng / ml (IEC18, IEC6, FaDu) or 50 and 200 ng / ml (NIH3T3) in basal media containing 0.1% serum (IEC18, IEC6, FaDu) or 1% serum (NIH3T3) Cells were treated with CG53135 at 16 hours (IEC18, IEC6, FaDu) or 1 hour (NIH3T3). Then, 2.5, 5, 7.5, 10, 12.5 and 15 ky, 130 kVp (resulting in 50 cGy) using an X-ray irradiation device (Faxtron) (Wheeling, IL) equipped with an aluminum filter (0.5 mm). The cells were irradiated at a rate of 1 / min). Immediately after irradiation, cells were trypsinized and NIH3T3: 250, 500, 1000, 2000, 5000 and 10,000 cells per 60 mm dish; FaDu, IEC18 and IEC6: density of 500, 2500 cells per well of 6-well dishes Seeded (duplicate). Cells were grown for 1-2 weeks in complete growth medium until the average colony diameter was 2 mm, after which the colonies were stained with crystal violet and counted.

result:
Results are shown as the slope described below:
D0—curve slope between the last two points, indicating the rate of cell killing at high radiation doses. The value is interpreted as the radiation dose required to reduce the fraction of viable cells to 37% of the previous value on the graph. A smaller number indicates a more rapid cell kill rate.

  D1—Curve slope between the first two points, indicating the rate of cell killing at low radiation doses. The value is interpreted as the radiation dose required to reduce the fraction of viable cells to 37% of the previous value on the graph. A smaller number indicates a more rapid cell kill rate.

  Dq-shoulder width of the curve before an exponential decrease in cell survival is seen. This is essentially a threshold for the amount of radiation needed before the occurrence of cell killing is seen. A higher Dq value indicates that the cells are more fully protected at lower radiation doses.

  The results of the IEC18 clonogenic assay are shown in FIG. Although no trend was seen in D0 and D1, Dq showed that these cells were more protected at lower radiation doses compared to untreated (both dose 10 ng / ml and 100 ng / ml) Has a wider shoulder width). Therefore, it is concluded that at both doses of CG53135-05 IEC18 appears to be more protected at lower radiation doses and requires higher radiation doses until cell killing is seen compared to untreated cells. It is done.

The results of the NIH3T3 clonogenic assay are shown in FIG. The interpretation of these results is as follows:
The final slopes of Do-dose 50 ng / ml and 200 ng / ml both slope towards larger values, approaching significance for a dose of 50 ng / ml. This indicates that the rate of cell death is slower at higher doses compared to untreated.
Dq—no trend is seen between shoulder widths of 50 ng / ml and 200 ng / ml, mainly due to untreated dispersion.
The initial slope with D1-dose of 100 ng / ml tends to a smaller value. This indicates that cell death has a slower rate at lower radiation doses compared to untreated.

  Thus, in NIH 3T3 cell clonogenic assays, there is no difference in protection at low doses, but the rate of cell death is delayed by treatment with CG53135-05 at both low and high doses.

The results of the HUVEC clonogenic assay are shown in FIG. Interpretation of these results is as follows:
The final slope of the Do-dose 100 ng / ml tends to a larger value. This shows that the rate of cell death is slower at higher doses compared to no treatment and 10 ng / ml.
The shoulder width of the Dq-dose 100 ng / ml is wider. This indicates that these cells are protected with a lower radiation dose compared to untreated ones. There was no trend in D1.

  Thus, high doses of CG53135-05 significantly reduce the rate of cell killing at high radiation doses. The cells also appear to be better protected at low radiation doses compared to untreated cells.

  FaDu cells and IEC6 showed no trend at D0, Dq or D1 (data not shown). These results indicate that CG53135-05 has a protective effect against radiation in vitro.

To further optimize these experiments,
(A) provides better cell killing than currently seen at higher radiation doses; and (b) provides conditions for maximum protective effect by CG53135-05 by adjusting treatment conditions and time. .

5.6. Example 6: Effect of CG53135-05 on cytokine release Cytokines are important cell signaling proteins that mediate a wide range of physiological responses. Ionizing radiation can induce a series of changes in gene expression and cytokine properties. The purpose of this study is to evaluate the cytokine properties over time for treatment with CG53135-05 in cell culture over time.

  BioPlex cytokine assay, a multiplex bead-based assay designed to quantify multiple cytokines derived from tissue culture supernatants, was used for cytokine detection. The principle of the assay is the same as the capture sandwich immunoassay. 3T3 cells were seeded in 96 well plates. Cells were washed with DMEM + 0.1% calf serum (SFM). 10 ng / ml and 100 ng / ml CG53135-05 were added to the cells. Cell supernatants were collected after 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours and 24 hours. TNF 50 ng was used as a positive control. Bioplex 18-Plex cytokine assay (BioRad Laboratories Inc, CA) was performed according to the manufacturer's protocol.

Results: FIG. 16 shows the effect of CG53135-05 on Mo KC release. Mo KC is also known as the chemokine CXCL1 (also described as Gro1, melanoma growth stimulating activity (MSGA) or neutrophil activating protein-3 (NAP3)). It functions as a neutrophil chemoattractant that signals through the CXCR1 receptor. It is also involved in the response to whole body irradiation and increases the possibility of having radioprotective properties per se (see Radiat. Res. 160: 637-46, 2003). FIG. 16 shows a constant dose (p = 0.0085) and a time-dependent increase (p = 4.6 × 10 ⁻⁶ ) in the measured response. Furthermore, both concentrations of CG53135-05 showed a significantly higher response than the control (no CG53135-05).

  Further testing can be performed to determine if CG53135-05 when combined with CXCL1 is synergistic in both in vitro and in vivo radioprotection experiments. In addition, other cytokines (eg IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, MCP-1, GM) in different cell lines (eg HUVEC, CCD-18, NIH3T3). -CSF, RANTES) induction can also be tested in the presence of CG53135-05 (those skilled in the art will understand this).

5.7. Example 7: Measurement of scavengers of reactive oxygen intermediates after radiation exposure
As the first step of the CM-H ₂ DCFDA method , cells with increased reactive oxygen species up-regulate superoxide dismutase-Cu, Zn-SOD, Mn-SOD and extracellular-SOD to produce superoxide radicals. Is scavenged into hydrogen peroxide. These enzyme activities can be measured indirectly by the by-product of H ₂ 0 ₂ using acetoxymethyl ester. A derivative of this class, also known as _CM-H 2 DCFDA, 5- (and -6) - chloromethyl-2 ', 7'-dichlorodihydrofluorescein acetate is effectively retained within the cell, When oxidized with H ₂ 0 ₂ , it emits fluorescent green.

IEC18 (rat intestinal epithelial cells) and CCD-18Co (human colon fibroblast) cells were seeded in 60 mm dishes at a density of 1 × 10 ⁵ cells per dish. After attachment, the medium was switched to medium containing 0.1% serum and a predetermined dose of CG53135. After 18 hours of incubation, the cells were irradiated with X-ray 2 or 4 Gy using an X-ray irradiation apparatus (Faxtron) (Wheeling, IL), and incubated for 15 minutes with 5 mM CM-H ₂ DCFDA (Molecular Probes, Eugene, OR). did. Cells were then washed, trypsinized, and analyzed on the FL1 channel of FACSCalibur (Becton Dickinson, San Jose, Calif.).

The results show that IEC18 and CCD18Co cells have increased intracellular H ₂ O ₂ in a dose-responsive manner after treatment with CG53135-05 (FIGS. 17, 18 and 19). This is thought to be due to the enhanced expression of superoxide dismutase (mainly MnSOD) induced by CG53135-05.

Intracellular H ₂ O ₂ production by CG53135-05 in IEC18 cells is enhanced with increasing ionizing radiation dose (FIG. 18). This result reflects that irradiation increases the production of more reactive oxygen species, such as superoxide and hydroxyl, thus increasing the substrate of superoxide dismutase induced by CG53135-05. .

Using Red CC-1 method ionizing radiation exposure, the cells react as a result of ionizing H ₂ O to hydroxyl radical (OH), superoxide (0 ₂ ⁻ ) or hydrogen peroxide (H ₂ O). Accumulate oxygen species. Because large amounts of these ions can have deleterious effects on cells, the pathway is up-regulated, scavenging these molecules into more stable converters. It is hypothesized that CG53135-05 can protect cells from ionizing radiation by up-regulating pathways that reduce the cytosolic redox capacity. The die, Redox Sensor 1 (Red CC-1), can monitor the redox level of the cytosol upon oxidation by changing to a red fluorescent agent that can be measured by FACS (FL2 channel).

Cytosol reactive oxygen species + Red CC-1 → Oxidized Red CC-1

IEC18 (rat intestinal epithelial cells) and CCD-18Co (human colon fibroblast) cells were seeded in 60 mm dishes at a density of 1 × 10 ⁵ cells per dish. After attachment, the cell culture medium was switched to medium containing 0.1% serum and a predetermined dose of CG53135. After 18 hours of incubation, the cells were irradiated with X-ray 2 or 4 Gy using an X-ray irradiator (Faxtron) and incubated with 5 mM Red CC-1 (Molecular Probes, Eugene, OR) for 15 minutes. The cells were then washed, trypsinized and analyzed on the FL2 channel of FACSCalibur (Becton Dickinson).

  Results: IEC18 and CCD18Co cells were found to have reduced cytosolic redox capacity in a dose-responsive manner after treatment with CG53135-05 as shown in FIGS. The data presented here is thought to result from increased expression of superoxide dismutase induced by CG53135-05, which scavenges more reactive species of superoxide and hydroxyl radicals. . Furthermore, CG53135-05 increases the expression of Nrf2, a major anti-oxidant-regulating transcription factor, which is shown to contribute to this decrease in cytosolic reactivity in other ways.

6). Equivalents and Cited References All publications, including patents and patent applications cited herein, are hereby incorporated by reference in their entirety.
Numerous modifications and variations of the present invention may also be within the spirit and scope of the invention, as will be apparent to those skilled in the art. The specific embodiments described herein are provided for purposes of illustration only. Thus, we do not intend to be limited to the exact terms and limitations described herein, but we wish to make use of such modifications and variations that can be made to adapt the invention to various uses and conditions. . Such modifications and variations differ in the composition for administering the polypeptide to the subject according to the present invention; in the amount of the polypeptide; in the number of administrations and in the means; (Including, but not limited to, combinations of different peptides or combinations of compounds and peptides having different biological activities). Such modifications and variations are modifications in the amino acid sequence of a particular polypeptide described herein (where such variations do not alter the functionality of the polypeptide, but do not alter the peptide in the composition to be administered to the subject). It alters the sequence in a manner that alters solubility, absorption of the peptide by the body, within the shelf life, or the protection of the polypeptide by the time the physiological action of the peptide in the body can produce the desired effect. As well as such similar modifications. Accordingly, such modifications and variations are suitably intended to be within the full scope of equivalents of the present invention.

FIG. 1 shows the effect of CG53135-05 on the survival of mice after a single exposure to acute radiation, a dose of 600 cGy. FIG. 2 shows the change in average body weight of mice after a single exposure to acute radiation, a dose of 600 cGy. FIG. 3 shows the effect of phosphate buffered saline (PBS) controls on the survival of mice after exposure to radiation doses of 484 cGy, 534 cGy, 570 cGy, 606 cGy or 641 cGy. FIG. 4 shows the effect of prophylactic administration (day -1) of CG53135-05 on the survival of mice after exposure to radiation doses of 484 cGy, 534 cGy, 570 cGy, 606 cGy or 641 cGy. FIG. 5 shows the effect of prophylactic administration (-2 and -1 days) of CG53135-05 on the survival of mice after exposure to radiation doses of 484 cGy, 534 cGy, 570 cGy, 606 cGy or 641 cGy. FIG. 6 shows the cell location of the crypts. FIG. 7 shows crypt survival curves comparing prophylactic administration of CG53135-05 treatment to the PBS control group after different irradiation. FIG. 8 shows the effect of prophylactic administration of CG53135-05 on the survival of intestinal crypts in mice after undergoing radiation damage. FIGS. 9A and 9B show that CG53135-05 induced scavenger, cyclooxygenase, trefoil factor and transcription factor expression in NIH3T3 cells. FIG. 10 shows that CG53135-05 induced scavenger, cyclooxygenase, trefoil factor and transcription factor expression in CCD1070sk cells. FIG. 11 shows that CG53135-05 induced scavenger, cyclooxygenase, trefoil factor and transcription factor expression in CCD18Co cells. FIG. 12 shows that CG53135-05 induced scavenger, cyclooxygenase, trefoil factor and transcription factor expression in human umbilical vein endothelial cells (HUVEC). FIG. 13 shows the effect of CG53135-05 on the survival of IEC18 cells irradiated with different X-ray doses. FIG. 14 shows the effect of CG53135-05 on the survival of NIH 3T3 cells irradiated at different X-ray doses. FIG. 15 shows the effect of CG53135-05 on the survival of HUVEC irradiated at different X-ray doses. FIG. 16 shows the effect of CG53135-05 on cytokine release in NIH 3T3. FIG. 17 shows the dose response of CM-H2DCFDA fluorescence from IEC18 cells treated with CG53135-05 after irradiation with 4 Gy. FIG. 18 shows the dose response of CM-H2DCFDA fluorescence from IEC18 cells treated with CG53135-05 after irradiation with 2 Gy and 4 Gy. FIG. 19 shows the dose response of CM-H2DCFDA fluorescence from CCD-18Co cells treated with CG53135-05 after irradiation with 4 Gy. FIG. 20 shows the dose response of Red CC-1 fluorescence from IEC18 cells treated with CG53135-05 after irradiation with 4 Gy. FIG. 21 shows the dose response of Red CC-1 fluorescence from IEC18 cells treated with CG53135-05 after irradiation with 4 Gy and 6 Gy. FIG. 22 shows the dose response of Red CC-1 fluorescence from CCD-18Co cells treated with CG53135-05 after irradiation with 10 Gy.

Claims (17)

  A method of preventing or treating a disease associated with radiation exposure or one or more symptoms thereof in a subject, comprising administering to the subject a composition comprising a CG53135-05 polypeptide.   The method of claim 1, wherein the subject is a human.   The method of claim 1, wherein the disease is neutropenia.   The method according to claim 1, wherein the disease is gastrointestinal mucositis.   2. The method of claim 1, wherein the symptom is diarrhea, skin burns, wounds, fatigue, dehydration, inflammation, hair loss, oral mucosal ulcers or bleeding from the nose, mouth or rectum.   2. The method of claim 1, wherein a prophylactically effective amount of said polypeptide is administered to a subject that is potentially exposed to radiation.   7. The method of claim 6, wherein the effective amount is a single dose of the polypeptide.   8. The method of claim 7, wherein the effective amount is greater than or equal to a single dose of the polypeptide.   2. The method of claim 1, wherein a therapeutically effective amount of the polypeptide is administered to the subject after exposure to radiation.   10. The method of claim 9, wherein the effective amount is a single dose of the polypeptide.   10. The method of claim 9, wherein the effective amount is greater than or equal to a single dose of the polypeptide.   The method of claim 1, wherein the polypeptide is administered by a parenteral route.   13. The method of claim 12, wherein the route of administration comprises intravenous, intramuscular, subcutaneous, intradermal or nasal administration.   2. The method of claim 1, wherein a therapeutically effective amount of said polypeptide is formulated in a pharmaceutical composition.   A method of recovering or protecting hematopoietic tissue from radiation damage comprising administering to a subject in need thereof a composition comprising a CG53135-05 polypeptide.   A method of recovering or protecting gastrointestinal tissue from radiation damage comprising administering to a subject in need thereof a composition comprising a CG53135-05 polypeptide. A method of preventing or treating a disease associated with exposure to mustard gas or one or more symptoms thereof in a subject, comprising administering to the subject a composition comprising a CG53135-05 polypeptide. Method.

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