AU2004271913A1 - Therapeutic use of G53135-05(FGF-20) in radiation protection - Google Patents

Description

WO 2005/025489 PCT/US2004/014622 5 THERAPEUTIC USE OF G53135-05 (FGF-20) IN RADIATION PROTECTION This application claims the benefit of U.S. Provisional Application Serial Nos. 60/469,353, filed May 9, 2003, and 60/544,308, filed February 26, 2004, as well as to U.S. Provisional Application identified by Attorney Docket No. Cura 57UN (entitled "Therapeutic Use of G53135-05 (FGF-20) in Radiation Protection", filed April 26, 2004, all of which are incorporated herein by reference in their entireties. 10 1. FIELD OF INVENTION The present invention relates to methods of preventing or treating one or more symptoms associated with radiation exposure. In particular, the present invention provides methods of preventing or treating one or more symptoms (e.g., gastrointestinal (GI) syndrome, hematopoletic (bone marrow) syndrome, and cerebrovascular (CNS) syndrome) associated with radiation exposure comprising 15 administering to a subject in need thereof a composition comprising CG53135-05, a Fibroblast Growth Factor-20 (FGF-20) protein. 2. BACKGROUND OF THE INVENTION The FGF family consists of more than 20 members, each containing a conserved amino acid core (see, e.g., Powers et al., Endocr. Relat. Cancer, 7(3):65-197 (2000)). FGFs regulate diverse cellular 20 functions such as growth, survival, apoptosis, motility, and differentiation (see, e.g., Szebenyi et al., Int. Rev. Cytol., 185:45-106 (1999)). Members of the 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 et al., Endocr. Relat. Cancer, 7(3):165-197 (2000); or Szebenyi.et al., Int. Rev. Cytol. 185:45-106 (1999)). 25 Through a homology-based genomic mining process, a new member of the FGF family, FGF-20 (also known as CG53135), has been identified (see e.g., Jeffers et al., Cancer Res., 61(7):3131-8 (2001)). A single, large 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: 30 cerebrovascular (CNS) syndrome, gastrointestinal (GI) 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 CNS syndrome, within 3-10 days for GI 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 35 loss, neutropenia, ulceration of the oral mucosa and GI system, and 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. Specifically affected stem cells in either the WO 2005/025489 PCTIUS2004/014622 5 gastrointestinal and/or hematopoietic systems represent a cellular target for pharmacologic intervention. Neutropenia (a decrease in the number of neutrophils resulting in increased risk of infection) and gastrointestinal mucositis represent two of the most significant causes of mortality resulting from ionizing radiation. Mustard agents, like radiation, cause similar effects at the cellular level; their use in combination will have a geometric effect on morbidity. 10 Palliative medical interventions, such as blood cell replacements, antibiotics, cytokines, and in high dose cases, hematopoietic stem cell transplants, could increase overall survival. Biological modification of radiation response has been an active area of investigation for many years. The development of both active radiation sensitizers and protectants could also provide therapeutic advantages to patients undergoing radiation therapy (see, e.g., Cancer Chemotherapeutic Agents, 15 1995:501-527). Additional indications for radioprotectants include the protection of personnel involved in accidental exposure to ionizing radiation in an industrial or military setting. Treatment with members of the FGF family have improved survival and hematopoietic recovery in mice following ionizing radiation to the whole body (see, e.g., Cytokine, 9(1):59-65 (1997); Acta Oncol.,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)). 20 Acidic FGF (aFGF or FGF-1 , 1-24 mg) increased 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)). The radioprotective effects of 3 other FGF family members, basic FGF (bFGF or FGF-2), keratinocyte growth factors 1 and 2 (KGF-1 or FGF-7, and KFG-2 or FGF-10), have been examined in both C3H/He and BalbC mice (see Am. J. Clin. Oncol., 24(5):491-5 (2001)). These studies showed that the LD50 for ionizing radiation was 25 increased by approximately 100 cGy with a single dose (6 pg) of growth factor. Ionizing radiation is called so because of the ability of the radiation particles to generate reactive ions when bombarded against another molecule within the cell or elsewhere. As one of the most common molecules within a cell is water, H 2 0, 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 30 (ROS), include superoxide (O2), hydroxyl (HO) and hydroge peroxide (H 2 0 2 ) (see Proc. NatI. Acad. Sci. USA, 78:1001-1003 (1981)). While these molecular, intermediates are useful to the cell, for example, the production of energy by means of the electron transport pathway in the mitochondria, an overabundance of them can cause tissue destruction due to their highly reactive nature. Breakage of genomic DNA, depolarization of the mitochondrial membrane and alteration of proteins are among the damages that 35 such radicals can cause. The body's first line of defense against these factors are enzymes that scavenge the radicals to less reactive species. These include the superoxide dismutases (MnSOD, Cu, ZnSOD, extracellular-SOD), glutathione peroxidase and genes induced by the transcription factor Nrf2 (see Free Radic. Biol. Med., 17:389-395 (1994); FASEB J., 7:361-368 (1993); Proc. Nati. Acad. Sci. USA 94:5361-5366). Indeed, many of these proteins have been shown to be radioprotective to cells when 40 overexpressed (see 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 the expression of Nrf2 (see l\/ol. Cell Biol., 22:5492-5505 (2002)). Therefore, since ROS play a major role 2 WO 2005/025489 PCTIUS2004/014622 5 in cellular damage after ionizing radiation, understanding the effect of FGF-20 on ROS-mediated pathways will delineate some of the mechanisms of radioprotective effect on the whole animal. 3. SUMMARY OF THE INVENTION The present invention provides methods of preventing or treating one or more symptoms associated with radiation exposure, chemotherapy, chemical warfare agents, and/or any other insults 10 affecting rapidly proliferating tissues in the body by administering to a subject in need thereof a composition comprising CG53135-05, a FGF-20 protein. The present invention is based, in part, upon the inventors' discovery that CG51 35-05 protects rapidly proliferating tissues, such as hematopoietic and gastrointestinal tissues, from insults such as radiation exposure, chemotherapy, and chemical warfare agents. While not limited by any theory, CG53135-05 is believed to protect stem cells associated with the 15 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 provides a method of treating a subject exposed to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical warfare agents) comprising 20 administering to the subject a therapeutically effective amount of a composition comprising CG53135-05 polypeptide. The present invention also 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 warfare agents) comprising administering to the subject a therapeutically 25 effective amount of a composition comprising CG53135-05 polypeptide. In one embodiment, the present invention provides a method of treating neutropenia of a subject exposed to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical warfare agents) comprising administering to the subject a therapeutically effective amount of a composition comprising CG53135-05 polypeptide. 30 The present invention also 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 warfare agents) comprising administering to the subject a therapeutically effective amount of a composition comprising CG53135-05 polypeptide. In one embodiment, the present invention provides a method of treating gastrointestinal mucositis of a subject exposed to an insult 35 affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical warfare agents) comprising administering to the subject a therapeutically effective amount of a composition comprising CG53135-05 polypeptide. The present invention further provides a method of preventing, treating, or ameliorating a symptom (such as but is not limited to, diarrhea, skin burn, sores, fatigue, dehydration, inflammation, hair 40 loss, ulceration of oral mucosa, and bleeding for the nose, mouth or rectum) associated with an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical warfare agents) 3 WO 2005/025489 PCTIUS2004/014622 5 comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a composition comprising CG53135-05 polypeptide. In one embodiment, the composition comprising CG53135-05 is administered prior to the subject's exposure to the insult. In another embodiment, the composition comprising CG53135-05 is administered after the subject's exposure to the insult. In yet another embodiment, CG53135-05 polypeptide is administered to a subject who is at risk of 10 exposing to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical warfare agents). The present invention provides a method of protecting and/or regenerating hematopoietic tissues of a subject comprising administering to the subject a prophylactically effective amount of a composition comprising CG53135-05 polypeptide prior to the subject's exposure to an insult affecting rapidly 15 proliferating tissues such as radiation, chemotherapy, and chemical warfare agents. In one embodiment, the present invention provides a method of preventing neutropenia of a subject comprising administering to the subject a prophylactically effective amount of a composition comprising CG53135-05 polypeptide prior to the subject's exposure to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical warfare agents). 20 The present invention also provides a method of protecting and/or regenerating gastrointestinal tissues of a subject comprising administering to the subject a prophylactically effective amount of a composition comprising CG53135-05 polypeptide prior to the subject's exposure to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical warfare agents). In one embodiment, the present invention provides a method of preventing gastrointestinal mucositis of a subject 25 comprising administering to the subject a prophylactically effective amount of a composition comprising CG53135-05 polypeptide prior to the subject's exposure to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical warfare agents). In one embodiment, 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 30 warfare agents) comprising administering to the subjects a prophylactically or therapeutically effective amount of a composition comprising CG53135-05 polypeptide. The therapeutically effective dose may be a single dose, two or more than two doses of CG53135-05. In another embodiment, a single prophylactic dose of CG53135-05 is administered to a subject followed by an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical 35 warfare agents), where such prophylactic dose causes a defined, short acting proliferative effect on various compartments in the proliferating tissues (e.g., intestinal villi). In another embodiment, more than a single prophylactic dose, which may be two or more than two doses of CG53135-05 polypeptide is administered to a subject exposed to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical warfare agents) to preventing, treating or ameliorating a symptom 40 associated with the insult. 4 WO 2005/025489 PCTIUS2004/014622 5 In some embodiments, an insult affecting rapidly proliferating tissues is radiation exposure. In some embodiments, an insult affecting rapidly proliferating tissues is one or more alkylating agents, one or more mustard agents, or one or more other chemotherapeutic agents, or a combination thereof. In some embodiments, an insult affecting rapidly proliferating tissues is a radiation exposure, one or more alkylating agents, one or more mustard agents, or one or more other chemotherapeutic agents, or a 10 combination thereof. In some embodiments, CG53135-05 polypeptide is used in combination with one or more other therapies known in the art to prevent, treat, or ameliorating one or more symptoms associated with an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical warfare agents). 15 Pharmaceutical compositions, formulations, and kits are also provided by the present invention. 3.1. Terminoloqv The invention is based on the identification of novel FGF-20 nucleic acid and amino acid and their variants referred to here and elsewhere as CG53135 (referred to as "FGF-C)C' in U.S. Apple. Serial. Nos. 09/494,585 and 10/174,393, both of which are incorporated herein by reference in toto). As used herein, 20 the term "CG53135-05 polypeptide" refers to an isolated polypeptide comprising the amino acid sequence consisting of 211 amino acids. Furthermore the invention also includes fragments, derivatives, variants, homologs or analogs of CG53135-05. I n some embodiments, the invention includes a variant of CG53135-05 polypeptide, in which some amino acids residues, e.g., no more than 1%, 2%, 3%, 5%, 10% or 15% of the amino acid sequences of CG53135-05 are changed. In some embodiments, the isolated 25 CG53135-05 polypeptide includes the amino acid sequence of a mature form of an amino acid sequence given by CG53135-05, or a variant of a mature form of an amino acid sequence given by CG53135-05. Preferably, no more than 1%, 2%, 3%, 5%, 10% or 15% of the amino acid sequences of CG53135-05 are changed in the variant of the mature form of the amino acid sequence. As used herein, the term "effective amount" refers to the amount of a therapy (e.g., a composition 30 comprising CG53135-05 polypeptide) which is sufficient to reduce and/or ameliorate the severity and/or duration of a disease or disorder characterized by an insult to rapidly proliferating tissues (such as radiation, chemotherapy, and chemical 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 35 improve the prophylactic or therapeutic effect(s) of another therapy (e.g., prophylactic or therapeutic agent). As used herein, the term "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 40 minutes, 1 hour, 2 hours, 4 hours, 6 ho urs, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 vveeks, 8 weeks, or 12 weeks before), concomitantly with, or 5 WO 2005/025489 PCTIUS2004/014622 5 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. As used herein, the term prophylacticallyy effective amount" refers to the amount of a therapy (e.g., CG53135-05 polypeptide) which is sufficient to result in the prevention of the development, 10 recurrence, or onset of a disease or disorder associated with an insult to rapidly proliferating tissues (such as radiation, chemotherapy, and chemical warfare agents) or one or more symptoms thereof, or to enhance or improve the prophylactic effect(s) of another therapy. As used herein, the terms "subject" and "subjects" refer to an animal, preferably a mammal, including a non-primate (e.g., a cow, pig, horse, cat, dog, rat, or mouse) and a primate (e.g., a monkey, 15 chimpanzee, or human), and more preferably a human. In a certain embodiment, the subject is a mammal, preferably a human, who has exposed to or is going to expose to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, or chemical warfare agents). In another embodiment, the subject is a farm animal (e.g., a horse, pig, or cow) or a pet (e.g., a dog or cat) which has exposed to or is going to expose to an insult affecting rapidly proliferating tissues (such as radiation, 20 chemotherapy, and chemical warfare agents). In another embodiment, the subject is a mammal, preferably a human, who may expose to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical warfare agents).. The term "subject" is used interchangeably with "patient" in the present invention. As used herein, the term "therapeutically effective amount" refers to the amount of a therapy 25 (e.g., 53135-05 polypeptide), which 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 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 30 warfare agents), or enhance or improve the therapeutic effect(s) of another therapy. The terms "therapies" and "therapy" can refer to any protocol(s), method(s), and/or agent(s) that can be used in the prevention, treatment, or amelioration of a disease or disorder characterized by an insult to rapidly proliferating tissues (such as radiation, chemotherapy, and chemical warfare agents) or one or more symptoms thereof. In certain embodiments, the terms "therapy" and "therapy" refer to anti 35 viral therapy, anti-bacterial therapy, anti-fungal therapy, biological therapy, supportive therapy, and/or other therapies useful in treatment or amelioration of a disease or disorder characterized by an insult to rapidly proliferating tissues (such as radiation, chemotherapy, and chemical warfare agents) or one or more symptoms thereof known to skilled medical personnel. 4. BRIEF DESCRIPTION OF THE FIGURES 40 Figure 1 shows the effect of CG53135-05 on mouse survival after single exposure to acute radiation dose of 600 cGy. 6 WO 2005/025489 PCTIUS2004/014622 5 Figure 2 shows the mean weight change in mice after single exposure to acute radiation of 600 cGy. Figure 3 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. Figure 4 shows the effect of prophylactic administration of CG53135-05 (day-1) on mice survival 10 after exposure to radiation doses of 484 cGy, 534 cGy, 570 cGy, 606 cGy, or 641 cGy. Figure 5 shows the effect of prophylactic administration of CG53135-05 (day-2 and -1) on mice survival after exposure to radiation doses of 484 cGy, 534 cGy, 570 cGy, 606 cGy, or 641 cGy. Figure 6 shows the cell positions in the crypt. Figure 7 shows the crypt survival curve comparing prophylactic administration of CG53135-05 15 treatment to PBS control group following different radiation dosages. Figure 8 shows the effect of prophylactic administration of CG53135-05 on mice intestinal crypt survival after radiation insult. Figure 9A and 9B show CG53135-05 induced expression of scavengers, cycloxegenase trefoil factor, and transcription factors in NIH 3T3 cells. 20 Figure 10 shows CG53135-05 induced expression of scavengers, cycloxegenase, trefoil factor, and transcription factors in CCD1 070sk cells. Figure 11 shows CG53135-05 induced expression of scavengers, cycloxegenase, trefoil factor, and transcription factors in COD1 8Co cells. Figure 12 shows CG53135-05 induced expression of scavengers, cycloxegenase, trefoil factor, 25 and transcription factors in Human Umbilical Vein endothelial cells (HUVEC). Figure 13 shows the effect of CG531 35-05 on the survival of IEC 18 cells irradiated with different X-ray doses. Figure 14 shows the effect of CG53135-05 on the survival of NIH 3T3 cells irradiated with different X-ray doses. 30 Figure 15 shows the effect of CG53135-05 on the survival of HUVEC irradiated with different X ray doses. Figure 16 shows the effect of CG53135-05 on the release of cytokine in NIH 3T3 cells. Figure 17 shows dose response of CM-H2DCFDA fluorescence from IECl 8 cells treated with CG53135-05 after 4Gy irradiation. 35 Figure 18 shows response of CM-H2DCFDA fluorescence from IECI8 cells treated with CG53135-05 after 2Gy and 4Gy irradiation. 7 WO 2005/025489 PCTIUS2004/014622 5 Figure 19 shows dose response of CM-H2DCFDA fluorescence from CCD-18Co cells treated with CG53135 after 4Gy irradiation. Figure 20 shows dose response of Red CC-1 fluorescence from IEC1 8 cells treated with CG53135 after 4Gy irradiation. Figure 21 shows response of Red CC-1 fluorescence from IEC1 8 cells treated with CG53135-05 10 after 4Gy and 6Gy irradiation. Figure 22 shows response of Red CC-1 fluorescence from CCD-18Co cells treated with CG53135 before and after 10Gy irradiation. 5. DETAILED DESCRIPTION OF THE INVENTION The present invention provides methods of preventing or treating one or more symptoms 15 associated with radiation exposure, chemotherapy, chemical warfare agents, and/or any other insults affecting rapidly proliferating tissues in the body by administering to a subject in need thereof a composition comprising CG53135-05 polypeptide, which is a FGF-20 protein. CG53135-05 polypeptide and methods of making such a polypeptide are described in U.S. Application Serial Nos. 09/494,585 and 10/174,394, both of which are incorporated herein by reference in 20 their entireties. The present invention provides a method of 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 warfare agents) comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a composition comprising CG53135-05 polypeptide. 25 In some embodiments, an insult affecting rapidly proliferating tissues is radiation exposure. In a specific embodiment, the insult is ionizing radiation. In another embodiment, the insult may be one or more chemotherapies or one or more chemical warfare agents, or a combination thereof. Non-limiting examples of chemotherapy and chemical warfare agent are alkylating agents and mustard agents. In some embodiments, an insult affecting rapidly proliferating tissues is one or more radiation exposures, 30 one or more chemotherapies, one or more chemical warfare agents, or a combination thereof. Organs and body systems most sensitive to the effects of insult such as ionizing radiation include the skin, hematopoietic and lymphatic systems, gonads, lungs, nerve tissues, and the GI tract. In one embodiment, the insult are particularly damaging to hematopoietic and/or gastrointestinal tissues of a subject. In a specific embodiment, the disorder to be prevented or treated is netropenia. In another 35 embodiment, the disorder to be prevented or treated is rnucositis. In some embodiments, the symptoms associated with an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical warfare agents) include, but are not limited to, diarrhea, skin burn, sores, fatigue, dehydration, inflammation, hair loss, ulceration of oral mucosa, and bleeding for the nose, mouth or rectum. 8 WO 2005/025489 PCTIUS2004/014622 5 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 warfare agents), subjects who are suspected to have been exposed an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical warfare agents), subject who will be exposed to an insult affecting rapidly proliferating tissues 10 (such as radiation, chemotherapy, and chemical warfare agents), and subject who are at risk to be exposed to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical warfare agents). In one embodiment, the composition comprising CG53135-05 is administered prior to the subject's exposure to the insult. In another embodiment, the composition comprising CG53135-05 is 15 administered after the subject's exposure to the insult. In yet another embodiment, CG53135-05 polypeptide is administered to a subject who are at risk for exposure to an insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical warfare agents). Compositions comprising CG53135-05 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 20 insult affecting rapidly proliferating tissues (such as radiation, chemotherapy, and chemical warfare agents). In a preferred embodiment, compositions comprising CG53135-05 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 warfare agents). Examples of such other therapies include, but 25 are not limited to, Mesna (sodium 2-mercaptoethene s ulfonate) 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. In one embodiment, during a combination therapy, CG53135-05 polypeptide and/or another therapy are administered in a sub-optimal amount, e.g., an amount that does not manifest detectable 30 therapeutic benefits when administered alone, as determined by methods known in the art. In such methods, co-administration of CG53135-05 polypeptide and another therapy results in an overall improvement in effectiveness of treatment, In one embodiment, CG53135-05 polypeptide and one or more other therapies are administered within the same patient visit. In another embodiment, CG53135-05 polypeptide is administered prior to 35 the administration of one or more other therapies. In yet another embodiment, the CG53135-05 polypeptide is administered subsequent to the administration of one or more other therapies. In a specific embodiment, CG53135-05 polypeptide and one or more other therapies are cyclically administered to a subject. Cycling therapy involves the administration of CG53135-05 polypeptide for a period of time, followed by the administration of one or more other therapies for a period of time and repeating this 40 sequential administration. Cycling therapy can reduce the development of resistance to one or more of 9 WO 2005/025489 PCTIUS2004/014622 5 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 (e.g., CG53135-05 polypeptide) 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 5 o (the dose therapeutically 10 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 LD 5 0

/ED

5 0 . 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. 15 In one embodiment, 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 ED50 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 20 professional (e.g., a physician). For any composition used in the method of the invention, 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 IC50 (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 25 humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. In some embodiments, the dosage of CG53135-05 polypeptide for administration in a human patient provided by the present invention is at least 0.1 mg/kg, at least 0.5 mg/kg, at least 1mg/kg, at least 2 mg/kg, at least 3 mg/kg, at least 4 mg/kg, at least 5 mg/kg, at least 6 mg/kg, at least 7 mg/kg, at least 8 mg/kg, at least 9 mg/kg, at least 10 mg/kg, at least 15 mg/kg, at least 20 mg/kg, at least 25 mg/kg, at 30 least 30 mg/kg, at least 35 mg/kg, at least 40 mg/kg, at least 45 mg/kg, at least 50 mg/kg, at least 60 mg/kg, at least 70 mg/kg, at least 80 mg/kg, at least 90 mg/kg, at least 1 00 mg/kg, at least 150 mg/kg, or at least 200 mg/kg. In some embodiments, the dosage of CG53135-05 polypeptide for administration in a human patient provided by the present invention is between 0.1-300 mg/kg, between 0.5-250 mg/kg, between 1-200 mg/kg, between 1-150 mg/kg, between 1-125 mg/kg, between 1-100 mg/kg, between 1-90 35 mg/kg, between 1-80 mg/kg, between 1-70 mg/kg, between 1-60 mg/kg, between 1-50 mg/kg, between 1 40 mg/kg, between 1-35 mg/kg, between 1-30 mg/kg, between 1-25 mg/kg, between 1-20 mg/kg, between 1-15 mg/kg, between 1-10 mg/kg, or between 1-5 mg/kg. The appropriate and recommended dosages, formulation and routes of administration for treatment modalities such as chemotherapeutic agents, radiation therapy and 40 biological/immunotherapeutic agents such as cytokines are known in the art and described in such literature as the Physician's Desk Reference (58th ed., 2004). 10 WO 2005/025489 PCTIUS2004/014622 5 Various delivery systems are known and can be used to administer a composition of the invention. 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, i ntraperitoneal, intrathecal, 10 intracerebroventricular, epidural, intravenous, subcutaneous, intranasal, intratumoral, transdermal, rectal, and oral routes. The compositions of the invention may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, virginal mucosa, rectal and intestinal mucosa, etc.), and rnay be administered together with other biologically active agents. Administration can be systemic or local. 15 In some embodiments, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment. This may be achieved by, for example, local infusion during surgery, or topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant (said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic 20 membranes, or fibers). In one embodiment, administration can be by direct injection at the site (or former site) of rapidly proliferating tissues which are most sensitive to an insult such radiation, chemotherapy, or chemical warfare agent. In some embodiments, where the composition of the invention is a nucleic acid encoding a prophylactic or therapeutic agent, the nucleic acid can be administered in vivo to promote expression of 25 their encoded proteins (e.g., CG53135-05 polypeptide), 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. Alternatively, a nucleic acid of the 30 invention can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination. The compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions that can be used in the preparation of unit dosage forms. In a preferred embodiment, a composition of the invention is a pharmaceutical composition. Such compositions 35 comprise a prophylactically or therapeutically effective amount of one or more compositions (e.g., CG53135-05 polypeptide) of the invention, and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical compositions are formulated to be suitable for the route of administration to a subject. In one embodiment, the term "pharmaceutically acceptable" rneans approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally 40 recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which 11 WO 2005/025489 PCTIUS2004/014622 5 the prophylactic or therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils (e.g., oils of petroleum, animal, vegetable o r 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, binders, tablet disintegrating agents, or encapsulating material. Water is a preferred carrier when the pharmaceutical 10 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, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, or a combination thereof. The composition, if desired, can also contain 15 minor amounts of wetting or emulsifying agents, or pH buffering agents. The compositions of the present invention may be formulated into any of many 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. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may 20 further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension rnay 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, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. 25 In some embodiments, the compositions of the present invention may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically sirnilar in nature, these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be 30 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 a specific embodiment, 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. Typically, 35 compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. If the compositions of the invention are to be administered topically, the compositions can be formulated in the form of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, 40 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 12 WO 2005/025489 PCTIUS2004/014622 5 useful. Preferred topical formulations include those in which the molecular complexes 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. Preferred lipids and liposomes include, but are not limited to, neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline), negative (e.g. dimyristoylphosphatidyl glycerol DMPG), and cationic (e.g. 10 dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). The molecular complexes of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, the molecular complexes may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic 15 acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1 dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C1-10 alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride, or pharmaceutically acceptable salt thereof. For non-sprayable topical dosage forms, 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 20 than water are typically employed. Other 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 in a squeeze bottle. 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. 25 If the method of the invention comprises intranasal administration of a composition, the composition can be formulated in an aerosol form, spray, mist or in the form of drops. In particular, prophylactic or therapeutic agents for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, 30 carbon dioxide or other suitable gas). In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, e.g., gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. If the method of the invention comprises oral administration, compositions can be formulated in 35 the form of powders, granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. 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 rnethylcellulose); fillers (e.g., 40 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). The tablets may be coated by methods well-knovvn in the art. Liquid 13 WO 2005/025489 PCTIUS2004/014622 5 preparations for oral administration may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cell ulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, 10 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). In one embodiment, the molecular complexes of the invention are orally administered in conjunction with one or more penetration enhancers, e.g., surfactants and chelators. Preferred 15 surfactants include, but are not limited to, fatty acids and esters or salts thereof, bile acids and salts thereof. Preferred bile acids/salts include, but are not limited to, chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24, 25 dihydro-fusidate, sodium glycodihydrofusidate. Preferred fatty acids include, but are not limited to, 20 arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1 monocaprate, 1 -dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium). In some embodiments, combinations of penetration enhancers are used, e.g., fatty acids/salts in combination with bile 25 acids/salts. In a specific embodiment, 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-cety ether. Molecular complexes 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 molecular complexes of the invention 30 include, but are not limited to, poly-amino acids, polyimines, polyacrylates, polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates, cationized gelatins, albumins, acrylates, polyethyleneglycols (PEG), polyalkylcyanoacrylates, 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, 35 polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). 40 The method of the invention may comprise pulmonary administration, e.g., by use of an inhaler or nebulizer, of a composition formulated with an aerosolizing agent. 14 WO 2005/025489 PCTIUS2004/014622 5 The method ofthe invention may comprise administration of a composition 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 10 dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use. In a preferred embodiment, the composition is formulated in accordance vith routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueo us buffer. Where 15 necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, 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 hermetically sealed container, such as an ampoule or sachette, indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an 20 infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. The compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include, but are not limited to, those formed with free amino groups such as those 25 derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. Non-limiting examples of pharmaceutically acceptable salts are acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium acetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, 30 estolate, esylate, fumarate, glucaptate, gluconate, glutamate, glycol lylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate, phosphateldiphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, triethiodide, benzathine, 35 chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminium, calcium, lithium, magnesium, potassium, sodium, and zinc. In addition to the formulations described previously, the compositions 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. Thus, for example, the compositions 40 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 15 WO 2005/025489 PCTIUS2004/014622 5 soluble salt. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophilic drugs. In one embodiment, the ingredients of the compositions of the invention are derived from a subject that is the same species origin or species reactivity as recipient of such compositions. The invention also provides kits for carrying out the therapeutic regimens of the invention. Such 10 kits comprise in one or more containers prophylactically or therapeutically effective amounts of the composition of the invention (e.g., CG53135-05 polypeptide) 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. Alternatively, the composition may be lyophilized or desiccated; in this instance, 15 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. In another embodiment, 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 20 optionally included for administration of the formulations of the invention by a clinician or by the patient. In some embodiments, the present invention provides kits comprising a plurality of containers each comprising a pharmaceutical formulation or composition comprising a dose of the composition of the invention (e.g., CG53135-05 polypeptide) sufficient for a single administration. As with any pharmaceutical product, the packaging material and container are designed to 25 protect the stability of the product during storage and shipment. In one embodiment, 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. Further, 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 30 disease or disorder in question. The present invention is further illustrated by the following examples. 5.1. Example 1: Protein Expression and Purification Recombinant human CG53135-05 was purified from Escherichia coiBLR (DE3) cells (Novagen, Madison, WI) as follows. The DE3 cells were transformed with full-length, codon-optimized CG53135-05 35 cloned in a pET24a vector (Novagen), and a manufacturing master cell bank (NvIMCB) of these cells was produced. 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 40 rrM citrate, pH 6.0, 2 mM ethylenediaminetetraacetic acid (EDTA), 200 mM sorbitol, 50 mM KCI, and 20% 16 WO 2005/025489 PCTIUS2004/014622 5 glycerol). Vehicle (without CG53135-05 protein) contains 30mM sodium citrate, pH 6.1, 2rn1M EDTA, 200mM sorbitol, 50mM KCI, 20% glycerol. 5.2. Example 2: Prophylactic Protective Effects of CG53135-05 From Radiation Exposure 5.2.1. Effect of CG53135-05 on Survival After Single Exposure to Acute Radiation (N-272) This study was performed to investigate the effect of CG53135-05 administered with different 10 schedules as a radioprotectant in mice after lethal total body ionizing radiation. Male C3H/He mice (total number "n" = 60) with an average weight of 22.1 gram at study initiation were used for treatment groups. Animals were fed with a standard commercial mouse diet. Food and water were provided ad /ibitum. Study Design 15 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. Anir-nals were randomly divided into 4 groups with a control group of 15 mice and 3 test groups of 15 mice each (Table 1). Mice were exposed to a single dose of 600 cGy of total body ionizing radiation. CG531 35-05 (12 mg/kg) was administered intraperitoneally (IP) daily on Days 2 and Day 3, Days 4-7 after radiation, or 20 Days 2 and 1 (-2 and -1) before radiation, respectively. Mortality (animal number, day of death) was recorded in each group. Table 1: Study Design Group Number of Induction Treatment Male Number Animals Day 0 Treatment Schedule 1 15 600 cGy Vehicle None 2 15 600 cGy CG53135-05 Daily, Days 2-3 12 mg/kg 3 15 600 cGy CG53135-05 Daily, Days 4-7 12 mg/kg 4 15 600 cGy CG53135-05 Daily, Days -2 and -1 12 mg/kg All animals were given CG53135-05 or vehicle (0.1 mL/1 0 g body weight) once daily. 25 Statistics Survival of treatment groups was compared using a Kaplan-Meier log-rank analysis. Weight change was analyzed by determining the area under the curve (AUC) using a trapezoidal rile transformation. The area under the curve was calculated using the equation, i = {yi(x+1 - xi) + (1/2)(yi+1 - yi)(x+1 - xi)} 17 WO 2005/025489 PCTIUS2004/014622 5 where x = time (d), y = percent weight change, and i = time point, from which it follows that xi is the value of x at time point i, yi is the value of y at that time point, and that xi+1 and yi+1 are the values of x and y at the next time point. Both the t-test and Mann-Whitney Rank Sum analysis were used to compare the AUC for weight change between different treatment groups. Results 10 Treatment of mice with CG53135-05 (12 mg/kg) on Days -2 and -1 before radiation significantly increased survival compared with untreated controls (P = 0.04), whereas treatment with CG53135-05 on Days 2-3 or Days 4-7 after radiation did not have a significant effect on survival (P> 0.05) (Figure 1). CG53135-05 treatment schedules of 2-3 and 4-7 did not have significant effect on survival. Furthermore, the prophylactic dose of CG53135-05 (on Days -2 and -1) also prevented weight loss post radiation 15 (Figure 2). Treatment with CG53135-05 on Days -2 and -1 (13.9%) and Days 4-7 (14.8%) also resulted in increased overall mean percent weight gains compared with untreated controls (9.7%), although statistical analysis of the area under the curve (AUC) for weight change by one-way ANOVA indicated no significant differences in the groups (P = 0.051) (Figure 2). The results of this study indicate that treatment of mice with CG53135-05 on Days -1 and -2 prior to total body irradiation is effective in 20 radioprotection and also supports the radioprotective effect on gastrointestinal or hematopoietic tissues from the damaging effects of total body irradiation. 5.2.2. Prophylactic Effect of CG53135-05 on Mice After Exposure to Acute lonizing Radiation (N-308) This study was performed to investigate the effect of CG53135-05 administered prophylactically 25 to mice that later were exposed to various doses of total body ionizing radiation. The procedures that were followed were the same as detailed in Section 5.2.1. Mice were exposed to ionizing radiation without anesthesia at a dose range of 484 to 641 cGy on day 0. Animals were dosed with PBS (control) or CG53135-05 (12 mg/kg, daily IP) on day -1, or days -2 and -1 before radiation. The schedule is represented in Table 2. The endpoints for the study were survival and weight changes. Survival was 30 followed for 30 days post-irradiation. Table 2. Study Design Group Number of Induction Treatment Treatment Number Animals Schecule* 1 16 males 484 cGy Day 0 PBS Day -2, -1 2 16 males 534 cGy Day 0 PBS Day -2, -1 3 16 males 570 cGy Day 0 PBS Day -2, -1 4 16 males 606 cGy Day 0 PBS Day -2, -1 5 16 males 641 cGy Day 0 PBS Day -2, -1 6 16 males 484 cGy Day 0 CG53135-05, 12 mg/kg, IP Day -1 18 WO 2005/025489 PCTIUS2004/014622 7 16 males 534 cGy Day 0 CG53135-05, 12 mg/kg, IP Day -1 8 16 males 570 cGy Day 0 CG53135-05,12 mg/lkg, IP Day -1 9 16 males 606 cGy Day 0 CG53135-05,12 mg/kg, IP Day -1 10 16 males 641 cGy Day 0 CG53135-05,12 mg/kg, IP Day -1 11 16 males 484 cGy Day 0 CG53135-05,12 mg/kg, IP Day -2, -1 12 16 males 534 cGy Day 0 CG53135-05, 12 mg/kg, IP Day -2, -1 13 16 males 570 cGy Day 0 CG53135-05,12 mg/kg, lP Day -2, -1 14 16 males 606 cGy Day 0 CG53135-05,12 mg/kg, IP Day -2, -1 15 16 males 641 cGy Day 0 CG53135-05, 12 mg/kg, IP Day -2, -1 5 Results: Survival decreased as radiation dose increased in all treatment groups. In animals receiving PBS, 30-day survival at the lowest dose of radiation (484 cGy) was 93.75%, and decreased to 50.0% at 534 cGy, 31.25% at 570 cGy, 12.5% at 606 cGy, and 6.25% at 641 cGy (Figure 3). In anirnals receiving 10 GC53135, 12 mg/kg IP on Day -1 only, the 30-day survival at the lowest dose of radiation (484 cGy) was 87.5%, compared to 87.5% at 534 cGy, 81.25% at 570 cGy, 43.75% at 606 cGy, and 31.25% at 641 cGy (Figure 4). In animals receiving GC53135, 12 mg/kg IP on Days -2 and -1, the 30-day survival at the lowest dose of radiation (484 cGy) was 87.5%, compared to 75.0% at 534 cGy, 37.5% at 570 cGy, 31.25% at 606 cGy, and zero at 641 cGy (Figure 5). 15 A multiple comparison test demonstrated a 4.8-fold increase in the odds of survival in animals treated on day -1 versus control animals (p=0.0001 6). LD50/30 values were calculated using a probit plot of survivorship with 95% confidence intervals calculated by bootstrapping. However, the odds of survival in animals treated on day -2 and -1 versus control animals were not significant (p=0.4162). The results are indicative of the therapeutic effect of prophylactic administration of CG53135-05 in radioprotection. 20 Further, one day treatment before radiation (day -1) also protected animals from weight loss in all but the highest radiation level (641 cGy, data not shown). In this particular system, single dose (one day -1) was significantly more effective than the two-dose regimen (on days -2 and -1, respectively) especially in higher radiation levels. In addition to the above results, the invention could be extended to additional dose regimens of 25 CG53135-05, such as prophylactically and/or therapeutically administer CG53135-05 prior and/or after the radiation exposure, which could be tested in the same animal model following the sarne 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 are not limited to, +1, +1, and +2 days after radiation exposure. 30 5.3. Example 3: Modulation of Intestinal Crypt Cell Proliferation and Apoptosis by CG53135-05 Administration to Mice (N-342) 19 WO 2005/025489 PCTIUS2004/014622 5 This study evaluates the effect of CG53135-05 on small intestinal crypt cell turnover in order to discriminate stem cell versus daughter cell effects, and to draw insights regarding the mode of action of CG53135-05 in syndromes associated with gastrointestinal stem cell damage (e.g., mucositis). Furthermore, the effect of CG53135-05 on stem cell radiosensitivity was also assessed. A "crypt" is a hierarchical structure with the stem cells towards the crypt base. As cells become 10 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 rnarked in Figure 6. Thus, the effects of CG53135-05 on the crypt microarchitecture was analyzed in the context of crypt cellularity. 15 Experimental Design Animals were sacrificed at various times after a single 12 mg/kg (IP) dose of CG53135-05. 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 CG53135-05 (12 20 mg/kg, single injection, ip) and another group was injected with a placebo control. Twenty four hours post injection, animals were irradiated with 1Gy X-ray (specifically to induce stem cell apoptosis) followed by routine in vivo BrdU labeling. Animals were sacrificed 4.5 hours later (at time of peak apoptosis). Mice were weighed and then dosed with CG53135-05 (12 mg/kg, 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 25 single injection of bromodeoxyuridine 40 minutes prior to sacrifice (see Table 3). Additional two groups of 6 mice were used to assess the effects of CG53135-05 on stern cell radiosensitivity (groups 8 and 9, see Table 3). One group was treated with CG53135-05 (12 mg/kg, 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. 30 Table 3. Study Design Group Number of Treatment Treatment Number Animals Schedule* 1 6 males CG53135-05, Injected and euthanize 3 hr later 12 mg/kg, IP 40mg/kg BrdU 40 min prior to sacrifice 2 6 males CG53135-05, Injected and euthanize 6 hr later 12 mg/kg, IP 40mg/kg BrdU 40 min prior to sacrifice 3 6 males CG53135-05, Injected and euthanize 9 hr later 12 mg/kg, IP 40mg/kg BrdU 40 min prior to sacrifice 4 6 males CG53135-05, Injected and euthanize 12 hr later 12 mg/kg, IP 40mg/kg BrdU 40 min prior to sacrifice 5 6 males CG53135-05, Injected and euthanize 24 hr later 20 WO 2005/025489 PCTIUS2004/014622 Group Number of Treatment Treatment Number Animals Schedule* 12 mg/kg, IP 40mg/kg BrdU 40 min prior to sacrifice 6 6 males CG53135-05, Injected and euthanize 48 hr la-ter 12 mg/kg, IP 40mg/kg BrdU 40 min prior to sacrifice 7 6 males Untreated 40mg/kg BrdU 40 min prior to sacrifice 8 6 males CG53135-05, Dose 24 hr prior to irradiation 12 mg/kg, IP Euthanize 4.5 hr post irradiation 1 Gy X ray 40mg/kg BrdU 40min prior to sac rifice 9 6 males PBS, IP Dose 24 hr prior to irradiation 1Gy X-ray Euthanize 4.5 hr post irradiation 40mg/kg BrdU 40 min prior to sacrifice 5 Intestinal Crypt Cell Proliferation and Apoptosis Modulation: Procedure All S-phase dividing cells incorporate the injected Bromodeoxyuridine (BrdU) and hence were marked as cycling cells. Animals that were irradiated were placed, unanaesthetised, in a perspex jig and subjected to whole body radiation of 1 Gy X-ray at a dose rate of 0.7Gy/min. This low level of radiation induced apoptosis in the small intestinal stem cell population, but not in the more mature cells. 10 The small intestine was removed, fixed in Carnoy's fixative, and processed for histological analysis (paraffin embedded). One set of 3 mm sections were immunolabeled for BrdU and one set of sections were stained with H&E. Longitudinal sections of small intestinal crypts were analyzed for the presence of either BrdU or apoptotic/mitotic nuclei. Fifty half crypts were scored per animal. Groups 1-7 (Group A in the results) were tested to determine the effect of CG53135-05 over a 48 15 hour period. Groups 8-9 (Group B in the results) were tested to determine whether CG53135-05 changes the number of apoptotic cells generated after low dose irradiation, i.e., whether CG53135-05 influences the radiosensitive stem cell population. The 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 20 hours after treatment with CG53135-05. Apoptosis, mitotic index, and proliferation were the end points for this study. Results: Group A. In groups 1-7 (Table 3), CG53135-05 had no significant effect on spontaneous apoptosis. Similar 25 results were obtained with the mitotic index (Table 4).. However, results of BrdU uptake as in Table 4, revealed the following: a) At 3 hour, there was extension/increase of proliferative region (positions 12-22). 21 WO 2005/025489 PCTIUS2004/014622 5 b) By 9 hours, large proliferative effects were noted in many positions. c) By 12 hours, only positions 4-8 showed increase in uptake (stem cells). d) By 24 hours, there was a significant inhibition of proliferation. e) By 48 hours, the uptake was comparable to control levels. Table 4. Summary of significant cell positions in the crypt after assessment of apoptosis, mitosis, 10 and proliferation Sample time Significant Cell Positions (hours) After treatment BrdU labeling Index Apoptotic Index Mitotic Index 3 12 to 22 None None 6 None None None 9 5to9&11 to20to21 None None 12 4to 8 None None 24 4to 8 None None 48 None None None The comparisons shown in Table 4 are between treated groups versus the untreated group. The cell positions shown are the ones that are significantly different from the untreated control (P<0.05). 15 Group B: In Group 8 and 9 (Table 3), stem cell radiosensitivity was assessed. As shown in Table 3, CG53135-05 or PBS was administered one day before dosing with 1 Gy radiation. Tissues were harvested 4.5 hours after radiation dosing. There was no significant effect on both radiation induced apoptosis and the mitotic index (data not shown). However, increased uptake in positions 4-8 by 12 20 hours and significant inhibition of proliferation were seen in mice pretreated with CG53135-05 and irradiated, consistent with the Group A results (Table 4). 5.4. Example 4: Effect of CG53135-05 Prophylactic Administration on Mice Intestinal Crypt Survival After Radiation Injury (N-343) The purpose of this study was to evaluate the efficacy of CG53135-05 against radiation- induced 25 crypt cell mortality in vivo using the Clonoquant T M assay. Mice were weighed and then dosed with CG53135-05 (12 mg/kg) or placebo. A single injection was given, intraperitoneally (ip), 24 hours prior to irradiation. Each group of 6 animals was irradiated as per table below. For each radiation dose, the response of a drug treated group and a placebo treated group was compared. 30 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, 35 were scored. 22 WO 2005/025489 PCTIUS2004/014622 5 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). 10 Table 5: Study design Group Number of Induction Treatment Treatment Number Animals Schedule* 1 6 males 10 Gy PBS Day -1 Day 0 2 6 males 11 Gy, PBS Day -1 Day 0 3 6 males 12 Gy, PBS Day -1 Day 0 4 6 males 13 Gy, PBS Day -1 Day 0 5 6 males 14 Gy, PBS Day -1 Day 0 6 6 males 10 Gy CG53135-05, Day -1 Day 0 12 mg/kg, IP 7 6 males 11 Gy, CG53135-05, Day -1 Day 0 12 mg/kg, IP 8 6 males 12 Gy, CG53135-05, Day -1 Day 0 12 mg/kg, IP 9 6 males 13 Gy, CG53135-05, Day -1 Day 0 12 mg/kg, IP 10 6 males 14 Gy, CG53135-05, Day -1 Day 0 12 mg/kg, IP 6 males Untreated Results: The crypt survival following prophylactic CG53135-05 administration showed inverse correlation 15 to the irradiation dose, the lesser the irradiation dose, the higher was the crypt survival (Figure 7 and 8). Prophylactic administration of CG53135-05 significantly increased the number of crypts (P<0.001). Table 6 shows the protection factor achieved for the radiation doses following prophylactic administration of the protein (CG53135-05). Protection factor (Table 6) represents the ratio between treated and untreated cells. On average, 1.55 times as many cells survived irradiation dose of 12 Gy, when animals were 20 administered with CG53135-05 prior to the radiation insult. 23 WO 2005/025489 PCTIUS2004/014622 5 Table 6: Radiation dose (Gy) Protection Factor 10 1.29 11 1.21 12 1.55 13 1.71 14 1.73 5.5. Example 5: Radioprotective Mechanism of CG53135-05 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 0. As this process produces the most reactive 10 molecules within the cell, in order to reduce cellular damage, the nucleus increases trasncription of enzymes that scavenge these radicals to less reactive intermediates. As CG53135-05 has been shown to be a radioprotectant, it is relevant to find if treatment of cells with CG53135-05 upregulates any of the genes known to be involved in radioprotection in the interest of "preloading" the cells with oxygen radial scavenging pathways. Evaluation of the effect of CG53135-05 on the intricate pathways involving ROS 15 scavengers and transcription factors will mechanistically describe the observed in vivo radioprotective effects of this agent. Thus, expression studies and survival studies were carried out at the cellular level. Expression Studies: To delineate the mechanism of radioprotection by CG53135-05, expression profile of free oxygen radical scavengers and transcription factor(s) were studied in fibroblast and endothelial cells. NIH3T3 20 (murine fibroblast), CCD-1 070sk (human foreskin fibroblast), CCD-18Co (human colonic finbroblast), and HUVEC (human umbilical cord vascular endothelial cells) cells were transferred to basal medium containing 0.1% FBS and the indicated concentration of CG53135. After 18 hours incubation, cells were harvested for total RNA using RNEasy (Qiagen, Valencia, CA). RNA was reverse transcribed using SuperScript First Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA) and amplified for the 25 gene of interest using the primers and cycles indicated below. Table: 7 Primers for RT-PCR (Human) Gene Name Primer SEQ ID NO No. of cycles COX2 5'- TTCAAATGAGAi-GTGGGAAAATTGCT -3' 1 30 5'- AGATCATCTCTGCCTGAGTATCTT -3' 2 TFF3 5'- GTGCCGGCCAAGGACAG -3' 3 40 5'- CGTTAAGACATCAGGCTCCAG -3' 4 SOD1/CuZn SOD 5'- TGGCCGATGTGTCTATTGAA -3' 5 30 5'- GGGCCTCAGACTACATCCAA -3' 6 SOD2/MnSOD 5'- CTGGACAAACCTCAGCCCTA -3' 7 28 5'- CTGATTTGGACAAGCAGCAA -3' 8 SOD3/ECSOD 5'- TCCATTTGTACCGAAACACCCCGCTCAC -3' 9 30 5'- CAAACATTCCCCCAAAGGAGCAGCTCTCAG 24 WO 2005/025489 PCTIUS2004/014622 -3' 10 Nrf2 5'- ATGGATTTGATTGACATACTT -3' 11 40 5'- CTAGT1TFFCTTAACATCTGG -3' 12 GPX1 5'- AAGGTACTACTTATCGAGAATGTG -3' 13 28 5'- GTCAGGCTCGATGTCAATGGTCTG -3' 14 Actin 5'- GGACTTCGAGCAAGAGATGG -3' 15 5'- AGCACTGTGTTGGCGTACAG -3' 16 5 Table: 8 Primers for RT-PCR (Mouse) Gene Name Primer SEQ ID NO No. of cycles MnSOD 5'- GGGAA1TCAGCGTGACTTTGGTCTTTT -3' 17 26 5'- GCGGATCCGAGCAGGCGGCAATCTGTAA -3' 18 Actin 5'- GCATCCATGAAACTACATT -3' 5'- 19 CACTTGCGGTGCACGATGG -3' 20 Results 10 Expression results are summarized in Table 9 and shown in Figures 9 through 12. Table 9 Gene NIH 3T3 CCD 1070 CCD 18Co HUVEC MnSOD Increased Moderately Increased No change Increased ECSOD Increased No change Moderately Absent Increased Cu, Zn-SOD No change No change Increased No change Nrf2 Increased Increased Increased Increased repressed at higher doses COX2 No change No change Increased Moderately Increased Tff3 No expression Increased Increased Increased GPX1 No change Moderately No change No change Increased Among the radioprotective superoxide dismutases, MnSOD, the most radioprotective one, was found to be induced by CG53135-05 consistently among cell lines (Table 9, Figures 9 through 12). 15 The Nrf2 trasncription factor, which is involved in regulation of several antioxidants that were thought to be the "antioxidant response element" (also termed as ARE), was induced by CG53135 in all cell lines studied (Table 9, Figures 9 through 12). 25 WO 2005/025489 PCTIUS2004/014622 5 It is well established that one mechanism by which cells are protected from ionizing radiation is through the induction of oxygen radical scavenging pathways. The gene expression studies detailed herein indicate that this may be one of the pathways by which CG53135-05 may modulate radioprotection. Furthermore, one of the primary target organs of total body irradiation (TBI) is the gastrointestinal tract. The data disclosed herein show that (1) the most responsive of all cell lines studied 10 was a gastrointestinal fibroblast (CCD1 8CO); and (2) a well characterized intestinal radioprotectant, Tff3, was strongly upregulated by CG53135-05. Considering that other radioprotectants known in the art strictly affect bone marrow survival and no other compartments, it is important to note that CG53135-05 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. 15 Survival Studies: Cells that receive a certain dose of radiation will have to brace against the onslaught of ionized radicals, repair the damage that the radicals perform or delay the onset of apoptosis in the face of irreperable harm, or, likely, a combination of all these mechanisms. Each of these pathways is thus important for the ultimate survival of a cell and its ability to replicate. This was assessed by a cell's ability 20 to form colonies in vitro after irradiation. Clonogenic assays were performed using CCD-18co cells, FaDu human squamous cell carcinoma cells, IEC6 and IEC18 rat colon crypt cells, and NIH3T3 cells, to assess the effect of CG53135-05 on radiation protection. Cell culture conditions are as follows: NIH3T3 cells were grown in DMEM + 10% Bovine serum + 50 pg/mI Pennicilin/Streptomycin; IEC6 and IEC18 cells were grown in 25 DMEM + 10% FBS + 0.1 U/ml Insulin + 50 pg/ml Pennicilin/Streptomycin; FaDu cells were grown in MEM + 10% FBS + 1mM Sodium Pyruvate + 50 pg/mI Pennicilin/Streptomycin + Non-essential amino acids. Cells were plated at a density of 5x1 05 per 10 cm dish (NIH3T3) or 5x105 per well of a 6-well dish (IEC1 8, IEC6, FaDu) and allowed to attach. Cells were then treated with CG53135 at doses of 10 or 100 ng/ml (IEC18, IEC6, FaDu) or at 50 and 200 ng/ml (NIH3T3) in basal media containing 0.1% serum (IEC18, 30 IEC6, FaDu) or 1% serum (NIH3T3) and incubated for 16 hours (IEC18, IEC6, FaDu) or 1 hour (NIH3T3). 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. Immediately after irradiation, cells were trypsinized and plated in duplicate at densities of NIH3T3: 250, 500, 1000, 2000, 5000 and 10,000 cells per 60mm dish; FaDu, IEC18 and IEC6: 500, 2500 cells per well of a 6-well 35 dish. Cells were grown in complete growth medium for 1-2 weeks until colonies of average diameter of 2mm, after which the colonies were stained with crystal violet and counted. Results: Results are represented as slope which are described as follows: DO - the slope of the curve between the final two points, indicating speed of cell killing at the 40 higher doses of radiation. The value is interpreted as the amount of radiation required to reduce the 26 WO 2005/025489 PCTIUS2004/014622 5 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 - 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 10 rapid rate of cell killing. Dq - 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. Results for the IEC1 8 clonogenic assays are shown in Figure 13. While DO and D1 had no 15 trends, Dq indicated that these cells are more protected at the lower doses of radiation compared to untreated (shoulder of both the long/ml and 1O0ng/ml doses is broader). Thus, it may be concluded that at both doses of CG53135-05, IEC1 8 appear more protected at the low doses of radiation, requiring a higher dose of radiation before cell killing is seen compared to untreated cells. Results for the NIH3T3 clonogenic assays are shown in Figure 14. The interpretation for these 20 results are as follows: Do -The final slope of both the 50 ng/ml and 200 ng/ml dose is trending towards larger values, approaching significance for the 50 ng/ml dose, indicating a slower rate of cell death at higher doses compared to untreated. Dq -There is no trends between the shoulders of the 50 ng/ml and 200 ng/ml, largely due to the 25 variance of the untreated. D1 -The initial slope of the 100 ng/ml dose is trending smaller values indicating a slower rate of the cell death at lower doses of radiation compared to untreated Thus, in the Clonogenic assays with NIH 3T3 cells, while there is no difference in protection at low doses, the rate of cell death is slowed by treatment with CG53135-05 at both low and high doses. 30 Results for the HUVEC clonogenic assays are shown in Figure 15. These results are interpreted as follows: Do -The final slope of the 100 ng/ml dose is trending towards larger values, indicating a slower rate of cell death at higher doses compared to untreated and 10 ng/ml. Dq -The shoulder of the 100 ng/ml dose is broader, indicating that these cells are more protected 35 at the lower doses of radiation compared to untreated. D1 did not show any trends. Thus, the higher dose of CG53135-05 provides a significant decrease in the speed of cell killing at the high dose of radiation. The cells also appeared more protected at the low doses of radiation as compared to untreated cells. 27 WO 2005/025489 PCTIUS2004/014622 5 FaDu cells and IEC6 did not show any trends in DO, Dq or D1 (data not shown). These results indicate that CG53135-05 has a protective effect against radiation in vitro. These experiments are being further optimized to (a) provide better cell killing than what is presently seen at the higher doses of irradiation; and (b) provide conditions for best protective effects by CG53135-05 by modulating treatment conditions and times. 10 5.6. Example 6: Effect of CG53135-05 on Cytokine Release 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 CG53135-05 treatment in cell culture over a time course. 15 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. 3T3 cells were plated in a 96 well plate. The cells were washed with DMEM+0.1% Calf Serum (SFM). CG53135-05, 10 ng/ml and 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 20 hours. 50 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. Results: Figure 16 shows the effect of CG53135-05 on Mo KC release. Mo KC is also known as the chemokine CXCL1 (which also has been described as Grol, Melanoma growth stimulatory activity (MSGA) or neutrophil-activating protein-3 (NAP3)). It functions as a chemoattractant for neutrophils, 25 signalling through the CXCR1 receptor. It has also been implicated in the response to whole body irradiation, raising the possibility that it possesses radioprotective qualities of its own (see Radiat. Res. 160:637-46, 2003). Figure 16 shows a consistent dose (p = 0.0085) and time dependent increase (p = 4.6x104) in the measured response. In addition both the concentrations of CG53135-05 showed significantly higher response than the control (no CG53135-05). 30 More experiments can be done to determine CG53135-05 in combination with CXCL1 is synergistic in radioprotection experiments, both in vitro and in vivo. Furthermore, induction of other cytokines (e.g., IL-2, IL-4, IL-5, IL-6, IL-8, IL-1 0, MCP-1, GM-CSF, RANTES) can also be tested, as a skilled person in art would recognize, in the presence of CG53135-05 in different cell lines (e.g., HUVEC, CCD-18, NIH3T3). 35 5.7, Example 7: Measurement of Scavengers of Reactive Oxygen Intermediates After Radiation Exposure CM-HpDCFDA method Cells with increased reactive oxygen species, as a first step, upregulate the Superoxide Dismutases - Cu, Zn-SOD, Mn-SOD, and Extracellular-SOD to scavenge the superoxide radical to 40 hydrogen peroxide. Activity of these enzymes can be indirectly measured by their by product of H 2 0 2 28 WO 2005/025489 PCTIUS2004/014622 5 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 0 2 . Cu, ZnSOD 02- C H 2 0 2 + CM-H 2 DCFDA - Fluorescein 10 MnSOD ECSOD IEC1 8 (rat intestinal epithelial) and CCD-1 Co (human colonic fibroblast) cells were plated to 60 mm dishes at a density of 1x10 5 cells per dish. After attachment, the cells were switched to medium containing 0.1% serum and the indicated dose of CG53135. After 18 hours of incubation, the cells were 15 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 FLi channel. Results indicate that IEC18 and CCD18Co cells possess increased intracellular H 2 0 2 after treatment with CG53135-05 in a dose responsive manner (Figures 17, 18 and 19). This is believed to be 20 due to enhanced expression of Superoxide dismutases, predominantly MnSOD induced by CG53135-05. Production of intracellular H 2 0 2 by CG53135-05 in IEC18 cells is enhanced with increasing dose of ionizing radiation (Figure 18). 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 CG53135-05. 25 Red CC-1 method Upon ionizing radiation exposure, cells accumulate reactive oxygen species as a result of radiation ionizing H 2 0 to the hydroxyl radical (OH), superoxide (02) or hydrogen peroxide (H 2 0). As these ions in abundance can have deleterious effects on the cell, pathways are upregulated to scavenge these molecules to more stable variants. It is hypothesized that CG53135-05 may protect the cell from 30 ionizing radiation damage by upregulating pathways that reduce the redox capacity of the cytosol. A dye called 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 on the FL2 channel. Cytosolic Reactive Oxygen Species + Red CC-1 -- > Oxidized Red CC-1 IEC1 8 (rat intestinal epithelial) and CCD-1 8Co (human colonic fibroblast) cells were plated to 35 60mm dishes at a density of 1 x10 5 cells per dish. After attachment, the cells were switched to medium containing 0.1% serum and the indicated dose of CG53135. After 18 hours of incubation, 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. 40 Results: IEC1 8 and CCD1 8Co cells were found to possess decreased cytosolic redox potential after treatment with CG53135-05 in a dose responsive manner as shown in Figures 20, 21, and 22. The 29 WO 2005/025489 PCTIUS2004/014622 5 data shown herein is believed to be the result of enhanced expression of superoxide dismutases induced by CG53135-05, which scavenge the more reactive species of superoxide and hydroxyl radicals. Also, CG53135-05 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. 6. EQUIVALENCE AND REFERENCE CITED 10 All references cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are 15 offered by way of example only. We therefore do not wish to be limited to the precise terms and limitations set forth herein, but desire to avail ourselves of such changes and alterations that may be made for adapting the invention to various usages and conditions. Such alterations and changes may include, but are not limited to, different compositions for the administration of the polypeptides according to the present invention to a subject; different amounts of the polypeptide; different times and means of 20 administration; different materials contained in the administration dose including, for example, combinations of different peptides, or combinations of peptides with different biologically active compounds. Such changes and alterations also are intended to include modifications in the amino acid sequence of the specific polypeptides described herein in which such changes alter the sequence in a manner as not to change the functionality of the polypeptide, but as to change solubility of the peptide in 25 the composition to be administered to a subject, absorption of the peptide by the body, protection of the polypeptide for either shelf life or within the body until such time as the biological action of the peptide 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 of the present invention. 30

Claims (17)

1. A method of preventing or treating a disorder or one or more symptoms thereof in a subject, wherein the disorder or one or more symptoms thereof are associated with exposure to radiation, said method comprising administering to the subject a composition comprising CG53135-05 polypeptide.

2. The method of claim 1, wherein the subject is a human. 10

3. The method of claim 1, wherein the disorder is neutropenia.

4. The method of claim 1, wherein the disorder is gastrointestinal mucositis.

5. The method of claim 1, wherein the symptoms are diarrhea, skin burn, sores, fatigue, dehydration, inflammation, hair loss, ulceration of oral mucosa, or bleeding from the nose, mouth or rectum. 15

6. A method of claim 1, wherein a prophylactic effective dose of said polypeptide is administered to the subject that may be exposed to radiation.

7. A method of claim 6, wherein the effective dose is a single dose of the said polypeptide.

8. The method of claim 7, wherein the effective dose is more than a single dose of the said polypeptide. 20

9. A method of claim 1, wherein an effective therapeutic dose of the said polypeptide is administered to the subject after the radiation exposure.

10. The method of claim 9, wherein the effective dose is a single dose of the said polypeptide.

11. The method of claim 9, wherein the effective dose is more than a single dose of the said polypeptide. 25

12. A method of claim 1, wherein the said polypeptide is administered by parenteral route.

13. The method of claim 12, wherein the said route of administration consists of intravenous, intramuscular, subcutaneous, intradermal or intranasal administration.

14. The method of claim 1, wherein a therapeutically effective amount of the said polypeptide is formulated in a pharmaceutical composition. 30

15. A method of recovery or protection of hematopoietic tissues from radiation damages comprising administering to a subject in need thereof a composition comprising CG53135-05 polypeptide. 31 WO 2005/025489 PCT/US2004/014622 5

16. A method of recovery or protection of gastrointestinal tissues from radiation damages comprising administering to a subject in need thereof a composition comprising CG53135-05 polypeptide.

17. A method of preventing or treating a disorder or one or more symptoms thereof in a subject, wherein said disorder or one or more symptoms thereof are associated with exposure to mustard gas agents, said method comprising administering to the subject a composition comprising CG53135-05 10 polypeptide. 32