HK1129861B - Use of peptides for the control of radiation injury - Google Patents

Description

Use of peptides for controlling radiation damage

Technical Field

The present invention relates to the field of drug development for acute radiation injury caused by exposure to high-energy electromagnetic waves (X-rays, gamma rays) or particles (alpha particles, beta particles, neutrons). To date there is no effective drug that can mitigate radiation damage following accidental exposure to ionizing radiation.

Background

Radiation damage is tissue damage caused by exposure to radiation. Here, the radiation refers to ionizing radiation generated from high-energy electromagnetic waves (X-rays, gamma rays) or particles (alpha particles, beta particles, neutrons). Such radiation is released by radioactive substances (radioisotopes) such as uranium, radon, and plutonium. Such radiation may also be generated by artificial radiation sources, such as X-ray and radiotherapy instruments. The radiation dose is measured using a number of different units, but they all relate to the amount of energy deposited. These units include roentgen (R), gray (Gy), and haworth (Sv). Xivor is similar to gray, except that it takes into account the biological effects of different radiation types. The two main types of radiation exposure are irradiation and contamination. Many radiation incidents expose people to both types simultaneously.

Irradiation is exposure to radiation waves that penetrate the body directly from outside the body. Irradiation can cause immediate morbidity (acute radiation sickness). In addition, irradiation, particularly high doses of radiation, can damage human genetic material (DNA), resulting in chronic (late-onset) diseases such as cancer and congenital defects. However, the irradiation does not cause the human or its tissue to be radioactive. Contamination is the contact and retention of radioactive materials, the latter typically in the form of dust or liquid. The radioactive material can remain on the skin and can fall or be wiped off the skin, contaminating other people and objects. These substances can also be absorbed by the body through the lungs, digestive tract, or skin lesions. The absorbed material is transported to various parts of the body, such as the bone marrow, where it continues to release radiation. Such internalized radiation can cause not only acute radiation diseases such as internal bleeding, but also chronic diseases such as cancer.

People are exposed to low levels of natural radiation (background radiation) for long periods of time. The radiation comes from outer space (cosmic radiation), but it is largely blocked by the earth's atmosphere. People living or working in high-radioactive element conditions are exposed to more cosmic radiation, in particular radon gas, which is also present in many rocks and minerals. These elements are ultimately found in a variety of substances, including food and building materials. In addition, people are also exposed to radiation from artificial sources of radiation, including environmental radiation from nuclear weaponry trials and radiation from various medical examinations and treatments. The average human is exposed to radiation from natural and artificial radiation sources in a total amount of about 3 to 4mSv (1 mSv-1/1000 Sv) each year. Persons in operation in contact with radioactive materials and X-ray sources are susceptible to exposure to higher levels of radiation. A person receiving radiation therapy for cancer may be exposed to very high levels of radiation. Nuclear weapons release a large amount of radiation. Such weapons have not been used for humans since 1945. However, many countries now own nuclear weapons, and some terrorist groups are also trying to acquire nuclear weapons, which increases the likelihood that such weapons will be reused day to day.

The damaging effects of radiation depend on a variety of factors, including the amount (dose) and duration of exposure. A single dose of radiation that is rapid across the body can be lethal, whereas exposure of the same total dose over weeks or months can be much less effective. For a given dose, rapid exposure is more likely to cause genetic damage. The effect of radiation also depends on the exposure range of the body. For example, radiation in excess of 6Gy often leads to death if dispersed throughout the body; but if concentrated on only a small area, as in cancer radiotherapy for example, 3 to 4 times the dose of radiation can be administered without serious harm to the subject as a whole. The distribution of the radiation is also important because certain parts of the body are more sensitive to radiation. Organs and tissues in which cells proliferate rapidly, such as the intestinal tract and bone marrow, are more susceptible to radiation damage than organs and tissues in which cells proliferate slowly, such as muscles and tendons. The genetic material of sperm and egg cells can be destroyed by radiation. Therefore, during cancer radiotherapy, these more fragile parts of the body are shielded as much as possible from the radiation so that high doses of radiation can be applied mainly to the cancer.

Radiation exposure produces two types of damage: acute (immediate) and chronic (delayed). Acute radiation injury causes inflammation by vascular endothelial injury leading to vascular leakage. Followed by a vascular response and a cellular response. Ionizing radiation inhibits immunity and damages the intestinal epithelium, both of which promote translocation of microorganisms from the intestine.

Cancer radiation therapy produces symptoms mainly in the areas of the body that receive the radiation. For example, in radiation therapy for rectal cancer, abdominal cramps and diarrhea are common due to the effects of radiation on the small intestine.

The search for non-toxic radioprotectors to protect normal tissues against radiation damage began shortly after world war ii. Extensive radiobiological studies have found a variety of protective agents that protect animals (mainly rodents) from radiation damage if administered prior to radiation exposure [ praladkn. CRC Press, 1995 ]. These studies have found that agents that scavenge free radicals and/or cause hypoxia have radioprotective value. However, most of these compounds are toxic to humans at radioprotective doses. With the reduced risk of nuclear challenges during cold wars, the interest in the subsequent research on radioprotectants has decreased significantly. Due to the rapid proliferation of X-ray based diagnostic instruments and the increasing use of radiological methods for early diagnosis of disease, there is a growing interest in increasingly frequent somatic and genetic mutations that can increase the risk of genetic-linked disease to present people and their offspring. Therefore, normal tissue must be protected from potential radiation damage, even if this damage is minimal.

Generally, radioprotectors are defined as compounds that, when administered prior to exposure to ionizing radiation, reduce the damaging effects of radiation, including radiation-induced death [ h.b. stone et al, Models for evaluating agents incorporated for the prophylylaxis, assessment and specification of radiation in resources. report of an NCI works, December3-4, 2003, radiation Res 162: 711-728.]. They can be used for radiological terrorism, military events, clinical oncology, space travel, radiation spot clearance [ r.h. johnson, Dealing with the terror of nuclear terrorism, Health Phys 87: s3-7, f.a.j.mettler, g.l.vollz, major exposure to exposure and how to response, N Engl J Med 346: 1554-: recommendations of the structural national stockpile Radiation Working Group, Ann Intern Med 140: 1037-1051.]. Recently, the U.S. department of Science and Technology Policy and the Homeland Security Council (Homeland Security Council) have listed the development of new radioprotective agents as the top priority research projects. Although synthetic radioprotective agents such as the aminothiols (aminothiols) produce the highest coefficient of protection, they are generally more toxic than naturally occurring protectants. Generally, the best radioprotectants are also believed to result in the highest toxicity.

Effective mitigation of radiation-induced health problems and fighting-force-reducing effects in military radiation events can reduce casualty loads at medical treatment sites, maintain more effective locomotion after radiation exposure events, allow commanders to conduct operations in the radiation field environment with a reduced risk of reduced mobility due to acute tissue injury, and reduce negative psychological trauma to those performing tasks in a contaminated environment. Ideally radioprotectors should be non-toxic, not detracting from working ability, and act after a single application, particularly if rapid access to areas at risk of external radiation is required.

A paper published in AFRRI CD05-2 by NATO human factors and medical Panel Research Task Group099, "Radiation Bioeffects and data organizations Research", filed by Bethesda, Md. 2005, was reported on the meeting (Landaur et al, NATO RTG-0992005) and suggested that genistein (genistein) mice could protect against gamma Radiation-induced death with a "dose reduction factor" (DRF) of 1.16 at the optimal dose (200 mg/kg; the highest survival rate was obtained at 24 hours prior to irradiation). No radioprotective effect was observed if administered 1 hour prior to total body irradiation (WBI). Other studies reported radioprotection of the drug identified as ON-01210 at the 51 st academy of radiation research (4 months 2004), indicating that this drug, ON-01210 (similar to other drugs currently under investigation for radiation exposure), is only protective until radiation exposure. The medicine has sulfhydryl component (4-carboxyystyrl-4-chlorobenzilsulfone), and can be used as antioxidant to remove free radicals generated during cell injury due to radiation.

Also, as described in the annual Report of the congress of the United states department of defense (March 2005; http:// medchembio. ame. army. mil/docs/CBDP _ Report _ To _ Congress. pdf), there are currently no commercially available nontoxic drugs or diagnostic capabilities suitable for use in the military operations environment. Amifostine (amifostine) is an aminothiol compound that has been approved by the FDA for use in patients receiving chemotherapy or radiation therapy, but its adverse toxicity profile reduces its potential for use in appropriate combat troops, and its intravenous route of administration requires specialized medical personnel. Other drugs such as hematopoietic cytokines for the treatment of bone marrow damage may be used by various physicians in an out-of-label dose depending on the specific circumstances of different cases, but regulatory limitations of such use make it difficult to handle the large numbers of casualties in military operations. Antibiotics are commonly used to treat the infectious sequelae of radiological injury, but must be properly selected to effectively treat both exogenous and endogenous systemic infections while minimally affecting the beneficial gut anaerobes. To address the limited scope of currently available medical strategies, the new compound 5-androstenediol (5-AED; Whitnall et al, Experimental Biology and Medicine 226: 625-. Also, administration of the compound prior to irradiation challenge in a mouse model may result in good radioprotectant efficacy. Subcutaneous administration of AED 24 hours before and 2 hours after gamma irradiation of mice improved survival. The dose reduction factor calculated from the probability value survival curve for a previous WBI administration was 1.3. Protection was observed in both male and female mice that were subsequently inoculated with or without lethal amounts of Klebsiella pneumoniae (Klebsiella pneumoniae). No protection was observed with various other steroids: dehydroepiandrosterone (DHEA), 5-androstene-3B, 7B, 17B-triol (AET), androstenedione, or estradiol. However, in the past year, intensive studies on non-human primate (NHP) models in preparation for the application of IND have demonstrated that 5-AED is far less effective when administered as a radioprotectant than in mouse models, but produces good efficacy on NHP models when administered therapeutically at continuous doses shortly after irradiation.

Acute radiation sickness. Acute radiation sickness usually occurs in people who are exposed systemically to radiation. The progression of acute radiation disease has multiple stages, beginning with early symptoms (prodrome) followed by an asymptomatic stage (latency). Different symptoms (symptom spectrum) then appear depending on the amount of radiation the person receives. The greater the amount of radiation, the more severe the symptoms and the faster the progression from early symptoms to acute syndromes. The symptoms and time course are consistent from person to person for a given radiation exposure. The physician can determine the radiation exposure of the person based on the time course and nature of the symptoms. Depending on the major organ system involved, physicians group acute radiation syndrome into three groups, although there is overlap between the groups.

Hematopoietic syndromes are caused by the effects of radiation on the bone marrow, spleen and lymph nodes, the major sites where blood cells are produced (hematopoiesis). Appetite decline (anorexia), lethargy, nausea and vomiting occur 2 to 12 hours after exposure to 2Gy or higher radiation. Within 24 to 36 after exposure, these symptoms are relieved and the person feels good for a week or more. In this asymptomatic phase, hematopoietic cells in the bone marrow, spleen and lymph nodes begin to deplete and not renew, resulting in a severe depletion of leukocytes, followed by a lack of platelets, followed by erythrocytes. The absence of leukocytes can lead to severe infections. Lack of platelets can lead to uncontrolled bleeding. Lack of red blood cells (anemia) causes weakness, pallor and dyspnea during physical activity. After 4 to 5 weeks, if a person can survive, blood cells begin to regenerate, but a person feels weak and weak for months.

Gastrointestinal syndromes are caused by the effect of radiation on the lining of the digestive tract by the cell lining the diagnostic track. Severe nausea, vomiting and diarrhea occur 2 to 12 hours after exposure to 4Gy or higher radiation. These symptoms can cause severe dehydration, but after 2 days these symptoms are relieved. The person feels good within the following 4 to 5 days, but the gut lining cell layer, which usually serves as a protective barrier, falls off in the dead disease. Thereafter, severe diarrhea, usually bloody diarrhea, reappears, resulting in dehydration. Bacteria from the digestive tract invade the body and cause serious infections. Persons receiving such intense radiation are also likely to develop hematopoietic syndromes, leading to bleeding and infection, and increasing their risk of death.

Cerebrovascular (brain) syndrome occurs when the total dose of radiation exceeds 20 to 30 Gy. People develop rapidly confusion, nausea, vomiting, bloody diarrhea and shock. Blood pressure drops within hours, with convulsions and coma. Cerebrovascular syndromes are considered to be all fatal.

Chronic effects of radiation. The chronic effects of radiation are caused by damage to genetic material in dividing cells. These changes can cause abnormal cell growth, such as cancer. In animals that are heavily irradiated, it has been found that germ cell damage causes defective offspring (birth defects). However, irradiation-induced deformities are rarely observed in the offspring of japanese bomb explosion survivors. The reason may be that radiation exposure below a certain (unknown) level is not sufficient to produce genetic material alterations that lead to birth defects.

Irradiation damage may be if a person is ill after receiving radiation therapy or accidental exposure to radiation. There is no specific test for diagnosing this condition, but some tests can be used to detect infection, low blood count, or organ dysfunction. To determine the severity of radiation exposure, the physician measures the number of lymphocytes (a type of white blood cells) in the blood. The lower the lymphocyte count 48 hours after exposure, the more severe the radiation exposure.

Unlike irradiation, radioactive contamination of the human body can be measured by a Geiger counter capable of detecting radiation. Swabs from the nose, throat and any wounds can also be used to check for radioactivity.

The consequences of radiation damage depend on the dose, dose rate (how rapidly exposure occurs), and distribution in the body, and also on the person's original state of health. In summary, the vast majority of people who experience WBI in excess of 6Gy die from gastrointestinal syndromes. Since it is difficult for doctors to know the exact amount of radiation a person receives, they generally judge the result according to the symptoms of the person. Cerebrovascular syndrome usually results in death within hours to days. Gastrointestinal syndromes usually lead to death in 3 to 10 days, although some people can survive for weeks. Many people who receive appropriate medical assistance do not die of the hematopoietic syndrome based on their total radiation; those who do not survive usually die after 8 to 50 days.

Irradiation currently has no emergency treatment, but physicians closely monitor the person for various symptoms and perform treatment at the time of symptom occurrence. At the same time, unfortunately, there are currently few medical products that address the various acute and long-term toxicities caused by nuclear or radiological attacks. When contamination occurs, the radioactive material needs to be immediately removed to prevent it from being taken up by the body. Skin contaminated with radioactive materials should be immediately scrubbed with large amounts of soap and water or with solutions designed for this purpose, if any. Small, broken wounds should be cleaned sufficiently to remove all radioactive particles, although scrubbing can cause pain. Contaminated hair is cut rather than shaved, and the shave can scratch the skin and allow contaminants to enter the body. Scrubbing was continued until the Geiger counter showed the disappearance of radioactivity. Emesis is induced if a person engulfs radioactive material. Some radioactive materials have special antidotes that prevent the ingested material from being absorbed. Most of these antidotes are only used for people exposed to significant radioactive contamination, such as large reactor accidents or nuclear explosions. Potassium iodide prevents the thyroid gland from absorbing radioactive iodine and reduces the risk of developing thyroid cancer. Other drugs, such as diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), and penicillamine, may be administered intravenously to scavenge some of the radioactive elements that have been absorbed.

When there is no suspected contamination, nausea and vomiting can be alleviated by the use of drugs that prevent vomiting (antiemetics); these drugs are routinely used by patients undergoing radiotherapy. Dehydration can be used for intravenous fluid infusion treatment.

Persons with gastrointestinal or hematopoietic syndromes should be isolated so that they do not come into contact with infectious microorganisms. Blood transfusions and injections of growth factors that stimulate blood cell production (e.g., erythropoietin and colony stimulating factors) may be performed to reduce bleeding and increase blood counts. These growth factors are ineffective if the bone marrow is severely damaged, sometimes requiring bone marrow transplantation, but with low success.

Patients with gastrointestinal syndromes require antiemetics, intravenous fluids, and sedatives. Some people may eat a light diet. Antibiotics such as neomycin may be administered to kill bacteria in the intestinal tract that may invade the body. Antibiotics and antifungal and antiviral drugs are administered intravenously as necessary. The goal of treatment of cerebrovascular syndrome is to make the patient comfortable by relieving pain, anxiety and dyspnea. Anti-convulsive agents may be administered.

People who present with chronic effects of radiation or disease caused by radiation therapy receive symptomatic treatment. The laceration or ulcer may be surgically removed or repaired and hyperbaric oxygen may be used to promote healing. Radiation-induced leukemia is treated with chemotherapy. Blood cells can be replenished by transfusion. Infertility is irreversible, but decreased sex hormone levels due to ovarian and testicular dysfunction can be treated by hormone replacement. Researchers are currently developing ways to prevent or reduce radiation-induced normal tissue damage using cytokines, growth factors, and various other therapies. Amifostine or pilocarpine hydrochloride has been found to reduce the dry mouth (xerostomia) symptoms in head and neck cancer patients receiving radiation therapy.

Clinical and experimental studies on the acute and delayed effects of radiation on cells have enhanced our knowledge in radiotherapy, enabling the optimization of radiation therapy protocols and the adoption of more precise radiation administration patterns. However, since normal and cancerous tissues have similar responses to radiation exposure, radiation-induced damage to normal tissues may occur during or after completion of radiation therapy. Studies do find some evidence that NSAIDs and prostaglandins have radioprotection. Both classes of drugs increase cell survival, but the mechanisms are completely different. Cell dynamics studies have found that cells in the mitotic phase (M) and late G2 of the cell cycle are generally most sensitive to radiation compared to cells in the S early and G1/G0 phases. In addition, radiation causes mitotic delay in the cell cycle. Thus, chemical agents that limit the proportion of cells in the M and G2 phases of the cell cycle or that enhance rapid cell growth could in principle be used to investigate their potential use as radioprotective agents for normal tissues during injection. NSAIDs have been found to exert anti-cancer effects by causing cell cycle arrest, which drives cells towards a quiescent state (G0/G1). The same mechanism of action is observed in the radioprotection of normal tissues. The increase in arachidonic acid concentration after NSAID exposure also results in the production of the apoptosis-inducing agent ceramide. NSAIDs also elevate intracellular levels of superoxide dismutase. Heat shock proteins activated by NSAIDs can enhance cell survival by altering cytokine expression. NSAIDs may also have the effect of inhibiting cell proliferation through an anti-angiogenic mechanism. Some in vivo studies provide evidence that NSAIDs can protect normal tissues from radiation damage. Prostaglandins do not regulate the cell cycle, but they have multiple effects on the growth and differentiation of cells. PGE2 mediates angiogenesis, increasing the supply of oxygen and nutrients necessary for cell survival and growth. Thus, PGE2 at sufficiently high plasma concentrations could enhance cell survival by inhibiting proinflammatory cytokines such as TNF-a and IL-1 β. Thus, PGE2 acts as a modulator, but not a mediator, of inflammation. Prospective studies suggest that administration of misoprostol (misoprostol), a PGE1 analogue, prior to irradiation has potential use in preventing radiation-induced adverse effects. Current understanding of NSAIDs and prostanoids pharmacology suggests that they may minimize adverse effects of radiation on normal tissues when used prophylactically.

In addition to transiently inhibiting cell cycle progression and killing those cells capable of proliferation, irradiation also interferes with homeostasis affected by endogenous mediators of cell-to-cell communication (bodily fluid components of tissues responding to radiation). Changes in mediator levels may modulate the effects of radiation by promoting restoration to normal (e.g., by elevating H-type cell line specific growth factors) or by exacerbating the damage. The latter model is illustrated by reports on changes in eicosanoid (eicosanoid) levels after irradiation and reports on results from empirical treatment of radiation injury with anti-inflammatory drugs. The prodromal, acute and chronic effects of radiation are accompanied by an overproduction of eicosanoids (prostaglandins, prostacyclins, thromboxanes and leukotrienes). These endogenous mediators of the inflammatory response can cause vasodilation, vasoconstriction, increased microvascular permeability, thrombosis, and chemotaxis following radiation exposure. Glucocorticoids inhibit eicosanoid synthesis by interfering with phospholipase a2, whereas non-steroidal anti-inflammatory drugs block prostaglandin/thromboxane synthesis by inhibiting cyclooxygenase. Empirically administered drugs belonging to both categories after irradiation help to mitigate the prodromal, acute and chronic effects of radiation in both humans and animals.

Herron, U.S. Pat. No. 5,380,668(Jan.10, 1995), discloses various compounds having the antigen binding activity of hCG, among others, which are incorporated herein by reference in their entirety. Wherein the diagnostic use of said oligopeptides is generally disclosed. Several patents and patent applications of Gallo et al (e.g., U.S. Pat. No. 5,677,275 (corresponding to WO96/04008A1), U.S. Pat. No. 5,877,148 (also corresponding to WO96/04008A1), WO97/49721A1, U.S. Pat. No. 6,319,504 (corresponding to WO97/49373), U.S. Pat. No. 2003/0049273A1 (also corresponding to WO97/49373), U.S. Pat. No. 5,968,513 (corresponding to WO97/49418), U.S. Pat. No. 5,997,871 (corresponding to WO97/49432), U.S. Pat. No. 6,620,416, U.S. Pat. No. 6,596,688, WO01/11048A2, WO01/10907A2. and U.S. Pat. 6,583,109) relate to different oligopeptides and their use, such as "inhibition of HIV infection", "treatment or prevention of cancer", "treatment or treatment of disorders associated with pathological angiogenesis", "treatment or prevention of a decrease of a somatic cell mass", "treatment, Expanding blood cells in vitro "and/or" providing blood cells to a patient ". Such as PCT International publication No. WO03/029292A2 (published: April10, 2003), PCT International publication No. WO01/72831A2 (published: October4, 2001) and U.S. patent application publication 20020064501A1 (published: May 1, 2002), 1A1 (published: June1, 2003) and 1A1 (published: September 1, 2003), U.S. patent application No.1, October1, 2005, International application No. PCT/EP 2005/1, April1, 2005, U.S. patent application No.1, April1, 2004, U.S. patent application No.1, September 1, PCT international application No. 1/002NL 1/3659 (published: International patent application No. WO 2001/1), and other immune modulating disease states, such as sepsis, e.g. immune disorders, such as oligopeptides, or immune disorders, such as oligopeptides, which have activity in PCT international publication, or which can be treated, the contents of all of the above documents are incorporated by reference into this application.

The present invention relates to the natural pathways by which the body regulates important physiological processes and is based on the knowledge from PCT International publications WO99/59617 and WO01/72831 and PCT International application PCT/NL02/00639, which are hereby incorporated by reference in their entirety. These applications disclose gene-regulatory peptides present in pregnant women resulting from proteolytic cleavage of placental gonadotropins, such as hCG. These cleavage products are usually only 2 to 6 amino acids in length and have been found to have superior immunological activity by modulating the expression of genes encoding inflammatory mediators such as cytokines. Unexpectedly, it was found that fragmentation of hCG produced a series of peptides that helped to maintain immunological homeostasis in pregnant women. These peptides balance the immune system to ensure that the mother remains immunologically stable, while its fetus is not rejected during pregnancy but is safely gestated until birth.

Furthermore, the present invention relates to U.S. application 10/821,240, which provides methods for screening and identifying other small gene regulatory peptides and using peptides from the results of such screening, e.g., from a reference peptide. For example, the peptide to be analyzed is from C-reactive protein (CRP) (e.g., human CRP), such peptides include LTSL, fvs, NMWD, LCFL, MWDF, FSYA, FWVD, AFTV, and WDFV; peptides from beta-catenin (e.g., human CTNB) such as GLLG, TAPS, VCQV, CLWT, VHQL, GALH, LGTL, TLVQ, QLLG, YAIT, LCEL, GLIR, APSL, ITTL, QALG, HPPS, GVLC, LCPA, LFYA, NIMR, NLIN, LHPP, LTEL, SPIE, VGGI, QLLY, LNTI, LWTL, LYSP, YAMT, LHNL, TVLR, and LFYA; peptides from β -hCG (e.g., human CG), such as GLLLLLLLS, MGGTWA, TWAS, TLAVE, RVLQ, VCNYRDV, FESI, RLPG, PRGV, NPVVS, YAVALS, LTCDDP, EMFQ, PVVS, VSYA, GVLP, FQGL, and AVAL; peptides from Bruton's tyrosine kinase (e.g., human BTK) such as LSNI, YVFS, LYGV, YVVC, FIVR, NILD, TIMY, LESI, FLLT, VFSP, FILE, TFLK, FWID, MWEI, QLLE, PCFW, VHKL, GVHLY, LESI, LSNI, YVFS, IYSL, and NILD; and peptides derived from matrix metalloproteinase-2 (e.g., human MM02), such as FKGA, FFGL, GIAQ, LGCL, YWIY, AWNA, ARGA, PFRF, APSP, CLLS, GLPQ, TFWP, AYYL, FWPE, CLLG, FLWC, RIIG, WSDV, PIIK, GLPP, RALC, LNTF, LSHA, ATFW, PSPI, AHEF, WRTV, FVLK, VQYL, KFFG, FPFR, IYSA, and FDGI, and the like.

Disclosure of Invention

The present invention relates to the field of drug development for acute radiation damage caused by exposure to high-energy electromagnetic waves (X-rays/photons and/or natural gamma rays) and/or other high-energy ionizing particles (alpha particles, beta particles, neutrons, protons, pi-mesons). To date there are no effective drugs that mitigate radiation damage after accidental exposure to ionizing radiation, or during therapeutic irradiation or after damage to normal tissue by radio-mimetic agents; there is also no effective prophylactic drug that can be administered prior to an event (e.g., a first responder) to prevent or minimize such damage. The inventors have unexpectedly observed that relatively small, non-toxic peptides can be effectively used as anti-radiation damage drugs. Importantly, the radiation resistant peptides of the present invention are not only useful as prophylactic agents, but also have a protective effect when administered hours after exposure to radiation. This makes them particularly suitable for use in military radiation events, such as for terrorist activities to combat nuclear terrorism. Accordingly, the present invention provides a method for preventing or treating radiation damage in a subject in need thereof, comprising administering to said subject a peptide of less than 30 amino acids or a functional analogue thereof. Preferably, the peptide or functional analogue thereof is administered to the subject after irradiation, i.e. after exposure of the subject to a radiation source. Furthermore, the present invention provides the use of a peptide of less than 30 amino acids or a functional analogue thereof for the manufacture of a pharmaceutical composition for the treatment of a subject suffering from or believed to suffer from radiation damage. In particular, the invention provides a radiation resistant peptide having a Dose Reduction Factor (DRF) for acute whole body irradiation of at least 1.10, which DRF can be determined as follows: testing what dose of Whole Body Irradiation (WBI) resulted in 50% mortality (LD50/30) in experimental rodents, such as mice, in the test group treated with the peptide immediately or up to 72 hours post WBI, 30 days post WBI, compared to WBI doses that resulted in 50% mortality (LD50/30) in untreated controls 30 days post WBI, where the DRF was calculated by dividing the LD50/30 radiation dose in the peptide-treated animals by the LD50/30 radiation dose in the vehicle-treated animals.

The present invention provides a method of treating a subject suffering from or believed to be suffering from radiation damage, the method comprising providing to the subject a pharmaceutical composition comprising a radiation resistant peptide of less than 30 amino acids. Current radioprotectors are non-peptidic or include large proteins such as cytokines. Peptides of less than 30 amino acids, such as MTRVLQGVLPALPQVVC, are disclosed that are useful for the protection and treatment of radiation damage. This is the first discovery that administration of peptide drugs after they have been exposed to radiation can reduce the damaging effects of radiation. For example, the radioresistant peptide consists of at most 29, at most 28, at most 27, at most 26, at most 25, at most 24, at most 23, at most 22, at most 21, at most 20, at most 19, at most 18, at most 17, at most 16, or at most 15 amino acids.

However, the peptide is preferably less than 15 amino acids. For example, the radioresistant peptide preferably consists of at most 14, at most 13, at most 12, at most 11, at most 10, at most 9 or at most 8 amino acids. Examples of some useful peptides are LPGCPRGVNPVVS, DINGFLPAL and QPLAPLVG. However, if the peptide is for self-treatment, for example self-treatment using an auto injector as described herein, it is preferred that the peptide is less than 7 amino acids for safety reasons. Such peptides do not normally bind to MHC receptors, thereby reducing the risk of developing autoimmunity triggered by an immune response against the administered peptide.

Another reason that less than 7 amino acids (aa) are particularly preferred is that peptides of size 7 amino acids (when comparing peptides from the proteasome of humans with peptides from the proteasome of pathogens, particularly viruses or bacteria (Burroughs et al, Immunogenetics, 2004, 56: 311-320)) were found to have only a 3% overlap between themselves and not themselves. For the 6 amino acid peptide, the overlap between human self and pathogen non-self was determined to be 30%, for the 5 amino acid peptide, the overlap between the peptides present in the human proteasome and the pathogen proteasome was determined to be 90%, and for the 4 amino acid and smaller peptides, the overlap was determined to be 100%. Based on these data, it is now recognized that the risk of adverse immune reactions such as anaphylactic shock is greatly reduced when there is no self-to-non-self difference, which is beneficial for medically untrained people to administer any drug to themselves or others.

Thus, in terms of preventing adverse reactions such as anaphylactic shock, preferably the peptide consists of 2 to 6 amino acids, more preferably 3 to 5 amino acids, and most preferably 3 or 4 amino acids. In terms of activity, from a general point of view, the greater the activity of the peptide, the more pronounced it is if it can undergo a complete proteolysis for a longer period, whereby a 3 amino acid metabolic fragment remains active, preferably the peptide consists of 4 amino acids. The compositions described above and below are preferably used for the treatment of acute radiation injury.

The use of peptides to protect against radiation damage has been mentioned in the prior art. Japanese patent applications JP09157291 and JP09157292 disclose specific 6-mer and 9-mer peptide sequences which have in vitro active oxygen inhibition, active oxygen radical scavenging and antioxidant activity. These peptides are believed to be useful in inhibiting adverse reactions in vivo to various events associated with reactive oxygen species formation, including radiation damage. But it was not subjected to in vivo irradiation experiments.

JP09176187 teaches histidine-containing 6-mer peptide analogues having active oxygen scavenging activity. Intraperitoneal administration of 660mg/kg body weight of peptide 20 minutes before irradiation increased the survival rate of mice from 10% in the control group to 70% in the treatment group. But it was not subjected to post-irradiation experiments in vivo.

WO2006/032269 discloses a blood cell homogenate from which components having a molecular weight of more than 3kDa have been removed. The homogenate is reported to be suitable for use in improving a cellular immune response in a subject. In a number of different immunological diseases and pathological situations, it is believed that the homogenate may be prophylactically administered to a patient in treatment with chemotherapy and/or radiotherapy to improve the patient's general condition. But the study did not involve any irradiation experiments. Furthermore, although the homogenate may comprise a mixture of proteins, the nature of the active ingredient is not at all clear, and the active ingredient may also be non-proteinaceous. In any event, this document does not isolate or identify any specific peptide.

EP0572688 discloses specific peptides comprising 14 amino acid residues which are capable of protecting mice against whole body irradiation at 20mg/kg body weight. Protection of the peptide was only observed when applied 1 hour prior to irradiation. No difference from the control group data was observed when the peptide was administered 1 hour after exposure to irradiation.

These prior art disclosures are in significant contrast to the present invention; the radioresistant peptides of the invention have a protective effect even when administered several hours after systemic irradiation.

Subjects receiving sub-lethal doses of radiation have benefited from the anti-inflammatory properties of some of the small peptides identified in the present invention, but unexpectedly, most of the benefit is derived from the anti-gastrointestinal syndrome activity of these small peptides, particularly from the 3-mer and 4-mer peptides at doses above 1mg/kg body weight, preferably above 5mg/kg body weight, more preferably above 10mg/kg body weight. Small peptide doses may be as high as 100mg/kg, given the low immunogenicity characteristics of small peptides (i.e. peptides of 3 to 4 amino acids), and in some cases as high as 200ng/kg, 500mg/kg, or even 1g/kg, given the emergency treatment needs to be given to the condition of the subject in need of treatment. Thus, it is now possible to treat subjects with radiation damage including lining lesions, so-called gastrointestinal syndromes; the peptide enables the epithelial lining to recover slowly.

In order to allow for better activity of the peptide at high radiation doses, the peptide is preferably selected to be placed in a pharmaceutical composition of the invention or an autoinjector of the invention, said peptide having a dose reduction coefficient (DRF) against acute gamma irradiation of at least 1.10, said DRF being determinable by: testing what dose of radiation resulted in 50% mortality (LD50/30) of test group mice treated with the peptide 30 days after total body irradiation (WBI), and testing what dose of radiation resulted in 50% mortality (LD50/30) of control group mice treated with the vehicle of the peptide only 72 hours after WBI, 30 days after total body irradiation (WBI), and wherein the DRF was calculated by dividing the LD50/30 of the peptide treated group animals by the LD50/30 of the vehicle treated group animals.

More preferably, the peptide used has a Dose Reduction Factor (DRF) of at least 1.20, more preferably of at least 1.25, especially if the radiation damage is irradiation damage. The peptides identified in the present invention are also referred to as radioresistant peptides. The present invention provides methods and pharmaceutical compositions for treating radiation damage from radioactive materials (radioisotopes) such as uranium, radon, and plutonium or from man-made radiation sources such as X-ray and radiotherapy instruments.

The invention also provides the use of a peptide of less than 30 amino acids in the manufacture of a pharmaceutical composition for treating a subject suffering from or believed to be suffering from radiation damage. As noted above, the peptide is preferably less than 15 amino acids and is used for self-treatment or administration by a non-professional, more preferably, the peptide is less than 7 amino acids. Some of the 3-mer peptides identified herein that can be used to prepare pharmaceutical compositions for the treatment of radiation injury are VVC, LAG, and AQG.

Similarly, some of the 4-mer peptides useful for treating radiation damage are LQGV, QVVC, MTRV, AQGV, LAGV, LQAV, PGCP, VGQL, RVLQ, EMFQ, AVAL, fvs, NMWD, LCFL, FSYA, FWVD, AFTV, LGTL, QLLG, YAIT, APSL, ITTL, QALG, GVLC, NLIN, SPIE, LNTI, LHNL, cpvqq, EVVR, MTEV, eal, EPPE, LGTL, VGGI, RLPG, LQGA, and LCFL, the 5-mer peptides useful for treating radiation damage are tlvave, vegll, and LNEAL, the 6-mer useful for treating radiation damage are vlpalparlp, MGGTWA, ddltcp, the 7-mer peptides useful for treating radiation damage are vlplqq, nyrdv, vcvgp, the 8-mer useful for treating radiation damage are vnvgp, and the 9-mer useful for treating radiation damage is flvg.

Other peptides, particularly 3-or 4-mer peptides, can be found by testing the peptides for anti-cell cycle activity in a proliferation assay, for example, by using the plant growth assay described herein. There is provided inter alia the use of a peptide consisting of 2 to 6 amino acids for the preparation of a pharmaceutical composition for the treatment of a subject suffering from or believed to be suffering from radiation damage. Also, the peptide used for preparing the pharmaceutical composition preferably consists of 2 to 6 amino acids, more preferably 3 to 5 amino acids, and most preferably 3 or 4 amino acids, in terms of preventing adverse reactions such as anaphylactic shock. If only in terms of activity, from a general point of view the greater the activity of the peptide is more pronounced as long as it is able (after administration) to undergo a complete proteolysis longer, whereby a 3 amino acid metabolic fragment remains active, preferably the peptide consists of 4 amino acids.

Furthermore, it is particularly useful that those subjects requiring treatment for radiation damage can now be treated by simple subcutaneous or intramuscular injection, thereby enabling self-treatment with self-use syringes, or by untrained or non-medical personnel, thereby making it extremely convenient to work with rescue organization in the event of an emergency where thousands of people require treatment. If only intravenous or equally dangerous intraperitoneal injections are effective, the subject to be treated will be more difficult to rescue than if there were a simple administration tool such as the self-contained injector provided by the present invention.

In particular, the invention also provides the use of a peptide of less than 30 amino acids for the preparation of a pharmaceutical composition for the treatment of radiation injury, wherein the pharmaceutical composition is placed in an autoinjector. An auto-injector is a medical device used to administer a single dose of a particular drug (typically a rescue medication), sometimes referred to as a pre-filled injector, for self-injection or injection by non-medical or non-professional persons. In the present application, the term "self-contained syringe" does not refer to a syringe used for automated application of biological samples (e.g., peptides) in analytical systems such as the chromatographic devices described in Husek et al (J.of Chromatography B: biological Sciences & Applications, Elsevier, Amsterda, Vol.767, No.1, (2002) pg.169-174).

By design, the self-use syringe is easy to use and can be administered to itself by the patient or to the patient by a non-professional. The injection site is typically in the thigh or in the buttocks, wherein the treatment comprises subcutaneous or intramuscular injection of the peptide. Since autoinjectors can be designed to automatically and reliably administer a desired dose of medication, they facilitate rapid, easy, and accurate administration of medication. In particular, self-contained syringes are well suited for use by those subjects who must administer therapeutic substances to themselves or by those medical personnel who must administer injections to multiple subjects in a relatively short period of time (as in the case of emergency medical services). Further, an auto-injector having a needle injection mechanism (needle injection mechanism) may be designed so that the needle is not visible before, during, or even after the injection operation, thereby reducing or eliminating any anxiety associated with the visible needle penetrating the subject tissue. Although the specific gauge of needled self-using syringes vary widely, they typically include a body or housing, a needled syringe or similar device, and one or more drive mechanisms for penetrating a needle into the tissue of a subject and delivering a desired dose of liquid drug through the penetrated needle. The drive mechanism in existing needle-stick autoinjectors typically includes an energy source capable of powering the drive mechanism. Such energy sources may be, for example, mechanical (i.e., spring-loaded), pneumatic, electromechanical, or chemical, see U.S. patents 6,149,626, 6,099,504, 5,957,897, 5,695,472, 5,665,071, 5,567160, 5,527,287, 5,354,286, 5,300,030, 5,102,393, 5,092,843, 4,894,054, 4,678,461, and 3,797,489, the contents of which are incorporated by reference herein. International publications WO01/17593, WO98/00188, WO95/29720, WO95/31235, and WO94/13342 also disclose syringes comprising different drive mechanisms. Most autoinjectors are (optionally spring-loaded) syringes.

The self-use syringe of the invention, particularly the body or housing in direct contact with the peptide, is preferably made of a material having minimal affinity for the peptide. This minimizes peptide adhesion or adherence to the autoinjector. One very suitable material is polypropylene, in particular substantially pure polypropylene.

Autoinjectors were originally designed to overcome the hesitation of needles to self-administer medication. An example of such an auto-injector is EpipenOr recently introduced TwinjectThe latter is often used for people at risk of allergic reactions. Another example of a self-use syringe is Rebiject for administration of interferon beta to treat multiple sclerosis. Self-use syringes are often used by the army to protect personnel against chemical warfare agents. In the army, there is a self-contained syringe in each biological or chemical weapons reaction package. The reaction package is distributed to each soldier who may be facing biological or chemical weapons. Once activated, the needle automatically injects the person, penetrating any clothing (even multiple layers of clothing) on the person. The self-using injector of the present invention includes not only the above-described injection device (typically spring-driven) to automatically perform skin penetration and/or drug injection, but also pre-filled injectors or self-using injection cartridges and the like.

The present invention provides such self-contained syringes that can be used to treat radiation damage, whether the radiation is emitted by radioactive substances (radioisotopes) such as uranium, radon, and plutonium, or generated by artificial radiation sources such as X-ray and radiotherapy instruments. The invention also provides an autoinjector of a protected pharmaceutical composition consisting of a peptide of less than 30 amino acids (herein referred to as radioresistant peptide) and a suitable excipient. Excipients are known in the art, see for example, Handbook of pharmaceutical Manufacturing Formulations (edited by Sarfaraz K Niazi; ISBN: 0849317460, which is incorporated herein by reference).

Excipients are composed of, for example, water, propylene glycol, ethanol, sodium benzoate and benzoic acid as buffers, and benzyl alcohol as preservatives; or mannitol, human serum albumin, sodium acetate, acetic acid, sodium hydroxide, and water for injection. Other exemplary compositions for parenteral administration by autoinjector include injectable solutions or suspensions containing, for example, a suitable non-toxic, parenterally acceptable diluent or solvent, for example, mannitol, 1, 3-butanediol, water, ringer's solution, isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono-or diglycerides, and fatty acids, including oleic acid.

In one embodiment, the autoinjector comprises as an active ingredient a radiation resistant peptide (or functional analog thereof) capable of reducing adverse effects of radiation when administered after exposure of the subject to radiation. Preferably, the peptide is capable of producing at least partial protection against radiation damage if administered at least 30 minutes, more preferably at least 1 hour, most preferably at least several hours or even days (e.g. 3 days) after irradiation. Such auto-injectors, also known as "emergency auto-injectors", represent their use in emergency situations.

In one embodiment, the present invention provides an auto-injector containing a sterile solution packaged in a syringe-like device that is capable of automatically delivering its entire 5mL contents upon actuation. Each mL contains 100mg, preferably 200mg, of the radioresistant peptide and excipients, for example excipients comprising propylene glycol, ethanol, sodium benzoate and benzoic acid as buffers, and benzyl alcohol as preservatives. In a preferred embodiment, the autoinjector for treatment of radiation injury carries a radiation resistant peptide of less than 15 amino acids, more preferably less than 7 amino acids.

Preferred autoinjectors for treating acute radiation injury carry peptides of 3 to 4 amino acids in length, preferably peptides having a Dose Reduction Factor (DRF) against acute gamma irradiation of at least 1.10, which can be determined as follows: testing what dose of radiation resulted in 50% mortality (LD50/30) of test group mice treated with the peptide 30 days after total body irradiation (WBI), and testing what dose of radiation resulted in 50% mortality (LD50/30) of control group mice treated with the peptide only vehicle 72 hours after WBI, 30 days after total body irradiation (WBI), and wherein DRF was calculated by dividing LD50/30 of the peptide treated group animals by LD50/30 of the control group animals.

More preferred autoinjectors carry peptides with a Dose Reduction Factor (DRF) of at least 1.20, more preferably at least 1.25. Also suitable peptides against placement in an autoinjector are those peptides which have anti-cell cycle activity in plants as defined by the invention. Peptides that are well suited for use in the self-injector of the invention are VVC, LAG, AQG, LQGV, QVVC, MTRV, AQGV, LAGV, LQAV, PGCP, VGQL, RVLQ, EMFQ, AVAL, FVLS, NMWD, LCFL, FSYA, FWVD, AFTV, LGTL, QLLG, YAIT, APSL, ITTL, QLGG, GVLC, NLIN, SPIE, LNTI, LHNL, CPVQ, EVVR, MTTV, EALE, EPPE, LGTL, VGGI, RLPG, LQGA, LCFL, TLAVE, VEGNL or LNEAL.

The present invention also provides a pharmaceutical composition for treating a subject suffering from or believed to be suffering from radiation damage, the pharmaceutical composition comprising: a pharmaceutically effective amount of a radioresistant peptide or functional analogue thereof, or a pharmaceutical composition as identified herein, and a pharmaceutically acceptable diluent. The invention herein provides a method of treating or preventing radiation damage in a subject in need or potential need thereof, the method comprising: administering to a subject a pharmaceutical composition comprising a means for treating or preventing radiation damage and a pharmaceutically acceptable excipient, wherein said means comprises a radiation resistant peptide or pharmaceutical composition identified herein, particularly wherein said radiation damage comprises radiation damage.

In one embodiment, the present invention provides a method for treating a subject having radiation damage comprising administering to the subject a composition comprising an oligopeptide obtained or derived from peptide MTRVLQGVLPALPQVVC or peptide LPGCPRGVNPVVS. Preferably, the oligopeptide is selected from the group consisting of MTR, MTRV, LQG, LQGV, VLPALP, VLPALPQ, QVVC, VVC, AQG, AQGV, LAG, LAGV, and any combination thereof. In another embodiment, preferably, the oligopeptide is selected from LPGC, CPRGVNP and PGCP. Such oligopeptides are particularly useful when the radiation damage comprises radiation damage. The present invention also provides a pharmaceutical composition for the treatment of radiation damage comprising an oligopeptide obtained or derived from peptide MTRVLQGVLPALPQVVC or peptide LPGCPRGVNPVVS, e.g. an oligopeptide selected from the group consisting of MTR, MTRV, LQG, LQGV, VLPALP, VLPALPQ, QVVC, VVC, AQG, AQGV, LAG, LAGV, LPGC, CPRGVNP and PGCP, and any combination thereof, and the use of said (oligo) peptide for the preparation of a pharmaceutical composition for the treatment of radiation damage.

We previously reported that a 6-mer oligopeptide derived from the beta-chain of human chorionic gonadotropin (VLPALP) inhibits septic shock in mice. Similarly, we have found that other short peptides derived from the beta-chain loop 2 of hCG (residues 41-57) (starting from the trimeric peptide) and modifications of the peptide by single amino acid substitution with alanine have similar anti-inflammatory activity. Furthermore, we have generated a reasoning that screens out some of these peptides to continue the development of therapeutic compounds for treating acute inflammation following accidental exposure to ionizing radiation.

Human chorionic gonadotropin (hCG) is a heterodimeric placental glycoprotein hormone produced by pregnant women. It exists in many forms, including cleavage products, in the urine of pregnant women and in commercial hCG preparations. Since it is postulated to have an effect on the prevention of fetal allograft rejection during pregnancy, several researchers have investigated the effect of heterodimeric hCG and variants thereof on the immune system. Some reports suggest that intact hormones anti-modulate the immune system, but such effects of cleavage products have not been reported. We previously (Khan et al, hum. Immunol.2002Jan; 63 (1): 1-7) reported that a 6-mer oligopeptide derived from the beta-chain of human chorionic gonadotropin (VLPALP) inhibits septic shock in mice. A single treatment with this hexapeptide after high dose Lipopolysaccharide (LPS) injection in mice inhibited septic shock in mice. Benner and Khan (Scand. J. Immunol. 2005Jul; 62Suppl 1: 62-6) investigated the immunological activity which a peptide fragment released in vivo might have, which was generated by cleavage of the sequence MTRVLQGVLPALPQVVC (residues 41-57) of hCG. beta. -subunit ring 2. It is reported herein that some peptides of 3 to 7 amino acids taken from loop 2 of the β -subunit, and some peptides derived therefrom by alanine substitution, exhibit significant anti-inflammatory activity (as determined by inhibition of septic shock syndrome in mice), and exceed the activity believed to be useful for the treatment of radiation injury, particularly radiation injury including gastrointestinal syndromes, and also exceed the activity believed to be useful for the preparation of pharmaceutical compositions for the treatment of radiation injury, particularly radiation injury including gastrointestinal syndromes.

The invention also provides pharmaceutical compositions having anti-cell cycle activity. The cell cycle is an ordered set of events, the result of which is that the cell grows and divides into two sister cells. The stage of the cell cycle is G1-S-G2-M. Stage G1 represents "GAP 1". S phase represents "Synthesis". This is the stage where DNA replication occurs. Stage G2 represents "GAP 2". The M phase represents "mitosis", which is when nuclear (chromatin separating) and cytoplasmic (cytokinesis) divisions occur. The term "anti-cell cycle activity" as used herein means that the peptide is capable of altering cell cycle kinetics. For example, it includes altering, i.e. increasing or decreasing, the frequency of cell division. In one embodiment, it refers to antiproliferative activity.

Also provided are pharmaceutical compositions having anti-cell cycle activity comprising PGCP; a pharmaceutical composition having anti-cell cycle activity comprising VGQL; a pharmaceutical composition having anti-cell cycle activity comprising RVLQ; a pharmaceutical composition having anti-cell cycle activity comprising EMFQ; a pharmaceutical composition having anti-cell cycle activity comprising AVAL; a pharmaceutical composition having anti-cell cycle activity comprising fvss; a pharmaceutical composition having anti-cell cycle activity comprising NMWD; a pharmaceutical composition having anti-cell cycle activity comprising LCFL; a pharmaceutical composition having anti-cell cycle activity comprising FSYA; a pharmaceutical composition having anti-cell cycle activity comprising FWVD; a pharmaceutical composition having anti-cell cycle activity comprising AFTV; a pharmaceutical composition having anti-cell cycle activity comprising LGTL; a pharmaceutical composition having anti-cell cycle activity comprising QLLG; a pharmaceutical composition having anti-cell cycle activity comprising YAIT; a pharmaceutical composition having anti-cell cycle activity comprising APSL; a pharmaceutical composition having anti-cell cycle activity comprising ITTL; a pharmaceutical composition having anti-cell cycle activity comprising QALG; a pharmaceutical composition having anti-cell cycle activity comprising GVLC; a pharmaceutical composition having anti-cell cycle activity comprising NLIN; a pharmaceutical composition having anti-cell cycle activity comprising SPIE; a pharmaceutical composition having anti-cell cycle activity comprising LNTI; a pharmaceutical composition having anti-cell cycle activity comprising LHNL; a pharmaceutical composition having anti-cell cycle activity comprising CPVQs; a pharmaceutical composition having anti-cell cycle activity comprising an EVVR; a pharmaceutical composition having anti-cell cycle activity comprising MTEV; a pharmaceutical composition having anti-cell cycle activity comprising an eal; a pharmaceutical composition having anti-cell cycle activity comprising EPPE; a pharmaceutical composition having anti-cell cycle activity comprising LGTL; a pharmaceutical composition having anti-cell cycle activity comprising VGGI; a pharmaceutical composition having anti-cell cycle activity comprising RLPG; a pharmaceutical composition having anti-cell cycle activity comprising LQGA; a pharmaceutical composition having anti-cell cycle activity comprising LCFL; a pharmaceutical composition having anti-cell cycle activity comprising TLAVE; a pharmaceutical composition having anti-cell cycle activity comprising VEGNL; a pharmaceutical composition having anti-cell cycle activity comprising LNEAL; a pharmaceutical composition having anti-cell cycle activity comprising MGGTWA; a pharmaceutical composition having anti-cell cycle activity comprising ltcc ddp; a pharmaceutical composition having anti-cell cycle activity comprising VCNYRDV; a pharmaceutical composition having anti-cell cycle activity comprising CPRGVNP; and a pharmaceutical composition having anti-cell cycle activity comprising DINGFLPAL.

Drawings

FIG. 1: total body irradiated mice treated with AQGV (peptide EA-230)

"WBI" stands for whole body irradiation. The in vivo protection against radiation injury was assessed after WBI (6.5 to 9.8Gy, Philips MG30, 81cGy/min) using anesthetized C57B1/6 mice, and the difference in survival was measured by Kaplan-Meirer analysis. Mice of all groups were injected first with peptide or vehicle 3 hours after WBI (control group animals). The group mortality rate with placebo injection was 80%, consistent with the prediction for this model. The radiation dose of 8.6 gray (═ 8.6Gy) is known to cause approximately 80% mortality in this species, and is therefore known as LD80 (80% lethal dose). Death begins around day 10-this is the usual case in WBI in animals or humans: on about day 10, damage and leakage of the lining of the intestine by radiation causes bacteria to enter the circulation and cause gastrointestinal syndromes, while damage to the bone marrow causes the failure to produce enough white blood cells to fight infection ("bone marrow syndrome"), with consequent death. The group represented by the symbol "x" received a first intravenous injection followed by a second subcutaneous injection (SC) 3 hours after the first injection. These animals survived 100%. They are not shown in the figure as they did not show any signs of disease at all. To an unknowing observer, they looked the same as fully normal mice. The group represented by the triangle symbols was injected with the peptide for the first time by the SC route, followed by additional SC injections every 48 hours for a total of 3 doses (except the first dose) -i.e., on days 3, 5, and 7. Only one of these animals died. The group represented by the square symbols is identical to the procedure for the triangle symbol group except that the 48 hour SC injection is continued for a total of 6 doses (except for the first dose). Thus its administration was continued up to day 13. This prolonged treatment resulted in complete protection (none of the groups died). None of the animals in this group showed any signs of disease. From these data we conclude that if animals receive twice the dose of peptide on the first day (the first dose is intravenous), complete protection is provided against a lethal dose of WBI. Extending treatment to the second week also resulted in complete protection if the animals received lower levels of treatment (SC only).

FIG. 2: second set of radioprotection experiments with peptide AQGV

With increasing doses of Whole Body Irradiation (WBI), there was a single exposure for each group, with subsequent groups being exposed to increasing doses. A single dose of the peptide EA-230(AQGV) was administered subcutaneously, but treatment was delayed until 3 days (72hr) after WBI. This test is called the dose reduction factor ("DRF") and is defined as the ratio between the LD50 of the treated group and the LD50 of the control group. LD50 represents the dose that caused 50% of the test animals to die. An acceptable DRF value is 1.20. To pass this test, a candidate drug must have an LD50 radiation dose at least 20% higher (a factor of 1.20 increase) than the LD50 dose in control animals on day 30 post WBI. For example, if the LD50 of a control animal is 8.2Gy, then the LD50 caused by the drug candidate should be at least 20% higher, i.e., in this case the dose should be 8.2x 1.20-10.4 Gy.

FIG. 3: oligopeptides play a role in Arabidopsis (Arabidopsis thaliana) cell cycle analysis. Compounds NAK4(LQGV) and NAK9(VVC) showed clear effects on the labeling of the mapping test. For the cell cycle marker (pCDG), a clear effect on the roots was observed at both time points. Time and/or dose dependent effects were observed in the transition zone and cotyledons. The same was observed for the plant growth hormone response marker (DR5:: GUS) as for the cell cycle marker. NAK26(DINGFLPAL) showed less consistent time dependent effects. The effect was only observed at the root in time. No effect was observed in the transition zone and cotyledons.

FIG. 4: representative oligopeptides were tested for their effect on cell proliferation during rapid growth of murine monocytes induced by CD3 when cells undergo rapid division. Mice (n ═ 5) were injected intraperitoneally with PBS, Nak4(LQGV), Nak47(LAGV), Nak46(AQGV) (Ansynth BV, supplied by The Netherlands), or Nak46(AQGV supplied by Diosynth BV, The Netherlands). Mice were treated with 0.5mg/kg or 5mg/kg of the peptide for 1 hour, followed by separation of spleen to prepare spleen cell suspensions. Spleen cell suspensions from each group were pooled and cultured in vitro (triplicate) in the presence of PBS or anti-CD 3 antibody, and proliferation was measured at 0, 12, 24, and 48 hours post culture.

Detailed Description

In the present application, a "purified, synthetic or isolated" peptide is a peptide that has been purified from its natural or biotechnological source or, more preferably, a synthetic peptide as described herein.

In the present application, "composition" refers to a variety of compounds containing or consisting of oligopeptides. Preferably, the oligopeptide is isolated and then added to the composition. Preferably, the oligopeptide consists of 2 to 6 amino acids, more preferably 3 to 4 amino acids.

For example, in one embodiment, a preferred compound may be: NT A Q G V CT, wherein NT at the N-terminal is selected from H-, CH3-, acyl, or general protecting group; and the C-terminal CT is selected from the group consisting of small peptides (e.g., 1 to 5 amino acids), - -OH, - -OR¹、--NH₂、--NHR¹、--NR¹R²or-N (CH)₂)_1-6NR¹R²Wherein R is¹And R²Independently selected from H, alkyl, aryl, (ar) alkyl, and wherein R is¹And R²May be connected to each other to form a ring.

In the present application, "alkyl" is preferably a saturated branched or straight chain hydrocarbon having 1 to 6 carbon atoms, such as methyl, ethyl and isoamyl.

In the present application, "aryl" is an aromatic hydrocarbon group, preferably having 6 to 10 carbon atoms, such as phenyl or naphthyl.

In this application, "(ar) alkyl" is an aromatic hydrocarbon group (having both aliphatic and aromatic moieties), preferably having from 7 to 13 carbon atoms, such as benzyl, ethylbenzyl, n-propylbenzyl, and isobutylbenzyl.

In the present application, an "oligopeptide" is a peptide of 2 to 12 amino acids linked together by peptide bonds. An equivalent of an oligopeptide is a compound having side chains identical or equivalent to the particular amino acids in the oligopeptide and arranged in the same order as the peptides, but linked together by non-peptide bonds, for example by isosteric linkages (isospecific linkages) such as ketone isosteres, hydroxyl isosteres, diketone isosteres or ketone-difluoromethyl isosteres.

"composition" also includes, for example, an acceptable salt of an oligopeptide or a labeled oligopeptide. In the present application, "acceptable salt" refers to a salt that retains the desired activity of the oligopeptide or equivalent compound, preferably without adversely affecting the oligopeptide or other components of the system in which the oligopeptide is used. Examples of such salts are acid addition salts formed from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like. Salts may also be formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, and the like. The salts may be formed with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, and the like, or with organic cations formed from N, N' -dibenzylethylenediamine or ethylenediamine, or combinations thereof such as zinc tannate.

Such pharmaceutical compositions may be administered to a subject parenterally or orally. Such a pharmaceutical composition may consist essentially of the oligopeptide and PBS. Preferably, the oligopeptide is synthetic. Suitable treatments, for example, entail administering the oligopeptide in a pharmaceutical composition intravenously to a patient in an amount of about 0.1 to about 35mg/kg body weight. The pharmaceutical composition may consist essentially of one to three different oligopeptides.

The chemical entities so produced may be administered and introduced into the body systemically, topically or locally. The peptide or a modification thereof may be administered as the entity itself or as a pharmaceutically acceptable acid or base addition salt formed by reaction with an inorganic acid such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid and phosphoric acid; or by reaction with organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid; or by reaction with an inorganic base (e.g., sodium hydroxide, ammonium hydroxide, potassium hydroxide); or by reaction with organic bases such as monoamines, diamines, triamines, and aromatic amines and substituted ethanolamines. The selected peptides and any derived entities may also be conjugated to sugars, lipids, other polypeptides, nucleic acids and PNAs; and act as a conjugate in situ or by local release after reaching the target tissue or organ.

With respect to various amino acids, "substitution" generally involves the replacement of a hydrogen present on an aromatic ring with a group such as alkoxy, halogen, hydroxy, nitrogen, or lower alkyl. Substitution may also be made on the alkyl chain connecting the aromatic moiety to the peptide backbone, for example by substituting hydrogen with lower alkyl. Other substitutions may also be made alpha to the amino acid, and alkyl groups may also be used.

Preferred substitutions use fluorine or chlorine as halogen and methoxy as alkoxy. As for the alkyl group and the lower alkyl group, generally, an alkyl group having a small number (1 to 3) of carbon atoms is preferable.

The compounds corresponding to the general formula (I) can be prepared by conventional methods for preparing these compounds. To this end, a suitable N-terminal alpha protected (and, if a reactive side chain is present, side chain protected) amino acid analogue or peptide is activated and coupled to a suitable carboxy protected amino acid or peptide derivative in solution or on a solid support. Protection of the alpha-amino function is typically performed with an urethane function, such as with acid-resistant t-butyloxycarbonyl ("Boc"), benzyloxycarbonyl ("Z") and substituted analogues, or alkali-resistant 9-fluorenylmethyloxycarbonyl ("Fmoc"). The Z group can also be removed by catalytic hydrogenation. Suitable additional protecting groups include Nps, Bmv, Bpoc, Aloc, MSC, and the like. For a review of amino protecting groups see The peptides, Analysis, Synthesis, Biology, Vol.3, E.Gross and J.Meiennhofer, eds. (Academic Press, New York, 1981). The carboxyl protection can be carried out by esterification, for example with alkali-resistant esters such as methyl or ethyl esters, with acid-resistant esters such as tert-butyl esters or substituted, benzyl esters or by hydrogenolysis. The protection of the side chain of side chain functional groups such as lysine and glutamic acid or aspartic acid can be carried out using the aforementioned groups. Protection of sulfhydryl (although not always required), guanidino, alcohol and imidazole groups can be achieved using a variety of reagents, see, for example, The Peptides, Analysis, Synthesis, Biology, id. or Pure and Applied Chemistry, 59(3), 331-344 (1987). Activation of the carboxyl group of a suitable protected amino acid or peptide may be carried out using the azide, mixed anhydride, active ester, or carbodiimide method, particularly with the addition of a catalytic racemization-inhibiting compound such as 1-N-N-hydroxybenzotriazole, N-hydroxysuccinimide, 3-hydroxy-4-oxo-3, 4-dihydro-1, 2, 3-benzotriazine, N-hydroxy-5-norbornene-2, 3-dicarboxylic acid imine. Anhydrides of phosphorus-containing acids may also be used. See, for example, The Peptides, Analysis, Synthesis, Biology, supra and Applied Chemistry, 59(3), 331-344 (1987).

The compounds can also be prepared by the solid phase method of Merrifield. Different solid supports and different strategies are known, see for example Barany and Merrifield, The Peptides, Analysis, Synthesis, Biology, vol.2, e.g. gross and j.meienhofer, eds. (acad.press, new york, 1980); Kneib-Cordonier and Mullen, int.J. peptide Protein Res., 30, 705-739 (1987); and Fields and Noble, int.j.peptide Protein res, 35, 161-214 (1990). Those compounds in which the peptide bond is isosterically substituted are generally synthesized using the protecting groups and activation methods described previously. Methods for synthesizing modified isosteres are described in the literature, e.g., in connection with- -CH₂- -NH- -isostere and- -CO- -CH₂The literature on isosteres.

Depending on the nature of the protecting group and the type of linker attached to the solid support used in the solid phase peptide synthesis process, different methods of removing the protecting group and cleaving from the solid support may be used. Deprotection is generally carried out under acidic conditions and in the presence of a scavenger. See, for example, volumes3, 5and9of The series on The Peptides Analysis, Synthesis, Biology, supra.

Another possible method is the synthesis of such compounds using enzymes. See, for example, review h.d. jakubke in The Peptides, Analysis, Synthesis, Biology, vol.9, s.udenfriend and j.meienhofer, eds. (acad.press, New York, 1987).

Although it may be less suitable from an environmental point of view, the oligopeptide of the present invention may also be prepared using recombinant DNA methods. Such methods involve the preparation of the desired oligopeptide by expressing in a suitable host microorganism a recombinant polynucleotide whose sequence encodes one or more oligopeptides of interest. The methods generally involve introducing a DNA sequence encoding one or more specific oligopeptides into a cloning vector (e.g., a plasmid, phage DNA, or other DNA sequence capable of replication in a host cell), introducing the cloning vector into a suitable eukaryotic or prokaryotic host cell, and culturing the host cell so transformed. If a eukaryotic host cell is used, the compound will contain a glycoprotein moiety.

In the present application, a "functional analog" of a peptide includes an amino acid sequence or other sequence monomer, the sequence of which has been altered such that the functional properties of the sequence are substantially identical in nature, but not necessarily identical in amount.

The function of a peptide or functional analog thereof can be determined using in vivo and/or in vitro assays. In vitro testing is preferred. In one embodiment, a comparative test is performed on functional peptide analogs in which a reference or control peptide, such as a peptide analog consisting of only L amino acids, is employed. Suitable assays include determining the ability of a candidate peptide to affect cell cycle dynamics. For example, the effect on cell cycle progression can be determined using plant model systems, such as the Arabidopsis system exemplified herein, or using cultured (mammalian) cells. In another aspect it relates to determining the ability of a candidate peptide to inhibit apoptosis, for example by inducing (transient) G2-M cell cycle arrest.

Analogs can be generated in a variety of ways, such as by "conservative amino acid substitutions. Furthermore, peptidomimetic compounds can be designed which are able to be functionally or structurally similar to the original peptide as starting point but which consist, for example, of unnatural amino acids or polyamides. By "conservative amino acid substitution", one amino acid residue is substituted with another residue of generally similar nature (size, hydrophobicity), whereby the overall function is not substantially severely affected. However, it is generally more desirable to be able to improve specific functions. Analogs can also be produced by comprehensively enhancing at least one desired property of the amino acid sequence. This can be achieved, for example, by Ala scanning (Ala-scan) and/or replacement net mapping (replacement net mapping) methods. Using these methods, many different peptides can be generated based on the original amino acid sequence, but each contains a substitution of at least one amino acid residue. The amino acid residues may be substituted by alanine (Ala-scan) or by any other amino acid residue (substitution net diagram). This synthesized many positional variants (positional variants) of the original amino acid sequence. Specific activity of each positional variant was screened. The data generated are used to design improved peptide derivatives of specific amino acid sequences.

Analogs can also be generated, for example, by replacing L-amino acid residues with D-amino acid residues. Such substitutions result in the production of non-naturally occurring peptides that may improve the properties of the amino acid sequence. A peptide sequence of known activity, consisting entirely of D amino acids, may be provided, for example, in a retro-inverted (retroinversion) form, thereby enabling its activity to be maintained and half-life to be increased. By generating a number of positional variants of the original amino acid sequence and screening for specific activity, improved peptide derivatives comprising such D amino acids can be designed with further improved properties. It has been found in the prior art that peptides protected at one or both ends with D amino acids are more stable than peptides consisting of L amino acids only. Other types of modifications include those known in the art to develop peptide drugs to have beneficial effects that can be used in pharmaceutical compositions. These effects may include increased efficacy, altered pharmacokinetics, increased stability and resulting in increased half-life, and reduced stringency requirements for cold chain treatment.

In one embodiment of the invention, the radiation-resistant peptide comprises a sequence of amino acids linked together by a peptide bond between their amino and carboxyl groups, wherein at least one of the amino acids is a D amino acid. For example, the radiation-resistant peptide is selected from the group consisting of: VVC, LAG, AQG, LQGV, QVVC, MTRV, AQGV, LAGV, LQAV, PGCP, VGQL, RVLQ, EMFQ, AVAL, FVLS, NMWD, LCFL, FSYA, FWVD, AFTV, LGTL, QLLG, YAIT, APSL, ITTL, QALG, GVLC, NLIN, SPIE, LNTI, LHNL, CPVQQ, EVVR, MTEV, EALE, EPPE, LGTL, VGGI, RLPG, LQGA, LCFL, TLAVE, VEGNL, LNEAL, VLPALP, MGGTWA, LTCP, VLPAPLPLQ, VCNYRDV, CPRGVNP, QPLAPLVG, and DINGFLPAL, wherein at least one of the amino acid residues represented by a standard one-letter code is a D amino acid.

One skilled in the art can generate analog compounds of the amino acid sequences. This can be done, for example, by screening peptide libraries. Such analogs have essentially the same functional properties as the original sequence, but need not be identical in amount. Furthermore, the peptides or analogues may be cyclized, for example by providing them with a (terminal) cysteine; dimerized or multimerized, for example by attachment to lysine or cysteine or other compounds having side chains that allow attachment or multimerization to occur; forming a tandem or repeating configuration; coupled or otherwise attached to carriers known in the art, so long as by a reliable linkage that allows dissociation. Compositions of these oligopeptides, as well as functional analogs or cleavage products, as described above, may be used in methods of treating radiation injury and subsequent disease.

In the present application, a "functional analogue" of a peptide is preferably smaller than the peptide from which it was derived, and is therefore preferably prepared by deletion and/or substitution, rather than increasing its length. Furthermore, in the present application, a "functional analog" of a peptide does not refer to larger proteins or peptides that contain an amino acid sequence identified herein as a radiation-resistant peptide with more amino acids on one or both sides.

The term "pharmaceutical composition" is intended herein to encompass both the active composition of the invention itself and compositions comprising the composition of the invention together with a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may contain a mixture of at least two of the radiation-resistant peptides or analogs described herein. Acceptable diluents for the oligopeptides detailed herein are, for example, physiological saline solutions or phosphate buffered saline solutions. In one embodiment, the oligopeptide or composition is administered systemically, e.g., intravenously, intramuscularly, or intraperitoneally, to an animal or human in effective concentrations. Another route of administration includes organ or tissue perfusion, which may be performed in vivo or in vitro using a perfusate comprising an oligopeptide or composition of the present invention. Administration may be in a single dose, discrete doses, or for a period of time sufficient to allow for sufficient modulation of gene expression. For sustained administration, the duration of sustained administration will vary depending on a number of factors, as will be readily understood by those skilled in the art.

The dose of active molecule administered can be of considerable extent. The concentration of active molecules that can be administered is generally limited by efficacy at lower concentrations and solubility of the compound at higher concentrations. The optimal dosage for a particular patient should and can be determined by the relevant physician or medical professional, taking into account well-known relevant factors such as the patient's condition, weight and age, etc.

The active molecule can be administered directly in a suitable vehicle, for example phosphate buffered saline ("PBS") or a solution in alcohol or DMSO. However, according to a preferred embodiment of the present invention, the active molecule is administered by single dose delivery using a drug delivery system. Suitable drug delivery systems should be pharmacologically inactive or at least tolerable. It is preferably neither immunogenic nor inflammatory and should be such that the release of the active molecule is able to maintain its effective level for the required time. Alternatives suitable for controlled release purposes are known in the art and are within the scope of the invention. Suitable delivery vehicles include, but are not limited to: microcapsules or microspheres; liposomes and other lipid-based delivery systems; sticky droplets (viscous actives); absorbable and/or biodegradable mechanical barriers and implants; and polymeric delivery materials such as polyethylene oxide/polypropylene oxide block copolymers, polyesters, crosslinked polyvinyl alcohols, polyanhydrides, polymethacrylates and polymethacrylamide hydrogels, anionic carbohydrate polymers, and the like. Useful delivery systems are well known in the art.

One formulation for achieving release of the active molecule includes injectable microcapsules or microspheres made from biodegradable polymers such as poly (dl-lactide), poly (dl-lactide-co-glycolide), polycaprolactone, polyglycolide, polylactic acid-co-glycolide, poly (hydroxybutyric acid), polyester or acetal resins. Injectable systems comprising microcapsules or microspheres having a diameter of about 50 microns to about 500 microns have advantages over other delivery systems. For example, they generally use less active molecules and can be administered by paramedical personnel. Furthermore, by selecting the size, loading and administration dosage of the microcapsules or microspheres, such systems have inherent flexibility in the design of different times and rates of drug release. In addition, it can be sterilized by gamma irradiation.

The design, preparation and use of microcapsules and microspheres are well known to those skilled in the art, and specific technical details regarding this can be found in the literature. Biodegradable polymers (such as lactide, glycolide, and caprolactone polymers) can also be used to prepare formulations other than microcapsules and microspheres; such as pre-fabricated and sprayed films containing reactive molecules, may be used in the present invention. Filters or fibers comprising active molecules are also within the scope of the invention.

Another formulation highly suitable for the delivery of the active molecules of the invention in a single dose is a liposome. Encapsulation of active molecules in liposomes or multilamellar vesicles is a well-known technique for targeted drug delivery and prolonged drug retention. The preparation and use of liposomes containing drugs is known to those skilled in the art and is described in detail in the literature.

Another suitable way of delivering the active molecules of the invention in a single dose involves a viscous drop. In this technique, high molecular weight carriers are used in admixture with active molecules, the resulting structure yielding a solution with high viscosity. Suitable high molecular weight carriers include, but are not limited to: dextran and cyclodextrin; a hydrogel; (cross-linked) viscous materials, including (cross-linked) viscoelastic materials; carboxymethylcellulose; hyaluronic acid; and chondroitin sulfate. The preparation and use of viscous drops containing a drug is known to those skilled in the art.

According to another approach, the active molecule may be administered in combination with an absorbable mechanical barrier such as oxidized regenerated cellulose. The active molecule may be covalently or non-covalently bound (e.g., by ionic bonding) to such a barrier, or simply dispersed thereon.

The invention is further explained by the following exemplary embodiments.

Examples

Selection of peptides

Selection was made based on known preferential cleavage sites on the sequence MTRVLQGVLPALPQVVC (residues 41-57) of hCG beta-subunit Loop 2 (Cole et al, J.Clin.Endocr.Metab.1993; 76: 704. 710; H.Alfthan, U.H.Stenman, mol.cell.Endocrinol.1996; 125: 107. 120; A.Kardana, et al, Endocrinology 1991; 129: 1541. 1550; Cole et al, Endocrinology 1991; 129: 1559. times.1567; S.Birken, Y.Maydelman, M.A.Gawinowicz, Methods 2000; 21: 3-14) and on amino acid sequences from C-reactive proteins (CRP) (beta-catenin, such as human CTNB), Bruton-type tyrosine kinases (such as human K), matrix metalloproteinases-2 and BTp-53).

Synthesis of peptides

The peptides described herein were prepared commercially by solid phase synthesis (Ansynth BV) either by proprietary methods (Diosynth BV) or using a 9-fluorenylmethyloxycarbonyl (Fmoc)/tert-butyl based method with 2-chlorotrityl chloride resin (2-chlorotrityl chloride resin) as the solid support. The side chain of glutamine was protected with a trityl function. These peptides were synthesized manually. Each coupling comprises the following steps: (i) removal of the Fmoc protection of the α -amino group with piperidine in Dimethylformamide (DMF), (ii) coupling of the Fmoc amino acid (3eq) with Diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) in DMF/N-methylformamide (NMP), and (iii) capping of the remaining amino function with acetic anhydride/Diisopropylethylamine (DIEA) in DMF/NMP. After the synthesis is finished, trifluoroacetic acid (TFA)/H is used₂The peptide resin was treated with a mixture of O/Triisopropylsilane (TIS)95:2.5: 2.5. After 30 minutes, TIS was added until decolorization. The solution is dehydrated in vacuo toThe peptide was precipitated with diethyl ether. The crude peptide was dissolved in water (50-100mg/ml) and purified by reverse phase high performance liquid chromatography (RP-HPLC). HPLC conditions: column: vydac TP21810C18(10x250 mm); an elution system: gradient system 0.1% TFA in water v/v (A) and 0.1% TFA in Acetonitrile (ACN) v/v (B); flow rate 6 ml/min; the absorption was measured at 190-370 nm. Different gradient systems are used. For example for peptides LQG and LQGV: 10 min 100% A followed by a linear gradient 0-10% B, 50 min. For example for peptides VLPALP and VLPALPQ: 5 min 5% B followed by a linear gradient 1% B/min. The collected fractions were concentrated to approximately 5ml by rotary membrane evaporation at 40 ℃ under reduced pressure. The residual TFA was exchanged for acetate by eluting twice on an anion exchange resin in acetate form (Merck II) column. The eluate was concentrated and lyophilized for 28 hours. The peptide was then dissolved in PBS for use.

Examples 1 and 2

In the first experiment, 12-week old female BALB/c mice were given a single intraperitoneal injection of PBS (n-9) or peptide (LGQV, VLPALP, LPGCPRGVPNPVVS, MTRVLQGVLPHALPHQVVC; n-8, 10 mg/kg). Half an hour after treatment mice were systemically exposed to a single dose of 10Gy¹³⁷Cs-gamma-irradiation. In a second experiment, 12-week old female BALB/c mice were first systemically exposed to a single dose of 10Gy¹³⁷Cs- γ -irradiation followed by a single intraperitoneal injection of PBS (n-9) or peptide (n-8 or 9, 10mg/kg) 1.5 hours after irradiation. Mortality and clinical signs were observed at various time points during the experiment (e.g., eye tears representing conjunctivitis, and weight loss). As can be seen from table 2, all the tested peptides had a good conjunctivitis-reducing effect in the treated group of mice, without an effect on mortality, which prompted us to select a peptide most suitable against acute inflammation for testing in lower irradiation at repeated doses performed at a later stage.

Example 3

6 oligopeptides (i.e., A: LAGV, B: AQGV, C: LAG, D: AQG, E: MTR, and F: MTRV) were tested in a double-blind animal experiment and the relative ability of each peptide to promote recovery in a mouse renal ischemia-reperfusion assay was tested in comparison to PBS (control). In the experiment, mice were anesthetized and one side of the kidney was excised. The kidney was ligated for 25 minutes on the other side and serum urea levels increased. Each different peptide (5mg oligopeptide/kg body weight) was administered intravenously to 30 different mice before and after ligation, and then the mortality and BUN concentration of each peptide-treated mouse were determined at 2 hours, 24 hours, and 72 hours. The results are shown in Table 3 (excluding the results for peptide A (LAGV (SEQ ID NO: 4)) obtained in example 3).

Under inhalation anesthesia, the left kidney was isolated with its arteries and veins and blocked with a microvascular clamp for 25 minutes. The animals were placed on a heating pad during surgery to maintain body temperature at 37 ℃.5 minutes before placing the vascular clamps and 5 minutes before releasing the vascular clamps, the animals were administered 5mg/kg of peptide intravenously, dissolved in 0.1mL of sterile saline. Left kidney reperfusion was followed by resection of right kidney. Renal function was assessed by measuring blood urea nitrogen before clamping and at 2, 24, and 72 hours after reperfusion.

results-Table 3 (mortality 72 hours after reperfusion)

PBS

A(LAGV)

B(AQGV)

C(LAG)

D(AQG)

E(MTR)

F(MTRV)

6/10

6/10

0/10

4/10

4/10

4/10

2/10

*P<(vs PBS)

NS

0.01

0.01

0.01

0.01

0.01

^*2x2 chi-square test. df is 1

Peptide A (SEQ ID NO: 4) was the first peptide to be administered in the renal ischemia reperfusion test. The person performing the experiment is familiar with the learning curve when using peptide a. During the course of the peptide administration to the inferior vena cava, some animals experienced moderate blood loss at the injection site, but others did not. These animals were returned to their cages unintentionally but no drinking water was placed in the cages the first night after the operation. In addition, these animals should be sacrificed at 72 hours, but were mistakenly sacrificed 48 hours after reperfusion. These and other problems did not occur during the experiments for peptides B-F.

It can be seen that animals administered the oligopeptide MTRV and in particular AQGV survived (significantly reduced mortality relative to the PBS control group) and had a reduced BUN concentration much better than the control group (PBS) or the group administered the other oligopeptides, more mice survived and serum urea levels were much lower than the other groups. However, oligopeptides LAG, AQG, and MTR did not reduce BUN concentration in this experiment, but significantly reduced mortality compared to the PBS control group, respectively, wherein MTR increased BUN levels in test mice even at 72 hours.

Example 4

For the reasons mentioned above, an oligopeptide (a) was retested for its ability to reduce BUN levels in mice. The results are shown in Table 4. It can be seen that mice receiving oligopeptide LAGV were much better than the control group (PBS) in both survival (significantly reduced mortality relative to the PBS control group) and reduced BUN concentration.

Example 5

Additional 4 oligopeptides (g (vlpalpq), h (vlpalp), i (lqgv) and j (lqg)) were tested for their ability to reduce BUN levels in the mouse experiments described above. The results are shown in Table 4. It can be seen that mice receiving oligopeptide LQG showed a reduction in BUN concentration early in the experiment (24 hours post-reperfusion), whereas mice receiving VLPALPQ showed a much better reduction in BUN concentration later in the experiment (72 hours post-reperfusion) than control (PBS) or other oligopeptide treated groups, with more mice surviving and serum urea levels much lower than the other groups.

Table 4: BUN after treatment with peptides A-J in mice with renal ischemia for 25 min

P-value obtained by 2-hour statistical analysis after reperfusion：

A p＝0.0491 NMPF-47 LAGV

-B p＝0.0008 NMPF-46 AQGV

-C p＝0.9248 NMPF-44 LAG

-D p＝0.4043 NMPF-43 AQG

-E p＝0.1848 NMPF-12 MTR

-F p＝0.0106 NMPF-11 MTRV

-G p＝0.1389 NMPF-7 VLPALPQ

-H p＝0.5613 NMPF-6 VLPALP

-I p＝0.9301 NMPF-4 LQGV

-J p＝0.0030 NMPF-3 LQG

P-value obtained by statistical analysis 24 hours after reperfusion：

A p＝0.0017 NMPF-47 LAGV

-B p<0.0001 NMPF-46 AQGV

-C p＝0.8186 NMPF-44 LAG

-D p＝0.2297 NMPF-43 AQG

-E p＝0.0242 NMPF-12 MTR

-F p＝0.0021 NMPF-11 MTRV

G p＝0.0049 NMPF-7 VLPALPQ

H p＝0.3297 NMPF-6 VLPALP

-I p＝0.8328 NMPF-4 LQGV

-J p＝0.9445 NMPF-3 LQG

P values were calculated by the Mann Whitney U-test (SPSS for Windows).

Example 6

To determine the dose response relationship, the dose response patterns of two peptides (D (AQG, showing good results for mortality in the mice tested in example 3) and B (AQGV, having superior results for BUN in the mice tested in example 3) were tested in the mouse renal failure experiment as described above the peptides were tested at doses of 0.3, 1, 3,10 and 30mg/kg as described in example 3 the P values (calculated by Mann Whitney U-test (SPSS for)) of the peptide D group at 72 hours after clipping relative to the serum urea level of PBS were 0.3mg/kg0.001, 1mg/kg0.009, 3mg/kg0.02, 10mg/kg0.000 and 30mg/kg0.23 for the peptide B group, these P values were 0.88, 0.054, 0.000, 0.001 and 0.003 for the AQGV of the peptides B group were shown to be reduced unexpectedly in the (the AQG) dosage of the peptides B) relative to the AQG, but the beneficial effect on mortality at the lower doses tested was also significant.

Table 6: urea levels in dose response experiments
				24h	72h
PBS	57.8	85.4
			Peptide D (AQG)0.3mg/kg	38.4	30.4
Peptide D (AQG)1.0mg/kg	48.4	38.4
			Peptide D (AQG)3.0mg/kg	39.3	40.3
Peptide D (AQG)10.0mg/kg	46.8	25.8
			Peptide D (AQG)30.0mg/kg	52.8	58.9
Peptide B (AQGV0.3mg/kg)	62.4	86.7
			Peptide B (AQGV1.0mg/kg)	50.0	52.6
Peptide B (AQGV3.0mg/kg)	37.4	19.6
			Peptide B (AQGV10.0mg/kg)	41.2	37.1
Peptide B (AQGVG 30.0 mg/kg)	47.8	38.0

Standard error of	24h	72h
PBS	7.1	14.7
			Peptide D (AQG)0.3mg/kg	8.6	3.5
Peptide D (AQG)1.0mg/kg	7.2	10.2
			Peptide D (AQG)3.0mg/kg	3.5	10.7
Peptide D (AQG)10.0mg/kg	8.0	3.4
			Peptide D (AQG)30.0mg/kg	9.5	12.9
Peptide B (AQGV)0.3mg/kg	10.8	14.1
			Peptide B (AQGV)1.0mg/kg	11.7	14.3
3.0mg/kg of lunate Pacific B (AQGV)	7.6	2.6
			Peptide B (AQGV)10.0mg/kg	8.5	6.9
Peptide B (AQGV)30.0mg/kg	5.8	7.8

Septic shock experiments were set up to determine which peptides were best suited to combat acute inflammation.

Mice used for sepsis or septic shock experiments: female BALB/c mice, 8-12 weeks old, were used for all experiments. Animals were kept in a facility free of specific pathogens according to the protocol of Report of European Laboratory Animal sciences associates (FELASA) Working group on Animal Health (Laboratory Animals 28: 1-24, 1994).

The injection scheme is as follows: for the endotoxine model, BALB/c mice were injected intraperitoneally with 150-. Control group was intraperitoneally injected with PBS only. To test the effect of the peptides, the peptides were dissolved in PBS and injected intraperitoneally at predetermined time points after PBS treatment.

Mice were scored for disease severity using the following measurement protocol:

0 has no abnormal condition

1 fur exudate, but no detectable behavioral differences compared to normal mice

Fur exudate, frizzy reflex, responding to stimuli (e.g., beating cage) and behaving actively during touch as healthy mice

3 the patting of the cage has slow reaction, is passive or gentle when touched, but is still curious when being in a new environment alone

4 lack of curiosity, little or no response to stimuli, little activity

5 breathe hard, do not move or slowly swing right after being overturned (dying)

D death

The first set of septic shock experiments was set up to determine which peptides LQG, LQGV, VLPALP, VLPALPQ, MTR, MTRV, VVC or QVVC were able to inhibit Lipopolysaccharide (LPS) -induced septic shock in mice treated with a single dose of peptide 2 hours after LPS treatment. The BALB/c mice were injected intraperitoneally with 5mg/kg body weight of peptide, and the dose of LPS (E.coli026: B6; Difco Lab., Detroit, MI, USA) was increasing, which was expected to cause 80-100% death of LPS over 24 to 72 hours. Control group was injected intraperitoneally with PBS only and no mortality occurred.

A second set of septic shock experiments was set up to determine which peptides LQG, LQGV, VLPALP, VLPALPQ, MTR, MTRV, VVC or AQG, AQGV, LAG and LAGV were able to suppress high dose LPS-induced septic shock in mice treated with double doses of peptide 2 and 24 hours after LPS treatment. In each treatment group, 5mg/kg body weight of the peptide was used. BALB/c mice were injected intraperitoneally with high doses of LPS (E.coli026: B6; Difco Lab., Detroit, MI, USA), which was expected to cause 80-100% of the deaths in 24 to 72 hours. Control group was injected intraperitoneally with PBS only and no mortality occurred.

Another set of septic shock experiments was set up to determine which of the peptides LQG, LQGV, VLPALP, VLPALPQ, MTR, MTRV, VVC or AQGV studied was most suitable for use early and/or late in or throughout the onset of shock. To determine the percent survival of endotoxin shock with peptide post or early treatment, BALB/c mice were intraperitoneally injected with 300 μ g LPS (E.coli026: B6; Difco Lab., Detroit, MI, USA) and were expected to cause 100% death at 48 hours without peptide treatment. Control group was injected intraperitoneally with PBS only and no mortality occurred.

Comparative experiments were set up to compare peptides MTR and AQGV, both of commercial origin. The comparative experiment had 6 groups of 6 animals each, of which two groups (1A and 1B) received Placebo (PBS), one group (2) received the peptide MTR (origin: Pepscan), one group (3) received the peptide MTR (origin: Ansynth), one group (4) received the peptide AQGV (origin: Pepscan), and the other group received AQGV (origin: Ansynth). Peptide/placebo was administered 2 hours after LPS. LPS (source) dosage is 10-11 mg/kg. Disease scoring was performed at 0, 2, 22, 26, 42 and 48 hours after LPS injection.

Results

Selection of peptides

We selected the synthetic peptides MTR, MTRV, LQG, LQGV, VLPALP and VLPALPQ, and QVVC and VVC. In the later stages of the study, we also selected to synthesize alanine-substituted peptide variants derived from LQG and LQGV, in which one amino acid was substituted for alanine, for 4 of them (AQG, AQGV, LAG, and LAGV), with the following results.

Infectious shock test

To test the effect of the peptides in the early stages of shock development, mice were injected intraperitoneally with the test peptides 2 or 24 hours after treatment with different doses of LPS, at a dose of 5mg/kg body weight. For mice without peptide treatment, the dose of LPS resulted in 100% death at 48-72 hours. The results are shown in Table 8. Of the 7 peptides tested, the peptides vlpalpalap and LQGV showed significant protection against LPS-induced sepsis.

To evaluate the effect of peptide treatment in the early or late phase of shock development, mice were injected intraperitoneally with test peptide at a dose of 5mg/kg body weight 2 hours or 24 hours after the LPS treatment. Mice were observed for 84 hours rather than 48 hours as in previous experiments. The results are shown in Table 10. Of all the peptides tested, AQGV alone was able to maintain 100% survival at 84 hours post-LPS treatment with no remaining clinical signs when administered early or late in the onset of shock.

Both MTR and AQGV were tested in comparison, and both peptides were from both sources and were administered at a dose of 5 mg/kg.

TABLE 11

LPS＝10-11mg/kg

#970 As 5mg/kg LPs Compound

Treatment No. 971 to 5mg/kg

#Ansynth12

Disease score # Ansynth46

TABLE 11

LPS＝10-11mg/kg

#970 As 5mg/kg LPS Compound

Treatment No. 971 to 5mg/kg

#Ansynth12

Disease score # Ansynth46

Some reports suggest that intact hCG can modulate the immune system, but such cleavage products are not reported in the scientific literature. Benner and Khan (Scand. J. Immunol. 2005Jul; 62Suppl 1: 62-6) investigated the immunological activity that an in vivo-releasing peptide fragment generated by cleavage of sequence MTRVLQGVLPALPQVVC (residues 41-57) of hCG. beta. -subunit loop 2 might have, despite the fact that peptides as small as 3 to 7 amino acids are generally considered to have no significant biological activity.

We designed peptides that were able to completely block LPS-induced septic shock in mice, and in some cases were still effective even with these peptides starting at 24 hours after LPS injection. These peptides are also capable of inhibiting MIF production. These findings provide the possibility for therapeutic use of these peptides for treating patients suffering from radiation damage.

Example 7

This example shows experimental results for the peptide AQGV on 8.6Gy Whole Body Irradiated (WBI) mice, where all groups of mice received the first injection 3 hours after TBI. Animals receiving the placebo-injected group died 80% as expected for this model. The radiation dose of the administered radiation (8.6 gray-8.6 Gy) is known to cause approximately 80% mortality in this species and is therefore known as LD80 (80% lethal dose). Death begins around day 10-this is the usual case in WBI in animals or humans: around day 10, damage and leakage of the lining of the intestine by radiation causes bacteria to enter the circulation and sepsis due to gastrointestinal syndrome, while damage to the bone marrow causes the failure to produce enough white blood cells to fight infection ("bone marrow syndrome"), with consequent death.

A first group of peptide-treated mice, represented by the symbol "x" (figure 1), received a first intravenous injection of AQGV, which was followed 3 hours after the first injection by a second subcutaneous injection (SC). Unexpectedly, these animals were 100% alive. Furthermore, these animals did not show any signs of disease. To an unknowing observer they looked the same as fully normal mice, in particular these peptide-treated mice did not develop GI syndrome.

The second group of mice received a first injection of peptide by the SC route, followed by additional SC injections every 48 hours for a total of 3 doses (in addition to the first dose) -i.e., on days 3, 5, and 7. Only one of these animals died, and the others did not show any symptoms of GI syndrome.

The third group of mice was identical to the second group of mice except that the SC injection was continued for a total of 6 doses (except for the first dose) over 48 hours. Thus its administration was continued up to day 13. This prolonged treatment resulted in complete protection (none of the groups died). None of the animals of this group exhibited any signs of disease, as well as no symptoms of GI syndrome.

From these data we conclude that AQGV is fully protective against lethal doses of WBI, and in particular protective against GI syndrome associated with that dose, if the animals receive twice the dose of peptide on the first day (i.v. first dose). Extending treatment to the second week also results in complete protection if the animal receives lower levels of treatment (SC only), and in particular protection against GI syndrome associated with this high dose.

When these results are compared with studies ON radioprotection of the drug with code ON-01210 reported at the 51 st academy of radiation research (4 months 2004), it was found that the drug ON-01210 (similar to other drugs currently under investigation for radiation exposure) only protects when administered prior to radiation exposure. This makes the drug not very useful in protecting against "dirty bombs". The medicine has sulfhydryl component (4-carboxyystyrl-4-chlorobenzilsulfone), and can be used as antioxidant to remove free radicals generated during cell injury due to radiation. But if the drug is not present in the body at the time of radiation exposure, it will not be effective in any way. In contrast, treatment with the peptides of the invention may be effected after exposure.

Similarly, reviewing treatment with other existing drugs, all data on these drugs (i.e., on treatment with anabolic steroids) shows that existing non-peptide drugs need to be administered prior to WBI (i.e., 24 hours) and that subsequent administration has no supporting effect on protection from acute radiation injury.

Example 8: DRF study

In this example, we report studies of increasing doses of whole body radiation (WBI), with a single exposure for each group followed by increasing doses for each group. The single dose peptide AQGV was administered subcutaneously, but treatment was delayed until 3 days (72hr) after WBI. This test is called the dose reduction factor ("DRF") and is defined as the ratio between the LD50 of the treated group and the LD50 of the control group. LD50 represents the dose that caused 50% of the test animals to die.

Acceptable DRF ratios are those for which the factor is at least 1.10; but is preferably at least 1.20 or even at least 1.25. To pass the DRF1.20 test, a candidate drug must have an LD50 radiation dose at least 20% higher (a factor of 1.20 increase) than the LD50 dose of control animals on day 30 post WBI. For example, if the LD50 of a control animal is 8.2Gy, then to pass the test the LD50 caused by the drug candidate should be at least 20% higher, i.e. in this case the dose should be 8.2x 1.20-10.4 Gy.

The number of animals tested in the DRF test and the results are shown in table 12.

TABLE 12

Dose (Gy)	“n”	Absolute number of surviving animals 30 days after WB1	Percent survival in 30 days	Percent death in 30 days
					7.4	50	45	90％	10％
8	100	60	60％	40％
					8.6	120	24	20％	80％
9.2	30	0	0％	100％
					8.6+ AQGV, day 3	20	20	100％	0％
9.2+ AQGV, day 3	10	10	100％	0％
					9.8+ AQGV, day 3	10	10	100％	0％
10.4+ AQGV, day 3	10	4	40％	60％
					11.0+ AQGV, day 3	10	0	0％	100％

It is important to discuss the reason for this decision to delay treatment by 72 hours: in some cases of radioactive exposure (e.g., exposure to nuclear fission units of a transportation ship, or an aircraft hitting a nuclear reactor in a city near the center, etc.), the destructive power may be so great that it may take several days to send all the victims to the treatment center. Military scientists (who are concerned with protecting the first responders) and civilian scientists (who are concerned with treating a large number of casualties) will therefore naturally need to determine whether a drug candidate will in any way eliminate the toxicity of acute radiation (GI syndrome, bone marrow syndrome), or it will suddenly break out after such a long delay.

DFR test results for AQGV. The dose of radiation to kill 50% of the control animals was 8.2 Gy. The protective effect of the peptide AQGV was so good that the radiation dose had to be increased by 25% (factor 1.25) until-10.4 Gy was reached to kill 50% of the animals, which was the result obtained with a 3-day delay of treatment. If such treatment could be administered more quickly, for example at 24hr or 48hr, then the radiation killing 50% of the animals would be higher.

Example 9

To further investigate the anti-cell cycle activity of the different oligopeptides, proliferation experiments were performed in arabidopsis seedlings. The objective was to test the effect of a group of 140 oligopeptides of different lengths on the expression of a biomarker gene during rapid growth of rapidly dividing cells. Both marker genes are involved in cell cycle progression, with high marker activity representing high cell cycle activity and no marker activity representing no cell cycle activity and therefore no proliferation. An example of the role of oligopeptides in the arabidopsis cell cycle assay is shown in figure 3.

Method of producing a composite material

The peptide was resuspended in 1 Xphosphate buffer (PBS) pH8 to a final concentration of 5 mg/ml. The resulting solution was dispensed into 96-well round bottom plates (corning Incorporated) at 40 microliters per well. The plates were stored at-20 ℃ for 4 days for use. Seeds of the Arabidopsis thaliana ecovariety Ws-0 were surface sterilized in 2% commercial bleach (Glorix) for 10 minutes and washed 5 times with sterile MQ water. The seeds were then resuspended in 0.1% agar and plated on MS20 plates, which were supplemented with 80mg/l kanamycin.

The plates were left at 4 ℃ overnight and then transferred to a climatic chamber at 210 ℃ for 16/8 hours light cycle. After 4 days of growth, the seedlings were transferred to 96-well plates containing peptide solution (4 seedlings per well) and incubated for 4 to 8 hours.

For this experiment, Arabidopsis homozygous seedlings carrying two reporter genes fused to GUS were used. The first reporter gene used was The Cell cycle marker, pCDG (Carmona et al, The Plant Journal, 1999, 20(4), 503-. After incubation with compounds, seedlings were stained for GUS. The staining reaction was performed in 100mM sodium phosphate buffer (pH7.0) (containing 10mM EDTA, 10% DMSO, 0.1% Triton X-100, 2mM X-Gluc, 0.5mM K)₃Fe(CN)₆And 0.5mM K₄Fe(CN)₆) At 37 ℃ for 16 hours. To terminate the GUS reaction and remove chlorophyll, seedlings were subsequently treated with 96% ethanol for 1 hour and then stored in 70% ethanol. The stained seedlings were observed under a stereomicroscope, and sections were prepared from the seedlings in which the compound treatment occurred. Seedlings were fixed and cleaned in chloral hydrate solution for microscopic observation and photographed under a microscope equipped with DIC lenses.

Results

The effect of the peptides on gene expression of rapidly growing Arabidopsis seedling markers was tested. This was monitored by changes in GUS distribution within different organs: root, root-hypocotyl transition zone and cotyledon.

Of the 140 compounds tested, a total of 43 showed a clear effect on the expression of the two markers tested. FIG. 3 shows in detail an example of the apparent changes caused by the test compounds, the changes at the microscopic level caused by the peptides LQGV, VVC and DINGFLPAL. Unexpectedly, this effect clearly correlated with the length of the different peptides tested. As can be seen from table 13, anti-cell cycle activity was very strong in the short peptides, whereas none of the peptides longer than 9 amino acids was able to reduce cell cycle activity. Approximately 22% of peptides 5 to 9 amino acids in length showed a decrease, but more than 50% of the tested 3-and 4-mers showed a decrease in cell cycle activity.

Table 13: frequency distribution of positive test peptides/peptide lengths found in the arabidopsis cell cycle test. Length of peptide # AA (amino acid); #, number of tests; number of positives; percent + positive

Example 10

To further investigate the anti-cell cycle activity of the different oligopeptides, in vitro experiments were performed using mouse peripheral blood cells stimulated with anti-CD 3 antibody. The aim was to test the effect of some representative oligopeptides on cell proliferation during CD 3-induced rapid growth of murine monocytes when cells undergo rapid division. Mice (n ═ 5) were injected intraperitoneally with PBS, Nak4(LQGV), Nak47(LAGV), Nak46(AQGV) (Ansynth BV, supplied by The Netherlands), or Nak46^*(AQGV supplied by Diosynth BV, the Netherlands). Mice were treated with 0.5mg/kg or 5mg/kg of the peptide for 1 hour, followed by separation of spleen to prepare spleen cell suspensions. Spleen cell suspensions from each group were pooled and cultured in vitro (triplicate) in the presence of PBS or anti-CD 3 antibody, and proliferation was measured at 0, 12, 24, and 48 hours post culture. All tested peptides were shown to be able to reduce proliferation (see figure 4).

Results of examples 9 and 10

3-mer peptides identified as useful for treating radiation injury are VVC, LAG, AQG, based on cell cycle studies in plants and in vitro studies to reduce peripheral blood cell proliferation. Similarly, the 4-mer peptide useful for treating radiation damage is LQGV, QVVC, MTRV, AQGV, LAGV, LQAV, PGCP, VGQL, RVLQ, EMFQ, AVAL, fvs, NMWD, LCFL, FSYA, FWVD, AFTV, LGTL, QLLG, YAIT, APSL, ITTL, QALG, GVLC, NLIN, SPIE, LNTI, LHNL, cpvqq, EVVR, MTEV, eal, EPPE, LGTL, VGGI, RLPG, LQGA, LCFL, the 5-mer peptide useful for treating radiation damage is TLAVE, vegrl, LNEAL, the 6-mer peptide useful for treating radiation damage is vlpapllp, MGGTWA, LTCDDP, the 7-mer peptide useful for treating radiation damage is VLPAPLQ, vcnryrdv, vnrgp, the 8-mer peptide useful for treating radiation damage is lapvg 9, and the flpgg is flpgg.

Reference to the literature

Khan N.A.，A.Khan，H.F.Savelkoul，R.Benner.Inhibition of septic shockin mice by an oligopeptide from the beta-chain of human chorionic gonadotropinhormone.Hum.Immunol.2002Jan；63(1)：1-7.

Benner R.，N.A.Khan.Dissection of systems，cell populations andmolecules.Scand.J.Immunol.2005Jul；62Suppl1：62-6.

Cole L.A.，A.Kardana，S.-Y.Park，G.D.Braunstein.The deactivation ofhCG by nicking and dissociation.J.Clin.Endocr.Metab.1993；76：704-710.

AlfthanH.，U.H.Stenman.Pathophysiological importance of variousmolecularforms of human choriogonadotropin.Mol.Cell Endocrinol.1996；125：107-120.

Kardana A.，M.M.Elliott，M.A.Gawinowicz，S.Birken，L.A.Cole.Theheterogeneity of human chorionic gonadotropin(hCG).I.Characterization ofpeptide heterogeneity in13individual preparations of hCG.Endocrinology1991；129：1541-1550.

Cole L.A.，A.Kardana，P.Andrade-Gordon，M.A.Gawinowicz，J.C.Morris，E.R.Bergert，J.O’Connor，S.Birken.The heterogeneity of human chorionicgonadotropin(hCG).III.The occurrence and biological and immunologicalactivities ofnicked hCG.Endocrinology1991；129：1559-1567

Birken S.，Y.Maydelman，M.A.Gawinowicz.Preparation and analysis ofthe common urinaryforms of human chorionic gonadotropin.Methods2000；21：3-14

Claims (6)

1. Use of a peptide for the manufacture of a pharmaceutical composition for treating a subject suffering from or believed to be suffering from radiation damage, the peptide being selected from the group consisting of: LQGV, LAGV and AQGV, said radiation damage being gastrointestinal syndrome.

2. The use of claim 1, wherein all amino acids of said peptide are L-amino acids.

3. The use of claim 1 or 2, wherein the peptide is AQGV.

4. The use of claim 1 or 2, wherein the treatment comprises subcutaneous or intramuscular injection of the peptide.

5. The use of claim 1 or 2, wherein the pharmaceutical composition is placed in an autoinjector.

6. The use of claim 1 or 2, wherein the treatment comprises administration of the peptide at least 30 minutes after irradiation.