AU2012269740A1 - Method and composition for treatment or inhibition of mucositis associated with chemotherapy or radiation damage - Google Patents

Description

WO 2012/171077 PCT/AU2012/000700 1 METHOD AND COMPOSITION FOR TREATMENT OR INHIBITION OF MUCOSITIS ASSOCIATED WITH CHEMOTHERAPY OR RADIATION DAMAGE Field [0001] This invention relates to a method of treatment or inhibition of the development of alimentary mucositis arising from chemotherapy or radiation damage, to a composition for use in such treatment, to the use of such composition in manufacture of a medicament for treatment or inhibition of the development of alimentary mucositis arising from chemotherapy or radiation damage. In particular the invention is directed to treatment of alimentary mucositis arising from chemotherapy or exposure to radiation, including cancer therapy. Background [0002] Exposure to radiation or chemotherapeutic agents often results in the destruction of normal tissue, especially hematopoietic cells and epithelial cells in the gastrointestinal tract. [0003] Furthermore, damage to normal tissues can also result from environmental sources of radiation that include accidental exposure to radiation or contact with radionuclides in, for example, the release of radioactive material following a nuclear accident or the purposeful use of 'dirty" bombs during a terrorist attack. [0004] Such an emergency may arise from a nuclear event, which, as the accidents at Chemobyl and Fukushima demonstrate, can present enormous challenges even without detonation-related toxicities. Rapid exposure of extensive body surface area to significant doses of penetrating radiation results in acute radiation syndrome (ARS). ARS-which can affect the hematopoietic, gastrointestinal (GI), central nervous, and cardiovascular systems-can manifest within minutes and last for weeks.

WO 2012/171077 PCT/AU2012/000700 2 [0005] In humans and mice, both radiation dose and host characteristics determine the extent of injury after exposure. At doses that are frequently fatal within weeks (4 to 10 Gy), hematopoietic toxicity (hematopoietic syndrome) contributes to mortality as demonstrated by the success of bone marrow shielding, hematopoietic cell transplant (HCT), and G-CSF administration in supporting recovery. At higher doses, death occurs earlier and even HCT does not reduce mortality. Concomitant thermal and skin injuries compromise survival at all doses. [0006] Damage from radiation exposure can include fibrosis, vascular damage, aberrant angiogenesis, pneumonitis, atherogenesis, osteonecrosis, immunosuppression and functional impairment of the gastrointestinal tract, lungs, kidneys, and other organs. Epithelial cells in the upper and low gastrointestinal tract are especially vulnerable to damage by radiation and chemotherapeutics agents used in cancer, and by analogy to radiation in the form of environmental exposure to radionuclides or high dose radiation from accidental or purposeful exposure. Damage to the epithelial tissue, directly and indirectly can lead to gastrointestinal symptoms and chronic conditions including mucositis, enteritis, and proctitis. [0007] The epithelial tissue of the mouth and oesophagus are particularly sensitive to radiation and chemotherapeutic agents. For example, after radiation treatment or treatment with chemotherapeutic agent for head and neck cancers, oral ulcerations characteristic of mucositis are a major clinical problem causing considerable pain, increased susceptibility to infection and inability to eat. Treatment of abdominal or pelvic cancers with radiation causes radiation enteritis, damage to the intestinal lining occurring typically in the small bowel, and less frequently in the large bowel. [0008] In the case of radiation therapy of cancer localized in the pelvis or abdomen, such as ovarian cancer, radiation enteritis is one of the most difficult to treat complications of abdominal and pelvic radiation. It is thought, for example, radiation treatment or exposure causes depletion of epithelial cells with each successive dose of pelvic radiation and/or chemotherapy, WO 2012/171077 PCT/AU2012/000700 3 which results in the clinically observed acute gastrointestinal symptoms. The incidence of radiation enteritis is increasing because of the current trend of combined high dose chemotherapy and high dose radiation. By an equivalent process, localized radiation treatment of prostate cancer results in lower bowel proctitis. [0009] An almost identical process of intestinal and epithelial injury can occur with use of certain chemotherapeutic agents, such as 5-fluorouracii, Cisplatin, methotrexate, doxorubicin, hydroxyurea, cytosine arabinoside. and irinotecan. Injury to the gastrointestinal tract following radiation therapy, chemotherapy or accidental exposure to ionizing radiation, resulting in enteritis, mucositis, or proctitis, has a significant role in patient survival or mortality as well as quality of life. [0010] Mucositis is the major clinical side-effect of exposure to radiation (e.g. radiation associated with cancer therapies) and treatment with chemotherapeutic agents, causing pain, weight loss, diarrhoea and in some cases patients suffer serious infections and may even elect to withdraw from cancer treatment, (Keefe, 2007, Current Opinion in Oncology 19:323-327). (0011] Clinically mucositis is seen as oral mucositis, called stomatitis, and as intestinal mucositis. The symptoms are caused by the cytotoxic effects associated radiation, and with cancer chemotherapy and radiation. These therapies are particularly damaging to epithelial cells because those cells are rapidly dividing. Epithelial cells border the lumen of the gastro-intestinal tract from the lips to the anus. [0012] Symptoms of alimentary mucositis include pain, discomfort, inability to tolerate food or liquid and diarrhoea. Mucositis can be limiting in the ability of a patient to undergo necessary cancer therapy. Mucositis of the intestinal lining will start to manifest 3-7 days after radiation exposure or chemotherapy while oral mucositis will appear 7-10 days after radiation exposure or chemotherapy.

WO 2012/171077 PCT/AU2012/000700 4 [0013] Post-radiation bacteraemia contributes to total body irradiation (TBI) toxicity. [0014] Many treatment options are used to prevent or treat oral mucositis, but there are fewer options which target intestinal mucositis which is more directly life-threatening due to septicaemia. [0015] The majority of current treatments for oral mucositis are palliative at best and many are of dubious effectiveness. (Niscola et al, Mucositis in patients with hematologic malignancies: an overview, in Haematologica, 2007; 92 (02)). The most effective treatment commonly used is oral care before treatment commences. This approach recommends dental work and the removal of caries before treatment starts. Cryotherapy (sucking on ice to reduce blood flow to the mouth lining) is used with success in bolus chemo therapies but has limited effectiveness in the case of continuous infusions. [0016] Mouthwashes are used relief of oral symptoms. For example, denzydamine hydrochloride has been used as a gargle in particular to relieve symptoms associated with head and neck cancer, but has less effectiveness in chemotherapy-induced mucositis, nor does it help counter intestinal mucositis. [0017] Topical anaesthetics have been used to some effect, but while they temporarily relieve pain, they do not change the course of the tissue damage. [0018] Antiseptic gargles such as chlorhexidine, hydrogen peroxide and povidone iodine are used; however they appear to be of limited effectiveness in the mouth and of no use in the intestine. [0019] Antibacterial agents such as antibiotic lozenges are used and release low amounts of antibiotic into the mouth. While these seem to have effectiveness in chemotherapeutic patients, they have little effectiveness in radiotherapy patients and have the added risk of selecting for antibiotic resistant strains of microorganisms.

WO 2012/171077 PCT/AU2012/000700 5 [0020] Monoclonal antibodies have the disadvantages when used as oral treatments that (a) they are more difficult to make at large scale leading to high expense on a per patient basis, (b) they have narrow specificity which prevents the capture of a broad spectrum of toxic agents, (c) they are generally more susceptible to degradation in the harsh gastric environment, (d) monoclonals present a limited ability to cross-link to form agglutinations which facilitate excretion from the body. [0021] In 1998, Congress appropriated funds for the CDC to acquire a pharmaceutical and vaccine stockpile to counter potential biological and chemical threats and threats from widespread diseases that could affect large numbers of persons in the civilian population. Subsequently reconfigured as the Strategic National Stockpile (SNS) and managed by the Department of Health and Human Services, SNS is a national repository of antibiotics, chemical antidotes, antitoxins, life-support medications, IV administration, airway maintenance supplies, and medical/surgical items. [0022] The agents in the SNS that could be used for medical problems related to radiological/nuclear events are calcium and zinc DTPA (diethylenetriamine pentaacetic acid) which are chelating agents for the transuranium elements plutonium, americium, and curium), Prussian Blue which is an oral ion exchange drug is indicated for decorporation of cesium and thallium), and the growth factors G-CSF and GM-CSF which are to minimise the severity of neutropenia-associated complications and shorten the duration of severe neutropenia. [0023] However, there remains a need for compositions that treat the damage associated with exposure to chemotherapeutic agents or radiation, for example the radiation associated with cancer therapy or radiological/nuclear events. [0024] The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a WO 2012/171077 PCT/AU2012/000700 6 context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application. Summary [0025] We provide a method for treating or inhibiting the development of alimentary mucositis arising from chemotherapy or radiation damage in a subject comprising: providing a hyperimmune dairy material or egg derived material comprising anti-lipopolysaccharide (anti-LPS) antibody raised against a vaccine comprising LPS; and administering to the subject a composition comprising the anti-LPS antibody or fragment thereof which antibody or fragment binds LPS associated with commensal bacteria. [0026] In one aspect, the subject is a patient suffering cancer. In another aspect, the subject is a person exposed to radiation. [0027] In one aspect the LPS in the vaccine is derived from gram negative bacteria. [0028] In another aspect the LPS in the vaccine is at least partly separated from bacterial cell walls. [0029] In a further aspect the LPS in the vaccine has been separated by mechanical means. [0030] In another embodiment, the radiation damage is associated with cancer therapy. (0031] In another embodiment, the present invention provides a method of treatment of a subject exposed to radiation to reduce the effects of radiation damage comprising: forming hyperimmune colostrum by vaccinating cows; and administering the hyperimmune material to said patient wherein the step WO 2012/171077 PCT/AU2012/000700 7 of vaccinating cows to produce hyperimmune material comprises vaccination with a vaccine comprising LPS. [0032] In another embodiment, the present invention provides a method of treatment of a patient suffering cancer to reduce the effects of cancer therapy comprising: forming hyperimmune colostrum by vaccinating cows; and administering the hyperimmune material to said patient wherein the step of vaccinating cows to produce hyperimmune material comprises vaccination with a vaccine comprising LPS. [00331 In one embodiment, the hyperimmune colostrum formed comprises material selected from the group consisting of anti-LPS antibodies and fragments thereof wherein the material binds commensal bacteria. (0034] In another embodiment, the present invention provides a medicament composition for treatment of a subject exposed to radiation to.reduce the effects of radiation damage comprising anti-LPS antibodies which bind commensal bacteria. [0035] In another embodiment, the present invention provides a medicament composition for treatment of a patient suffering cancer to reduce the effects of cancer therapy comprising anti-LPS antibodies which bind commensal bacteria. [0036] In another embodiment, the present invention provides a use of a hyperimmune colostrum in manufacture of a medicament for treatment or inhibition of alimentary mucositis arising from radiation damage in a subject exposed to radiation wherein the hyperimmune material is raised by vaccination with an antigen comprising LPS and the medicament comprises material selected from the group consisting of anti-LPS antibodies and fragments thereof wherein the material binds commensal bacteria. [0037] In another embodiment, the present invention provides a use a hyperimmune colostrum in manufacture of a medicament for treatment or WO 2012/171077 PCT/AU2012/000700 8 inhibition of alimentary mucositis arising from cancer therapy in a patient suffering cancer wherein the hyperimmune material is raised by vaccination with an antigen comprising LPS and the medicament comprises material selected from the group consisting of anti-LPS antibodies and fragments thereof wherein the material binds commensal bacteria. [0038] In another embodiment, the present invention provides a use of LPS in manufacture of a medicament for treatment or inhibition of alimentary mucositis arising from radiation damage in a subject exposed to radiation by administration of a composition comprising one or more of anti-LPS antibodies and fragments thereof obtained from hyperimmune colostrum raised by vaccination of cows with LPS wherein the anti-LPS antibodies and fragments thereof bind commensal bacteria. [0039) In another embodiment, the present invention provides a use of LPS in manufacture of a medicament for treatment or inhibition of alimentary mucositis arising from cancer therapy in a patient suffering cancer by administration of a composition comprising one or more of anti-LPS antibodies and fragments thereof obtained from hyperimmune colostrum raised by vaccination of cows with LPS and wherein the anti-LPS antibodies and fragments thereof bind commensal bacteria. [0040] In another embodiment, the present invention provides a method of cancer radiotherapy which comprises administering to a subject in need of such therapy an amount of a composition comprising anti-LPS antibodies which bind commensal bacteria effective to minimise damage to subject to radiation. [0041] In one embodiment, the subject is a patient suffering cancer, In another embodiment, the radiation damage is associated with cancer therapy. [0042] In another embodiment, the radiation damage is alimentary mucositis. Brief Description of the Drawings WO 2012/171077 PCT/AU2012/000700 9 [0043] Figure 1. A: Western Blot with anti-LPS antibodies (dairy derived) made as described in Example 3. B is a Western Blot with antibodies contained in non-hyperimmune colostrum. This data demonstrates colostrum containing polyclonal arti-LPS antibodies of the present invention binds LPS of commensal bacteria. (0044] Figure 2. Effect of colostrum containing polyclonal anti-LPS antibodies in combination with oral antibiotic enrofloxacin on survival of mice after 7 gray Total Body Irradiation (TBI). Mice were irradiated with 7 gray then 24 hours later treated with nothing ("7Gy"), oral antibiotic enrofloxacin ("ABX") thru day 30 (first 5 days of which was administered via gavage with our without 14 days of ad libitum skim milk, or colostrum containing polyclonal anti-LPS antibodies produced by the methods described in Example 3 ("ETEC"). The percentage of animal surviving to Day 30 are indicated in the legend. N = 12 mice per group. Mice receiving 7 gray all died by day 16. In contrast, mice receiving oral antibiotic show significantly increased time to death, with 100% mortality by day 23. Mice receiving both antibiotic and colostrum containing polyclonal anti-LPS antibodies ("ETEC") demonstrate survival to Day 30. These data indicate colostrum containing polyclonal anti-LPS antibodies decrease the morbidity mortality and mortality associated with exposure to radiation. Detailed Description [0045] The present invention is based in part on the surprising discovery that compositions comprising anti-LPS antibodies protect against damage caused by exposure to radiation or chemotherapeutic agents. [0046] This result is surprising, since it has been demonstrated previously that microbial products protect against damage caused by exposure to radiation. For example, it has been shown previously that-flagellin protects intestinal epithelial cells (IEC) from radiation-induced apoptosis and increases hose survival when injected into mice before lethal doses of radiation (Burdelya LG, Krivokrysenko VI, Tallant TC, et al. An agonist of toll-like receptor 5 has radioprotective activity in mouse and primate models. Science 2008; 320:226- WO 2012/171077 PCT/AU2012/000700 10 230.). LPS increased both endogenous intestinal prostaglandin (which increases IEC proliferation and decreases IEC apoptosis) production and intestinal crypt survival in mice exposed to 14 Gray radiation when injected parenterally 2-24 hours prior to exposure (Riehl T, Cohn S, Tessner T, et al. Lipopolysaccharide is radioprotective in the mouse intestine through a prostaglandin-mediated mechanism. Gastroenterology 2000; 118:1106 1116). [0047] Importantly, it has also been demonstrated that blocking pathways that are activated by bacterial products have been shown to have a detrimental effects on the ability of IECs and crypts to survive radiation insults (Packey and Ciorba, Current Opinion in Gastroenterology, 2010, 26(2): 88-94). [0048] Therefore, blocking LPS activity is expected to increase apoptosis and decrease survival of subjects. [0049] In contrast, the present inventors have shown colostrum containing polyclonal anti-LPS antibodies decrease the morbidity mortality and mortality associated with exposure to radiation and chemotherapeutic agents. [0050] Accordingly, in one aspect, the present invention provides a method for treating or inhibiting the development of alimentary mucositis arising from chemotherapy or radiation damage in a subject comprising: providing a hyperimmune dairy material or egg derived material comprising anti lipopolysaccharide (anti-LPS) antibody raised against a vaccine comprising LPS; and administering to the subject a composition comprising the anti-LPS antibody or fragment thereof which antibody or fragment binds LPS associated with commensal bacteria. [0051] The subject is may be a patient suffering cancer and/or a person exposed to radiation. [0052] The hyperimmune material is raised against LPS.

WO 2012/171077 PCT/AU2012/000700 11 [0053] The "hyperimmune material" in one embodiment is hyperimmune dairy derived material such as milk particularly colostral milk (colostrum) and the like which is enriched in antibodies or fragments thereof and which is derived from an animal source. The hyperimmune dairy material is preferably hyperimmune colostrum. In another embodiment the hyperimmune material is derived from bird eggs. The hyperimmune material is enriched when compared with corresponding material in which the animal has not been challenged with the antigen in question namely LPS. [0054] The term colostrum where used herein includes colostral milk; processed colostral milk such as colostral milk processed to partly or completely remove one or more of fat, cellular debris, lactose and casein; and colostral milk or processed colostral milk which has been dried by for example, freeze drying, spray drying or other methods of drying known in the art. Colostral milk is generally taken from a mammal such as a cow within five days after parturition. Preferably, the mammalian colostrum is bovine colostrum retained from the first 4 days post parturition, more preferably bovine colostrum retained from the first 2 days post parturition, even more preferably bovine colostrum retained from the first day post parturition, and most preferably bovine colostrum retained from the first milking post parturition. [0055] Preferably the colostrum collected from the cow comprises at least 4% total protein (weight %), more preferably 5%, more preferably at least 8%, more preferably at least 10%. (0056] Preferably the ratio of IgG to total protein of the colostrum collected from the cow is at least 10%, more preferably 20%. [0057] Lipopolysaccharide (LPS) is a molecule consisting of a lipid and a polysaccharide (carbohydrate) joined by a covalent bond. LPS is a major component, for example, of the outer membrane of Gram-negative bacteria, contributing greatly to the structural integrity of the bacteria, and protecting the membrane from certain kinds of chemical attack. The only Gram-positive WO 2012/171077 PCT/AU2012/000700 12 bacteria that possesses LPS is Listeria monocytogenes, the common infective agent in unpasteurized milk. [0058] The term "radiation" includes a radiation source, such as, ionising radiation, radiation therapy, accidental radiation exposure, and exposure to individuals at risk for radiation exposure. Accordingly, the methods can benefit individuals who are exposed to radiation inadvertently or who are exposed to radiation as part of their work, including, for example, individuals working in nuclear power plants or nuclear fuel and/or nuclear waste processing facilities, individuals transporting nuclear material, researchers working with radioactive materials, individuals responsible for handling hazardous materials, members of the military, medical care workers, and the like. In some embodiments, the subject is a patient receiving X-rays for purposes of medical monitoring and/or diagnosis. In some embodiments, the subject is a patient radiation purposes cancer therapy. [0059] The term "ionising radiation" as used herein refers to photons having enough energy to ionise a bond, such as, a, P and y rays from radioactive nuclei and x-rays. Examples of ionizing subatomic particles include alpha particles, beta particles and neutrons. Electromagnetic waves with shorter wavelengths (higher frequencies) possess higher energy and are more likely to be ionizing. Examples of high energy, or high frequency, ionizing electromagnetic waves include ultraviolet (UV) rays, x-rays and gamma-rays. Exposure to ionizing radiation is commonly known to cause damage to living tissue, including breaks in DNA molecules. [0060] Exposure to a significant dose of ionizing radiation can also result in systemic effects. For example, high doses of radiation can induce radiation sickness, also referred to as Acute Radiation Syndrome (ARS). ARS is induced as a result of the effect of radiation on various systems of the body, including the hematopoietic system, digestive system, cardiovascular system, reproductive system, and the like. Bone marrow and circulating immune cells, such as leukocytes, are particularly sensitive to the effects of ionizing radiation. Radiation results in immunosuppression leading to opportunistic WO 2012/171077 PCT/AU2012/000700 1 3 infection, including bacterial translocation from a damaged gastrointestinal system. In addition, neutropenia and thrombocytopenia are characteristics of exposure to high but acute dose of whole body. [0061] The mean lethal dose of radiation required to kill 50% of humans 60 days after whole-body irradiation (LD50/60) is between 3.25 and 4Gy without supportive care, and 6-7Gy when antibiotics and transfusion support are provided. The mortality is largely attributed to the haematopoietic syndrome, a consequence of hypoplasia or aplasia of the bone marrow. Cytopenias develop as a result of radiation-induced and normal attrition of mature functional cells, combined with the failure of replacement because of radiation-induced depletion of haematopoietic stem cells and progenitors. The time and extent of cytopenia generally correlate with radiation dose and prognosis, but the kinetics of depletion and recovery of blood cells also varies between the erythropoiesis, myelopoiesis and thrombopoiesis lineages, thrombopoiesis being the slowest. [0062] The gastrointestinal syndrome arising from chemotherapy or radiation damage with radiation damage may result from ablation of stem cells in intestinal crypts, which in turn leads to denudation of the intestinal mucosa. With regard to radiation damage, this injury occurs after whole-body doses in the range of 3-15Gy and in rodents doses at the upper end of this range usually result in death within about 1 week after irradiation. [0063] The term "radiation damage" as used herein includes damage at the molecular level (e.g., DNA mutagenesis or alteration, or chromosomal damage), the cellular level (e.g., apoptosis, or uncontrolled cell proliferation, including tumour formation and metastasis), the tissue level (e.g., tissue damage, including degeneration, atrophy, fibrosis, and necrosis), the organ level (e.g., organ failure), and/or the system or organism level (e.g., mortality). [0064] The term "chemotherapeutic agent" as used herein refers to agents that have the property of inhibiting the growth-or proliferation (e.g., a cytostatic agent), or inducing the killing, of tumour cells (and interchangeable terms as WO 2012/171077 PCT/AU2012/000700 14 discussed above). The chemotherapeutic agent inhibits or reverses the development or progression of a cancer, such as for example, solid tumour, or a soft tissue tumour. A chemotherapeutic agent may reduce, prevent, mitigate, limit, and/or delay the growth of metastases or neoplasms, or kill neoplastic cells directly by necrosis or apoptosis of neoplasms or any other mechanism, or that can be otherwise used, in a pharmaceutically-effective amount, to reduce, prevent, mitigate, limit, and/or delay the growth of metastases or neoplasms in a subject with neoplastic disease. [0065] Chemotherapeutic agents include, for example, fluoropyrimidines; pyrimidine nucleosides; purine nucleosides; anti-folates, platinum agents; anthracyclines/anthracenediones; epipodophyllotoxins; camptothecins; hormones; hormonal complexes; antihormonals; enzymes, proteins, peptides and polyclonal and/or monoclonal antibodies; vinca alkaloids; taxanes; epothilones; antimicrotubule agents; alkylating agents; antimetabolites; topoisomerase inhibitors; antivirals; and various other cytotoxic and cytostatic agents. The skilled person would be familiar with a variety of chemotherapeutic agents available for treatment of cancer. [0066] The term "chemotherapy" as used herein refers to administration of at least one chemotherapeutic agent to a patient having a cancer. [0067] In some embodiments, a composition described herein provides a clinically significant decrease in tissue damage. The nature,. extent, time course, and/or other aspects of chemotherapy or radiation-induced tissue damage can be measured using various methods and clinical indicators known in the art. For example, in some aspects, the protective effect of a composition described herein is assessed by measuring levels and/or recovery of haematological endpoints, such as lymphocyte, neutrophil, and/or platelet counts. In further aspects, a protective effect can be assessed at the organ or system level by, for example, measuring the occurrence of opportunistic infection and/or bacterial translocation. In yet further aspects, a protective effect can be assessed at the organism level by measuring survival WO 2012/171077 PCT/AU2012/000700 15 rates, such as the thirty day survival in mice, which is a robust measurement of recovery from radiation, as exemplified in Example 4, below. [0068] In some embodiments, a composition of the present invention is administered to a subject following inadvertent exposure to radiation. [0069 Cancer therapy, including cancer radiotherapy, is' a very significant public health activity. Given the incidence of cancer in the population and the international assessment that more than 50% of cancer patients benefit from inclusion of radiotherapy in their treatment, more than 10% of the population are likely to experience cancer radiotherapy in their lifetime. [00701 In one embodiment, the subject is a patient suffering cancer. In another embodiment, the radiation damage is associated with cancer therapy. [0071] In another embodiment, the radiation damage is alimentary mucositis. [0072] In another embodiment, the present invention provides a method of treatment of a subject exposed to radiation to reduce the effects of radiation damage comprising forming hyperimmune colostrum by vaccinating cows; and administering the hyperimmune material to said patient wherein the step of vaccinating cows to produce hyperimmune material comprises vaccination with a vaccine comprising LPS. [0073] In another embodiment, the present invention provides a method of treatment of a patient suffering cancer to reduce the effects of cancer therapy comprising: forming hyperimmune colostrum by vaccinating cows; and administering the hyperimmune material to said patient wherein the step of vaccinating cows to produce hyperimmune material comprises vaccination with a vaccine comprising LPS. [0074] In one embodiment, the hyperimmune colostrum formed comprises material selected from the group consisting of anti-LPS antibodies and fragments thereof wherein the material binds commensal bacteria.

WO 2012/171077 PCT/AU2012/000700 16 [0075] In another embodiment, the present invention provides a medicament composition for treatment of a subject exposed to radiation to reduce the effects of radiation damage comprising anti-LPS antibodies which bind, commensal bacteria. [0076] In another embodiment, the present invention provides a medicament composition for treatment of a patient suffering cancer to reduce the effects of cancer therapy comprising anti-LPS antibodies which bind commensal bacteria. [0077] In another embodiment, the present invention provides a use of a hyperimmune colostrum in manufacture of a medicament for treatment or inhibition of alimentary mucositis arising from radiation damage in a subject exposed to radiation wherein. the hyperimmune material is raised by vaccination with an antigen comprising LPS and the medicament comprises material selected from the group consisting of anti-LPS antibodies and fragments thereof wherein the material binds commensal bacteria. [0078] In another embodiment, the present invention provides a use a hyperimmune colostrum in manufacture of a medicament for treatment or inhibition of alimentary mucositis arising from cancer therapy in a patient suffering cancer wherein the hyperimmune material is raised by vaccination with an antigen comprising LPS and the medicament comprises material selected from the group consisting of anti-LPS antibodies and fragments thereof wherein the material binds commensal bacteria. [00791 In another embodiment, the present invention provides a use of LPS in manufacture of a medicament for treatment or inhibition of alimentary mucositis arising from radiation damage in a subject exposed to radiation by administration of a composition comprising one or more of anti-LPS antibodies and fragments thereof obtained from hyperimmune colostrum raised by vaccination of cows with LPS wherein the anti-LPS antibodies and fragments thereof bind commensal bacteria.

WO 2012/171077 PCT/AU2012/000700 17 [0080] In another embodiment, the present invention provides a use of LPS in manufacture of a medicament for treatment or inhibition of alimentary mucositis arising from cancer therapy in a patient suffering cancer by administration of a composition comprising one or more of anti-LPS antibodies and fragments thereof obtained from hyperimmune colostrum raised by vaccination of cows with LPS and wherein the anti-LPS antibodies and fragments thereof bind commensal bacteria. [0081] In another embodiment, the present invention provides a method of cancer radiotherapy which comprises administering to a subject in need of such therapy an amount of a composition comprising anti-LPS antibodies which bind commensal bacteria effective to minimise damage to subject to radiation. [0082] In one embodiment, the subject is a patient suffering cancer. In another embodiment, the radiation damage is associated with cancer therapy. [0083] In another embodiment, the radiation damage is alimentary mucositis. [0084] A composition of the present invention can be administered in single or multiple doses within a few hours after exposure to chemotherapy or radiation. In some embodiments, the composition is administered within 24 hours of exposure to chemotherapy or radiation. In further embodiments, the composition is administered within 48 hours, 72 hours, or 96 hours or more following exposure to chemotherapy or radiation. [0085] The medicament composition for administration in treatment of a patient suffering cancer to reduce the effects of chemotherapy or radiation damage comprises anti-LPS antibodies which bind commensal bacteria. [0086] The term "commensal" refers to non-pathogenic bacteria which form part of the normal flora of a healthy human alimentary tract. Examples of commensal gram negative genera may be selected from the group of genera WO 2012/171077 PCT/AU2012/000700 18 consisting of Enterobacter, Escherichia, Klebsiella, Bacteroides, Proteus, Salmonella, Serratia, Veillonella and Fusobacteria. [0087] The determination of anti-LPS antibodies Which bind LPS may be determined by conducting an appropriate assay. The binding of LPS associated with commensal bacteria will generally result from the use of LPS from commensal bacteria in the vaccine used to prepare the anti-LPS. In some cases, however, LPS from non-commensal bacteria may produce antibodies which bind LPS associated with commensal bacteria and the above referenced test may be used to determine whether such anti-LPS antibodies have the requisite binding ability of LPS associated with commensal bacteria. [0088] The LPS antigen used vaccination to produce anti-LPS antibodies may and preferably is derived from gram negative bacteria. Further as described above it is particularly preferred to use an antigen comprising LPS from gram negative commensal bacteria. The antigen may comprise LPS in any of a range of forms. It may be in the form of whole live, attenuated or killed bacteria or may be in the form at least partly separated from bacterial cell walls. [0089] In one embodiment, the anti-LPS immunoglobulin preparation may be prepared by immunizing a mammal with LPS from multiple E. coli strains. [0090] The mammal or avian may be immunized with LPS selected from the group consisting of 06, 08, 015, 025, 027, 063, 078, 0114, 0115, 0128, 0148, 0153, 0159, and other LPS associated with enterotoxigenic E. coli. [00911 The mammal or avian may be immunized with LPS selected from the group consisting of 078, 06, 08, 0129 and 0153 LPS. The. LPS may comprise 078 LPS. [00921 In one embodiment, the mammal is immunised with 06, 08, 015, 025, 027, 063, 078, 0114, 0115, 0128, 0148, 0153, and 0159 LPS.

WO 2012/171077 PCT/AU2012/000700 19 [0093] It is preferred the bacteria from which each type of 0 antigen is isolated are grown in separate bacterial culture systems, and after separation of the 0 antigen from the bacteria, the component antigens are added together to form a component of the vaccine. [0094] LPS may be separated, at least in part, by a range of methods using for example heat, detergents, lysis or mechanical means. Methods of separating LPS from cell walls of bacteria are described in our application WO/2004/078209 (with reference to separation of O-antigen) the contents of which are herein incorporated by reference. In particular the preferred method of separating LPS from cell walls is by application of shear. The LPS antigen used in vaccination can be separated from the bacterial cell walls by application of an effective amount of shear, homogenisation or heat or by effective combinations thereof. Methods of preparing hyperimmune bovine colostrum (HIBC) are also described in WO/2004/078209. [0095] In one embodiment the method involves the use of colostrum or a colostrum extract, further characterised in that the colostrum is enriched in anti-LPS antibodies when compared with colostrum obtained without vaccination. [0096] The anti-LPS antibodies are polyclonal immunoglobulins or chimeric antibodies or dendrimer presented immunoactive fragments or immunoactive fragments such as F(ab) and F(ab) 2 fragments or recombinant immunoactive fragments, or affinity purified immunoglobulins or immunoactive fragments thereof. Fragments thereof which bind LPS associated with commensal bacteria may be identified by methods known in the art. 100971 Studies of dairy products show low levels of LPS antibodies are naturally present in these materials. For example in normal colostrum there are no significant LPS antibodies (<100mg per litre of liquid colostrum of IgG ligand or equivalent molar amount of other LPS ligand. This corresponds to <1g per kg of colostrum solids of IgG ligand or equivalent molar amount of other LPS ligand).

WO 2012/171077 PCT/AU2012/000700 20 [0098] The hyperimmune dairy material preferably contains at least 3 g per kilogram of product which is IgG anti-LPS antibody, or an equivalent molar concentration of other anti-LPS antibody. For example the hyperimmune material may contain at least 5g, at least 1Og or at least 15g anti-LPS antibody per kg of hyperimmune material on the basis of the dry weight of components. The upper end of the range of antibody concentration will depend on factors such as the dose, the disease state and the health of the patient. The hyperimmune material may, for example contain no more than 80g such as no more than 60g, no more than 50g or no more than 40g anti-LPS antibody per kg of hyperimmune material on the basis of the dry weight of components. 10099) The composition to be administered to the patient preferably contains at least 3 g per kilogram of product which is IgG anti-LPS antibody or fragment which binds LPS of commensal bacteria (based on the dry weight of components). For example the composition may contain at least 5g, at least 1 Og or at least 15g anti-LPS antibody and fragments thereof per kg of composition on the basis of the dry weight of components. The upper end of the range of antibody concentration will depend on factors such as the dose, the disease state and the health of the patient. The hyperimrnune material may, for example contain no more than 80g such as no more than 60g, no more than 50g or no more than 40g anti-LPS antibody and fragments thereof which bind LPS of commensal bacteria per kg of composition on the basis of the dry weight of components. (0100] The effective amount of a composition to be administered can be dependent on any number of variables, including without limitation, the species, breed, size, height, weight, age, overall health of the subject, the type of formulation, the dose of radiation received or anticipated, or the amount of time before or the amount of time elapsed since exposure to radiation. The appropriate effective amount can be routinely determined by those of skill in the art using routine optimization techniques, the skilled and informed judgment of the practitioner, and other factors evident to those skilled in the art. Preferably, a therapeutically effective dose of the compounds described WO 2012/171077 PCT/AU2012/000700 21 herein will provide therapeutic benefit without causing substantial toxicity to the subject. [0101] In one embodiment the anti-LPS immunoglobulin preparation is administered at a dose of about 5 mg to about 25000 mg per day, 10 mg to about 20000 mg per day, 25 mg to about 15000 mg per day, or about 100 mg to about 2000 mg per day. [0102] In one embodiment, the anti-LPS immunoglobulin preparation is administered at a dose of about 200 mg to about 2000 mg per day. In another embodiment, the anti-LPS immunoglobulin preparation is administered at a dose of about 50 mg to about 500 mg one to four times per day. [0103] In one embodiment, the anti-LPS immunoglobulin preparation is administered at a dose of 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, or 2000 mg per day. [0104] In one embodiment, the anti-LPS immunoglobulin preparation is formulated for administration at a dose of about 5 mg to about 25000 mg per day, about 10 mg to about 20000 mg per day, about 25 mg to about 15000 mg per day, or about 100 mg to about 2000 mg per day. [0105] In one embodiment, the anti-LPS immunoglobulin preparation is formulated for administration at a dose of about 200 mg to about 2000 mg per day. In another embodiment, the anti-LPS immunoglobulin preparation is formulated for administration at a dose of about 50 mg to about 500 mg one to four times per day. [0106] In one embodiment, the anti-LPS immunoglobulin preparation is formulated for administration at a dose of 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, WO 2012/171077 PCT/AU2012/000700 22 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, or 2000 mg per day. [0107] Toxicity and therapeutic efficacy of agents or compounds can be assayed using standard pharmaceutical procedures in a variety of systems and environments, including cell-free environments, cellular environments (e.g., cell culture assays), multicellular environments (e.g., in tissues or other multicellular structures), and/or in vivo (e.g., in experimental animals), e.g., by determining the LD50 (the dose lethal to 50% of the population) and/or the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. [0108] Data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in the subject. The dosage of such agents or compositions lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. [0109] The composition used in the treatment of mucositis may in one embodiment comprise a carrier admixed with the anti-LPS antibody prior to administration, for example, by mixing a composition of hyperimmune colostrum from immunised cows or one or more processed components thereof with conventional foods and/or pharmaceutically acceptable excipients. The ratio of enriched product relative to conventional dairy material from unvaccinated animals may, for example, be at least 4, such as at least 10 in a comparative ELISA assay. [0110] In another embodiment part or all of the LPS antibodies are extracted from the colostrum and used to prepare a composition for administration to treat mucositis.

WO 2012/171077 PCT/AU2012/000700 23 [01111 In one embodiment the hyperimmune material binds LPS taken from at least one Gram negative organism selected from the group of genera consisting of Enterobacter, Escherichia, Klebsiella, Bacteroides, Proteus, Salmonella, Serratia, Veillonella and Fusobacteria. Preferably the hyperimmune material binds at least two of the above family, more preferably at least 3, even more preferably at least 4. [0112] The degree of enrichment in material selected from anti-LPS antibodies and fragments thereof which bind LPS may be at least 4 times, for example at least 10 times the level found in corresponding unvaccinated animals with respect each of 2 bacterial LPS, each of 3 bacterial LPS or each of 4 bacterial LPS as determined by standard ELISA. [0113] In one embodiment, low molecular weight moieties have been substantially removed from the colostrum or the colostrum extract. By substantially removed is meant that at least 75% and preferably 90% of the low molecular weight moieties are removed. [0114] In a preferred example of this embodiment at least 75% (such as at least 90% or substantially complete removal) of, moieties of molecular weight less than 30kDa have been removed from the colostrum or the colostrum extract. Preferably molecular weight moieties less than 60kDa have been substantially removed from the colostrum or colostrum extract. [0115] In one embodiment, the hyperimmune material comprises immunogenic material selected from antibody and antibody fragments which bind LPS associated with commensal bacteria. Preferably the antibody or antibody fragment is a polyclonal antibody or a polyclonal antibody fragment of bovine origin. [0116] In one embodiment the antibody or antibody fragment is generated by vaccinating a dairy cow, wherein the vaccine comprises LPS moieties substantially separated from bacterial wall fragments as a result of the WO 2012/171077 PCT/AU2012/000700 24 application of shear. This process is described in our co-pending application PCT/AU2004/00027, which is incorporated herein by reference. [0117] In one embodiment, the LPS ligands are provided in an oral formulation, and treatment of the patient by oral administration commences 3 to 6 days before cancer therapy takes place. [0118] In another embodiment, the LPS ligands are provided in an oral formulation, and treatment by oral administration commences within 1 to 48 hours after cancer therapy takes place. [0119] The vaccination regimen leading to the production of hyperimmune colostrum preferably involves the injection of an animal with 0.3 to 15 mL of vaccine on 2 to 8 occasions prior to parturition. The time period between successive vaccinations is 1 to 4 weeks, more preferably 2 to 3 weeks. Methods for production and processing of colostrum are provided in US Patent 5,780; 028 the contents of which are incorporated by reference. [0120] The processed hyperimmune colostrum can be formulated as a tablet or as a powder within a capsule or as an additive to a drink mix as described in US Patent 5,780, 028. [0121] In one embodiment, the composition is formulated for sequential, separate or co-administration with an antibiotic. [0122] In one embodiment the present invention provides for concurrent administration of a composition of the present invention with an antibiotic or combination of antibiotics. The antibiotics may include B-lactam antibiotics with and without B-lactamase inhibitors, aminoglycosides, tetracyclines, sulfonamides and trimethoprim, vancomycin, macrolides, fluoroquinolones and quinolones, polymyxins and other antibiotics. [0123] In one embodiment, the antibiotic is ciprofloxacin.

WO 2012/171077 PCT/AU2012/000700 25 [0124] Ciprofloxacin, the human-use equivalent of the veterinary fluoroquinolone ENR, was Food and Drug Administration-approved in 1987. Fluoroquinolones have excellent oral bioavailability, are well tolerated, and have been extensively used after myeloablative chemoradiotherapy. (0125] The compositions of the present invention have an advantage in that they are unlikely to produce toxicity in either minimally affected or critically ill populations. Furthermore, the compositions of the present invention have an advantage in that they are stable and may be stored and stockpiled for the treatment of populations of individuals. For example, the compositions of the present may be stored and stockpiled for the treatment of populations of individuals inadvertently exposed to radiation or chemical agents. [0126] Preferably the composition for administration to the patient further comprises a food-grade antimicrobial moiety, such as citrus extracts and iodine based antiseptics. In one preference the antimicrobial moiety is the grapefruit seed extract of the chemical family diphenol hydroxybenzene sold under the product name Citricidal by NutriBiotics of Ripton, Vermont, USA. [0127] The composition for administration to the patient may be the hyperimmune material but may and may be derived from the hyperimmune material. [0128] For example, in the case of colostrum the composition for administration to the patient may have been processed using a detailing operation, more preferably using a defatting operation and an operation to remove cellular debris, more preferably a defatting operation, an operation to remove cellular debris and an operation to remove salts, sugars, other low molecular weight entities and some water. {0129] In one embodiment the composition for administration to the patient comprises colostrum components which contain the anti-LPS in dried form. Other components such as selected from the group consisting of adjutants, carriers , drugs, and other actives may be present in the composition and may WO 2012/171077 PCT/AU2012/000700 26 be intimately mixed before, during or after the drying process. The composition comprising colostrum may be dried by lyophilisation or other method known in the art for drying colostrum. [0130] In one embodiment the composition for administration to the patient comprises at least three quarters of the lyophilised material by dry weight of the composition based -on the dry weight of lypholized hyperimmune colostrum. [0131] Preferably the bovine colostrum collected from the cow comprises at least 4% total protein (weight %), more preferably 5%, more preferably at least 8%, more preferably at least 10%. [0132] Preferably the ratio of IgG to total protein of the colostrum collected from the cow is at least 10%, more preferably 20%. [0133] The composition for administration to the patient maybe in the form of preparations such as food additives, aqueous solutions, oily preparations, emulsions, gels, etc. , and these preparations may be administered orally, topically, rectally, nasally, bucally, or vaginally. The preparations may be administered in dosage formulations containing conventional non-toxic acceptable carriers and may also include one or more acceptable additives, including acceptable salts, polymers, solvents, buffers, excipients, bulking agents, diluents, excipients, suspending agents, lubricating agents, adjuvants, vehicles, delivery systems, emulsifiers, disintegrants, absorbents, preservatives, surfactants, colorants, flavorants or sweeteners. A preferred dosage form of the present invention is a powder for incorporation into beverages, pills, syrup, capsules, tablets, granules, beads, chewable lozenges or food additives, using techniques known in the art. [0134] The composition for administration to the patient may, for example, contain additives such as described in our co-pending application WO/2006/053383 the contents of which are herein incorporated by reference.

WO 2012/171077 PCT/AU2012/000700 27 [0135] The composition may be administered to the patient in a range of forms depending on the area of the GI tract which is subject to mucositis (or likely to be subject to mucositis), the type of cancer treatment and condition of the patient. Examples of forms include mouth washes gargles, suppositories, tablets, caplets, pastes, syrups, or in powder or water dispensable powder or granular forms. Where the composition is administered in tablet form the tablet may be made by compressing or moulding the active ingredient, with one or more accessory ingredients optionally included. Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free- flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active, or dispersing agent. Moulded tablets may be made in a suitable machine, by moulding together a mixture of the powdered active ingredient and a suitable carrier, moistened with an inert liquid diluent. [0136] The treatment may be used in inhibiting and/or relieving mucositis in a range of cancer treatments. In an embodiment of the invention the patient is being treated for at least one cancer selected from the group consisting of bone marrow, neck, head, stomach/oesophagus, liver, pancreas and the chemotherapeutic agent is selected from the set of Alkylating agents (such as Cyclophosphamide), Anthrocyclines (such as Doxorubicin), Cytoskeletal disruptors (such as Paclitaxel), Epothilones, Nucleotide analogs and precursor analogs (such as Fluorouracil), peptide antibiotics (such as Bleomycin), Platinum based agents (such as Cisplatin), Retinoids and Alkyloids. {01371 The patient may be treated using the composition comprising anti-LPS antibodies or fragments prior to or during treatment for at least one of cancer of bone marrow, neck, head, stomach/oesophagus, liver, pancreas and the anti-cancer radioisotope agent is selected from the set teletherapy (external beam radiotherapy) including proton therapy, brachytherapy - sealed source radiotherapy, brachytherapy -unsealed source radiotherapy (this includes injection or ingestion of radioisotopes).

WO 2012/171077 PCT/AU2012/000700 28 [0138] In one embodiment, the composition is administered to the patient in a combination therapy with sucralfate paste. Preferably the sucralfate is freshly prepared prior to administration. [0139] In one embodiment, the composition and sucralfate are co administered in one or more medicament formulations. [0140] In another embodiment, the composition and sucralfate are separately administered. The composition may be administered to the patient in a dose of anti-LPS antibody (and /or fragment) suitable to relieve or inhibit mucositis. The dose will typically be repeated at intervals during and/or prior to cancer treatment which are effective to limit the effects of mucositis. In one example of this embodiment the composition is dosed 1 to 4 times per day at a dose of between 50 to 5000 mg of antibody such as from 200 to 3500 mg of antibody or fragment on an igG equivalent basis. The IgG equivalent of an antibody fragment is the original IgG moiety from which the fragment is taken. [0141] The antibody may-be delivered in a liquid, paste or solid and in one embodiment the antibody is in a liquid formulation and each dose comprises 20,to 200 ml total liquid volume such as from 50 to 150 ml total volume. [0142] Such a liquid formulation may comprise a thickening agent such as xantham gum and / or a flavouring agent such as vanilla. [0143] The invention will now be described with reference to the following examples. It is to be understood that the examples are provided by way of illustration of the invention and that they are in no way limiting to the scope of the invention. EXAMPLES Example 1 WO 2012/171077 PCT/AU2012/000700 29 [0144] In this example procedures are provided for confirming that hyperimmune sample material comprising anti - LPS antibody binds with LPS derived from commensal bacteria. [0145] This demonstration involves the following steps: 1. Procurement of a commensal Gram negative strain. The following strains were obtained from the University of Melbourne: Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa and Salmonella typhimurium. 2. Culturing the strain. Enterobacter aerogenes was cultured on horse blood agar (HBA) plates in 370C incubator for 16 hours, Kiebsiella pneumoniae was cultured on Luria agar (LA) plates in37*C incubator for 16 hours, Pseudomonas aeruginosa was cultured on horse blood agar (HBA) plates in 37*C incubator for 16 hours and Salmonella typhimurium was cultured on Luria agar (LA) plates in37 0 C incubator for 16 hours. 3. Purifying LPS from the culture. The procedure was based on Hitchcock, P. J. & Brown, T. M. (1983). Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver stained polyacrylamide gels. J. Bacterio. 154, 269-277, with modifications as follows: e Bacteria are collected from the plate the following day using a sterile cotton swab and suspended in phosphate buffered saline (PBS), to an optical density of 2.0 measured at 600 nm. * 1.0 ml of the suspension is transferred to microcentrifuge tube, centrifuged at 10 000 g for 3 minutes at room temperature. The supernatant is discarded. e Resuspend the bacterial pellet in 200 pl of lysis buffer (1 M Tris-HCI (pH 6.8), 2 % SDS, dH 2 0) and boil sample for 10 minutes.

WO 2012/171077 PCT/AU2012/000700 30 e Add 5 pl of proteinase K (stock 20 mg/ml), vortex and incubate at 60 *C for 60 minutes. 9 Add 300 p1 of phenol to the sample and incubate at 65 *C for 15 minutes. Vortex every 5 minutes. * Centrifuge samples at 10 000 g for 10 minutes at 4 *C. e Remove clear aqueous phase (top phase) to a clean tube. Add equal amount of chloroform to remove any residual phenol in the sample. * Centrifuge samples at 10 000 g for 5 minutes at 4 "C. e Remove top phase to a clean tube, store samples at - 20 *C until needed. 4. Electrophoresis of the purified LPS on SDS PAGE. The procedure was as follows * Prepare a 15 % acrylamide solution for. resolving gel and a 4 % acrylamide solution for separating gel. e Load equal volumes for all the samples into a total volume (in loading dye: 62.5 mM Tris-HCI pH 6.8, 10 % glycerol, 2 % SDS, 40 mM DTT, 0.125 5 bromophenol blue, dH20) of no greater than 15 pl per lane. e Load the samples into the wells and run them at 50 V until the dye has reached the resolving gel, then switch to 130 V and run the samples until the dye has left the gel. (Running buffer: 3.03 g Tris, 14.4 g Glycine, 1.0 g SDS, 1 L dH20). 5. Transfer of the LPS onto a PVDF membrane followed by a Western blot using a solution of the hyperimmune sample material - a replicate using non-hyperimmune material is also prepared. The procedure used was as follows: After running samples on Tris-Tricine-SDS-PAGE transfer the bands onto polyvinylidene difluorite (PVDF) membrane for 2.5 hours at 100 V at 4 0C in transfer buffer pH 8.3 (25 mM Tris, 192 mM Tricine, 10 % v/v MeOH, dH 2 0).

WO 2012/171077 PCT/AU2012/000700 31 * Block membranes overnight at 4 *C in PBS-Tween 0.1 % + 5 % skim milk powder. " Rinse blots with PBS/T 0.1 % for 5 minutes, shaking. " Incubate blots with hyperimmune material diluted 1/200 in PBSIT + 5 % skim milk powder (SMP) or non-hyperimmune material for 2 hours with gentle shaking. " Rinse blots and wash with PBS/T, two times for 5 minutes each wash. " Incubate blots with goat c bovine igG-HRP 1/20 000 in PBSIT + 5 % SMP for 1 hour, with gentle shaking. " Rinse and wash blots as above. " Drain excess PBS/T and place damp blots on an overhead sheet. Prepare ECL reagent; drop 1 ml on each membrane and leave for 1 minute. Place another overhead sheet on top of membrane and blot excess ECL reagent ensuring where the film is placed is dry. * Expose blots to film for required time (1-3 minutes) and develop film. [0146] The results of the above procedures will be discussed with reference to the attached drawings (Fig. 1). In the drawings: Figure A is a Western Blot with anti-LPS antibodies (dairy derived) made as described in Example 3, and Figure B is a Western Blot with antibodies contained in non hyperimmune colostrum. Example 2 [0147] 5-Fluorouracil mouse mucositis model: [0148] 30 Wild-type C57BL/6 mice may be treated with 5-fluorouracil (450mg/kg) by intra-peritoneal injection. 30 control mice may be treated similarly but with saline.

WO 2012/171077 PCT/AU2012/000700 32 (0149] Within each group of 30, after the above treatment, 10 mice may be placed in cages with ad lib access to drinking bottles containing 10% solution of anti-LPS colostrum, or 10 with ad lib access 10% solution of colostrum from non-vaccinated cattle, 10 may be given water in the drinking bottles. (0150] After 3 days the animals may be sacrificed and pieces of duodenum, jejunum and ileum harvested for assessment of epithelial damage by morphometry. 5-FU may be found to induce significant epithelial intestinal damage (reduction of villus height/crypts depth ratio). (0151] Animals with access to anti-LPS colostrum may have significantly less epithelial damage than animals in the other groups. Example 3 (0152] Production of Hyperimmune Colostrum containing polyclonal anti-LPS antibodies Step 1 - Production of vaccine for dairy cattle. The procedures for preparing LPS-containing antigen reported in Pub. No. W012004/078209 International Application No. PCT/AU2004/000277 (the contents of which are herein incorporated by reference) were used. In brief, cows were immunised with a vaccine comprising 06, 08, 015, 025, 027, 063, 078, 0114, 0115, 0128, 0148, 0153, and 0159 LPS, and the antibodies prepared according to the methods referred to below at Step 2. Step 2 - The procedures for preparing anti-LPS antibodies from vaccinated cattle reported in Pub. No. WO/2004/078209 International Application No. PCT/AU2004/000277 (the contents of which are herein incorporated by reference) were used. Example 4 WO 2012/171077 PCT/AU2012/000700 33 (0153] Effect of colostrum containing polyclonal anti-LPS antibodies in combination with oral antibiotic enrofloxacin on survival of mice after 7 gray Total Body Irradiation (TBI). [0154] To examine whether anti-ETEC bovine colostrum may provide benefit when administered 24 hours post-irradiation in conjunction with oral flouroquinolone antibiotic, mice were exposed to radiation and treated as follows. [0155] Powdered skim milk powder (Carnation Skim Milk Powder; Nestle) and colostrum containing polyclonal anti-LPS antibodies produced by the methods described in Example 3 ("ETEC") (Immuron; Melbourne, Australia) were reconstituted at a low (4mg/mouse/day) and high (40mg/mouse/day) dose in autoclaved tap water. [0156] Assuming each mouse drinks -5mL/day, bottle dosing was estimated at 0.8mg/mL of ETEC for low dose (4mg) and 8mg/mL of ETEC for the high dose (40mg). Fresh batches of skim and ETEC solution were made every 48 hours throughout the 14 days of milk product administration. J01571 Mice were irradiated with 7 gray then 24 hours later treated with nothing ("7Gy"), oral antibiotic enrofloxacin ("ABX") thru day 30 (first 5 days of which was administrered via gavage with our without 14 days of ad libitum skim milk, or colostrum containing polyclonal anti-LPS antibodies produced by the methods described in Example 3 ("ETEC"). The percentage of animal surviving to Day 30 are indicated in the legend. N = 12 mice per group. [0158] Mice receiving 7 gray all died by day 16. In contrast, mice receiving oral antibiotic show significantly increased time to death, with 100% mortality by day 23. Mice receiving both antibiotic and colostrum containing polyclonal anti-LPS antibodies ("ETEC") demonstrate survival to Day 30.

WO 2012/171077 PCT/AU2012/000700 34 [0159] These data indicate colostrum containing polyclonal anti-LPS antibodies decrease the morbidity mortality and mortality associated with exposure to radiation. [0160] Finally, it is understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein.

Claims (33)

1. A method for .treating or inhibiting the development of alimentary mucositis arising from chemotherapy or radiation damage in a subject comprising: providing a hyperimmune dairy material or egg derived material comprising anti-lipopolysaccharide (anti-LPS) antibody raised against a vaccine comprising LPS; and administering to the subject a composition comprising the anti-LPS antibody or fragment thereof which antibody or fragment binds LPS associated with commensal bacteria.

2. A method according to claim 1 wherein the subject is a patient suffering cancer

3. A method according to claim 1 wherein the subject is person exposed to radiation

4. A method according to claim 1 wherein the LPS in the vaccine is derived from gram negative bacteria.

5. A method according to claim 1 wherein the LPS in the vaccine is at least partly separated from bacterial cell walls.

6. A method according to any one of the previous claims wherein the LPS in the vaccine has been separated by mechanical means.

7. - A method according to any one of the previous claims wherein the hyperimmune material comprises at least 3g (preferably at least 1 Og) IgG anti LPS antibody per kilogram of hyperimmune material, or an equivalent molar concentration of other LPS antibody or antibody fragment.

8. A method according to any one of the previous claims wherein the composition for administration to the patient comprises at least 3g (preferably WO 2012/171077 PCT/AU2012/000700 36 at least 1 Og) IgG anti-LPS antibody per kilogram of composition based on the dry weight or an equivalent molar concentration of other LPS antibody or antibody fragment.

9. A method according to any one of the previous claims wherein the composition for administration to the subject comprises a colostrum derived from colostrum hyperimmune material using a defatting operation and an operation to remove cellular debris.

.10. A method according to any one of the previous claims wherein the hyperimmune material is enriched in anti-LPS antibodies by at least five times relative to the content of corresponding material from unvaccinated animals as determined in a comparative ELISA assay.

11. A method according to any one of the previous claims wherein the anti LPS antibodies or fragments thereof bind LPS from at least one commensal bacteria from a genus selected from the group consisting of Enterobacter, Escherichia, Klebsiella, Bacteroides, Proteus, Salmonella, Serratia, Veillonelia and Fusobacteria.

12. A method according to any one of the previous claims wherein composition for administration to the subject comprises material selected from the group of anti-LPS antibodies and fragments thereof which material binds LPS derived from two or more genera of gram negative bacteria.

13. A method according to any one of the previous claims wherein the composition for administration to the patient comprises a colostrum extract from which at least 75% and preferably 90% of components of less than 60kDa in hyperimmune colostrum material have been removed.

14. A method according to any one of the previous claims wherein the radiation damage is associated with cancer therapy. WO 2012/171077 PCT/AU2012/000700 37

15. A method according to claim 14 wherein hyperimmune material is provided in an oral formulation, and treatment of the subject by oral administration commences less than 6 days before cancer therapy takes place.

16. A method according to claim 14 wherein hyperimmune material is provided in an oral formulation, and treatment by oral administration commences within 1 to 48 hours after cancer therapy takes place.

17. A method according to any one of claims 14 to 16 wherein the patient is suffering.at least one cancer selected from the group consisting of bone marrow, neck, head, stomach/oesophagus, liver and pancreatic cancer.

18. A method according to any one claims 14 to 17 wherein the chemotherapeutic agent is selected from the group consisting of alkylating agents (such as.Cyclophosphamide), anthrocyclines (such as Doxorubicin), cytoskeletal disruptors .(such as Paclitaxel), epothilones, nucleotide analogs and precursor analogs (such as Fluorouracil), peptide antibiotics (such as Bleomycin), platinum based agents (such as Cisplatin), retinoids and alkyloids.

19. A method according to any one of claims 14 to 17 wherein the cancer therapy involves radiation therapy.

20. A method according to any one of claims 14 to 19 wherein the hyperimune material is administered in a combination therapy with sucralfate paste.

21. A method according to any one of the previous claims wherein the hyperimmune material is dosed 1 to 4 times per day at a dose of between 50 to 500 mg of antibody. WO 2012/171077 PCT/AU2012/000700 38

22. A method according to any one of the previous claims wherein the antibody is in a liquid formulation and each dose comprises 20 to 200 ml total liquid volume.

23. A method of treatment of a subject exposed to radiation to reduce the effects of radiation damage comprising: forming hyperimmune colostrum by vaccinating cows; and administering the hyperimmune material to said patient wherein the step of vaccinating cows to produce hyperimmune material comprises vaccination with a vaccine comprising LPS.

24. A method of treatment of a patient suffering cancer to reduce the effects of cancer therapy comprising: forming hyperimmune colostrum by vaccinating cows; and administering the hyperimmune material to said patient wherein the step of vaccinating cows to produce hyperimmune material comprises vaccination with a vaccine comprising LPS.

25. A method according to claims 23 or 24 wherein hyperimmune colostrum formed comprises material selected from the group consisting of anti-LPS antibodies and fragments thereof wherein the material binds commensal bacteria.

26. A method according to any one of the previous claims further comprising administering an antibiotic.

27. A medicament composition for treatment of a subject exposed to radiation to reduce the effects of radiation damage comprising anti-LPS antibodies which bind commensal bacteria.

28. A medicament composition for treatment of a patient suffering cancer to reduce the effects of cancer therapy comprising anti-LPS antibodies which bind commensal bacteria. WO 2012/171077 PCT/AU2012/000700 39

29. Use of a hyperimmune colostrum in manufacture of a medicament for treatment or inhibition of alimentary mucositis arising from radiation damage in a subject exposed to radiation wherein the hyperimmune material is raised by vaccination with an antigen comprising LPS and the medicament comprises material selected from the group consisting of anti-LPS antibodies and fragments thereof wherein the material binds commensal bacteria.

30. Use of a hyperimmune colostrum in manufacture of a medicament for treatment or inhibition of alimentary mucositis arising from cancer therapy in a patient suffering cancer wherein the hyperimmune material is raised by vaccination with an antigen comprising LPS and the medicament comprises material selected from the group consisting of anti-LPS antibodies and fragments thereof wherein the material binds commensal bacteria.

31. Use of LPS in manufacture of a medicament for treatment or inhibition of alimentary mucositis arising from radiation damage in a subject exposed to radiation by administration of a composition comprising one or more of anti LPS antibodies and fragments thereof obtained from hyperimmune colostrum raised by vaccination of cows with LPS wherein the anti-LPS antibodies and fragments thereof bind commensal bacteria.

32. Use of LPS in manufacture of a medicament for treatment or inhibition of alimentary mucositis arising from cancer therapy in a patient suffering cancer by administration of a composition comprising one or more of anti-LPS antibodies and fragments thereof obtained from hyperimmune colostrum raised by vaccination of cows with LPS and wherein the anti-LPS antibodies and fragments thereof bind commensal bacteria.

33. A method of cancer radiotherapy which comprises administering to a subject in need of such therapy an amount of a composition comprising anti LPS antibodies which bind commensal bacteria effective to minimise damage to subject to radiation.