CN101443080B - Use of peptides for the control of radiation injury - Google Patents

Abstract

The present invention relates to the field of drug development against acute radiation damage caused by exposure to high energy electromagnetic waves (X-rays, gamma rays) or particles (alpha particles, beta particles, neutrons). To date there are no effective drugs to reduce radiation damage following accidental exposure to ionizing radiation. The present invention provides methods for treating a subject suffering from radiation damage comprising administering to said subject a peptide of less than 30 amino acids or a functional analogue or derivative thereof. Furthermore, the present invention provides the use of a peptide of less than 30 amino acids or a functional analogue or derivative thereof for the manufacture of a pharmaceutical composition for the treatment of a subject suffering from or believed to be suffering from radiation damage. The invention specifically provides radioresistant peptides having a Dose Reduction Factor (DRF) for acute gamma irradiation of at least 1.10, which DRF can be determined by testing which dose of radiation results in a test group 30 days after whole body irradiation (WBI). 50% mortality (LD50/30) in mice, the test group of mice was treated with the peptide 72 hours after WBI; it was tested which dose of radiation resulted in control group 30 days after whole body irradiation (WBI) A 50% mortality rate (LD50/30) was observed in the mice in the control group treated with the peptide vehicle alone 72 hours after WBI; wherein the LD50/30 of the peptide-treated animals was divided by the vehicle-treated The DRF was calculated from the LD50/30 of the group of animals.

Description

Use of peptides in the management of radiation damage

technical field

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

Background technique

Radiation injury is tissue damage caused by exposure to radiation. Here, radiation refers to ionizing radiation produced by high-energy electromagnetic waves (X-rays, gamma rays) or particles (alpha particles, beta particles, neutrons). Such radiation is emitted by radioactive substances (radioisotopes) such as uranium, radon, and plutonium. Such radiation can also be produced by man-made radiation sources, such as X-rays and radiotherapy machines. Radiation dose is measured using a number of different units, but they all refer to the amount of energy deposited. These units include roentgen (R), gray (Gy), and sievert (Sv). Sievert 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 accidents expose people to both types at the same time.

Irradiation is exposure to radiation waves directly penetrating the body from outside the body. Exposure can cause immediate illness (acute radiation sickness). In addition, exposure, especially high doses, can damage a person's genetic material (DNA), leading to chronic (delayed) diseases such as cancer and birth defects. However, exposure does not render a person or his tissues radioactive. Contamination is exposure to and retention of radioactive material, typically in the form of dust or liquid. Radioactive material can remain on the skin and can fall or rub off from the skin, contaminating other people and objects. These substances can also be absorbed by the body through the lungs, digestive tract or broken skin. The absorbed material is transported to various parts of the body, such as the bone marrow, where it continues to release radiation. This internalized radiation can cause not only acute radiation sickness 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 is mostly blocked by the Earth's atmosphere. People who live or work in conditions of highly radioactive elements are exposed to more cosmic radiation, especially radon gas, which is also found in many rocks and minerals. These elements end up in a variety of substances, including food and building materials. In addition, people are exposed to radiation from man-made sources, including environmental radiation from nuclear weapons testing and from various medical tests and treatments. The average person is exposed to radiation from natural radiation and artificial radiation sources in a total amount of about 3 to 4 mSv (1 mSv = 1/1000 Sv) per year. People who work with radioactive materials and X-ray sources are prone to exposure to higher levels of radiation. People receiving radiation therapy for cancer can be exposed to very high levels of radiation. Nuclear weapons release large amounts of radiation. Such weapons have not been used against humans since 1945. However, many countries now possess nuclear weapons, and some terrorist groups are seeking to acquire them, raising the possibility that such weapons could one day be used again.

The damaging effects of radiation depend on many factors, including the amount (dose) and duration of exposure. A rapid single dose of radiation to the entire body can be lethal, whereas exposure of the same total dose over weeks or months can have significantly lesser effects. Rapid exposure is more likely to cause genetic damage for a given dose. The effects of radiation also depend on the extent of the body's exposure. For example, radiation in excess of 6 Gy usually results in death if dispersed throughout the body; but if concentrated only in a small area, as in cancer radiation, for example, 3 to 4 times that dose can be administered without serious harm to the subject as a whole . The distribution of radiation is also important because certain parts of the body are more sensitive to radiation. Organs and tissues with rapidly proliferating cells, such as the gut and bone marrow, are more susceptible to radiation damage than those with slow cell proliferation, such as muscles and tendons. The genetic material of sperm and egg cells can be damaged by radiation. Therefore, during cancer radiation therapy, it is important to shield these more vulnerable parts of the body from radiation as much as possible so that high doses of radiation can be delivered primarily to the cancer.

Radiation exposure produces two types of injury: acute (immediate) and chronic (delayed). Acute radiation injury triggers inflammation through vascular endothelial damage leading to vascular leakiness. Vascular and cellular responses follow. Ionizing radiation suppresses immunity and damages the gut epithelium, both of which facilitate translocation of microorganisms from the gut.

Radiation therapy for cancer produces symptoms mainly in the part of the body that received radiation. For example, in radiation therapy for rectal cancer, abdominal cramps and diarrhea are common due to the radiation's effects on the small intestine.

The search for non-toxic radioprotectants to protect normal tissues against radiation damage began shortly after World War II. Intensive radiobiology studies have identified a variety of protective agents that protect animals (mainly rodents) from radiation damage if administered prior to radiation exposure [Prasad KN. Handbook of radiobiology. 2nd edn. Boca Raton, FL: CRC Press , 1995]. These studies found that agents that scavenge free radicals and/or induce hypoxia have radioprotective value. However, most of these compounds are toxic to humans at radioprotective doses. As the danger of nuclear confrontation declined during the course of the Cold War, interest in the study of radioprotectants declined significantly later. Due to the rapid proliferation of X-ray-based diagnostic instruments and the increased use of radiological methods in the early diagnosis of disease, greater concern has been given to the increasing frequency of somatic and hereditary mutations that can predispose to genetically linked diseases in current people and future generations increased risk. Therefore, it is important to protect normal tissue from potential radiation damage, even if the damage is minimal.

In general, radioprotectants are defined as compounds that, 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 intended for the prophylaxis, mitigation and treatment of radiation injuries. Report of an NCI Workshop, December 3-4, 2003, Radiat Res 162: 711-728.]. They can be used in radiological terrorism, military incidents, clinical oncology, space travel, radiation point clearance [R.H. Johnson, Dealing with the terror of nuclear terrorism, Health Phys87: S3-7., F.A.J. Mettler, G.L. Voelz, Major radiation exposure what to expect and how to respond, N Engl J Med346:1554-1561, 2001] C.K.Nair, D.K.Parida, T.Nomura, Radioprotectors inradiotherapy, J Radiat Res (Tokyo) 42:21-37, J.K.Waselenko, T.J.MacVittie, W.F.Blakely , N. Pesik, A.L. Wiley, W.E. Dickerson, H.Tsu, D.L. Confer, C.N. Coleman, T. Seed, P. Lowry, J.O. Armitage, N. Dainiak, Medical management of the acute radiation syndrome: Recommendations of the Strategic National Stockpile Radiation Working Group, Ann Intern Med 140:1037-1051.]. More recently, the U.S. Office of Science and Technology Policy and the Homeland Security Council have made developing new radiation-protective drugs a top research priority. Although synthetic radioprotective drugs such as aminothiols yield the highest protection factors, they are generally more toxic than naturally occurring protectants. Often, the best radioprotectants are also considered to result in the highest toxicity.

In military radiation incidents, effectively mitigating radiation-induced health problems and combat effectiveness reduction effects can reduce the casualty load of medical treatment units, maintain more effective action after radiation exposure events, and allow commanders to guide operations in radiation environments and suffer acute Reduced risk of reduced mobility due to tissue damage and reduced negative psychological trauma for those performing tasks in contaminated environments. An ideal radioprotectant should be non-toxic, not reduce working capacity, and be effective after a single application, especially if rapid access to areas with external radiation hazards is required.

Presented at the NATO HumanFactors and Medicine Panel Research Task Group099 "Radiation Bioeffects and Countermeasures" Conference, Bethesda, MD, USA, June 21, 2005 (Landauer et al., NATO RTG-0992005) and published in AFRRI CD05-2 In this article, it is proposed that genistein (genisteine) can protect mice from gamma radiation-induced death at the optimal dose (200 mg/kg; the highest survival rate was obtained when this dose was administered 24 hours before irradiation). The "dose reduction factor" (dose reduction factor, DRF) is 1.16. No radioprotective effect was observed if administered 1 hour before whole body irradiation (WBI). At the 51st meeting of the Radiation Research Society (April 2004), other studies reported the radiation protection effect of the drug code-named ON-01210, stating that the drug ON-01210 (similar to other drugs currently being studied for radiation exposure ) are only protective if applied before radiation exposure. The drug has a sulfhydryl component (4-carboxystyrl-4-chlorobenzylsulfone) that acts as an antioxidant to scavenge free radicals produced when cells are damaged by radiation.

Also, as stated in the US Department of Defense Congress Annual Report (March 2005; http://medchembio.amedd.army.mil/docs/CBDP_Report_To_Congress.pdf), there are currently no commercially available wireless toxic drugs or diagnostic capabilities. Amifostine, an aminothiol compound, has been approved by the FDA for use in patients receiving chemotherapy or radiation, but its performance-degrading toxic side effects preclude its use in suitable combat units, and its intravenous administration The pathway requires professional medical personnel. Other drugs, such as hematopoietic cytokines used to treat bone marrow injury, may be used in off-label doses by individual physicians on a case-by-case basis, but regulatory limitations on such use make their use difficult to manage the high number of casualties in military operations. Antibiotics are commonly used to treat infectious sequelae of radiological injury, but must be appropriately selected to effectively treat both exogenous and endogenous systemic infections while barely affecting beneficial intestinal anaerobic bacteria. To address the limited medical options currently available, the Armed Forces Radiobiology Research Institute (AFRRI) has been investigating a new compound, 5-androstenediol (5-AED; Whitnall et al., Experimental Biology and Medicine 226:625-627( 2001)). Likewise, administration of this compound prior to irradiation challenge resulted in good radioprotectant efficacy in a mouse model. Subcutaneous administration of AED 24 hours before and 2 hours after gamma-irradiated mice improves survival. The dose reduction factor calculated from the probability-valued survival curve administered prior to WBI was 1.3. Protection was observed in both male and female mice subsequently vaccinated with or without a lethal dose of Klebsiella pneumoniae. No protective effect was observed with various other steroids: dehydroepiandrosterone (DHEA), 5-androstene-3B,7B,17B-triol (AET), androstenedione, or estradiol. Over the past year, however, intensive studies in nonhuman primate (NHP) models in preparation for IND applications have demonstrated that 5-AED is nowhere near as effective when administered as a radioprotectant as in mouse models , but produced good efficacy in the NHP model when administered therapeutically in continuous doses shortly after irradiation.

Acute radiation sickness. Acute radiation sickness usually occurs in people who have been exposed to radiation all over their body. The progression of acute radiation sickness has multiple stages, beginning with early symptoms (prodrome), followed by an asymptomatic phase (latency). Different symptoms (symptom spectrum) subsequently appear depending on the amount of radiation a person has received. The greater the radiation dose, the more severe the symptoms and the faster the progression from early symptoms to acute syndrome. Symptoms and time course are consistent between individuals for a given amount of radiation exposure. Doctors can tell a person's radiation exposure based on the time course and nature of symptoms. Doctors divide acute radiation syndrome into three groups based on the major organ systems affected, although there is overlap between the groups.

Hematopoietic syndrome is caused by the effects of radiation on the bone marrow, spleen, and lymph nodes—the main sites that produce blood cells (hematopoiesis). Decreased appetite (anorexia), lethargy, nausea, and vomiting occur 2 to 12 hours after exposure to 2 Gy or more. Within 24 to 36 days after exposure, these symptoms subside and the person feels fine for a week or more. During this asymptomatic phase, hematopoietic cells in the bone marrow, spleen, and lymph nodes become depleted and are not renewed, resulting in a severe deficiency of white blood cells, followed by platelets, and then red blood cells. A lack of white blood cells can lead to serious infections. A lack of platelets can lead to uncontrolled bleeding. A lack of red blood cells (anemia) causes fatigue, weakness, pallor, and difficulty breathing during physical activity. After 4 to 5 weeks, if the person survives, blood cells begin to regenerate, but the person feels weak and tired for several months.

Gastrointestinal syndrome is caused by the effects of radiation on the cells lining the digestive tract. Severe nausea, vomiting, and diarrhea appear 2 to 12 hours after exposure to 4 Gy or more. These symptoms can cause severe dehydration but resolve after 2 days. The person feels fine for the next 4 to 5 days, but the layer of cells that normally coat the digestive tract as a protective barrier falls off during death. Afterwards, severe diarrhea—usually bloody—causes dehydration again. Bacteria from the digestive tract invade the body and cause serious infections. People who receive such intense radiation are also more likely to develop hematopoietic syndrome, which causes bleeding and infection and increases their risk of death.

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

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

Radiation injury can occur if a person becomes ill after receiving radiation therapy or after being accidentally exposed to radiation. There are no specific tests for diagnosing this condition, although some tests can be used to detect infections, low blood counts, or abnormal organ function. To determine the severity of radiation exposure, doctors measure the number of lymphocytes (a type of white blood cell) in the blood. The lower the lymphocyte count 48 hours after exposure, the more severe the radiation exposure.

Unlike exposure, radioactive contamination of the human body is 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 injury depend on the dose, the dose rate (how quickly the exposure occurs), and its distribution in the body, as well as on the person's pre-existing state of health. In conclusion, the vast majority of people who receive a WBI greater than 6 Gy will die from gastrointestinal syndrome. Because it is difficult for doctors to know the precise amount of radiation a person has received, they generally base their judgments on the person's symptoms. Cerebrovascular syndrome usually results in death within hours to days. Gastrointestinal syndrome usually leads to death within 3 to 10 days, although some people survive for several weeks. Depending on their total radiation exposure, many people who received appropriate medical attention did not die from hematopoietic syndrome; those who did not survived usually died after 8 to 50 days.

There is currently no emergency treatment for exposure, but doctors closely monitor people for various symptoms and treat them when they occur. At the same time, unfortunately there are currently very few medicinal products for the various acute and long-term toxicities caused by nuclear or radiological attacks. Contamination requires immediate removal of radioactive material to prevent ingestion by the body. Skin contaminated with radioactive material should be immediately scrubbed with copious amounts of soap and water or a solution designed for this purpose, if available. Small broken wounds should be cleaned sufficiently to remove all radioactive particles, although scrubbing can be painful. Contaminated hair is cut rather than scraped, which can abrade the skin and allow contamination to enter the body. Scrubbing is to continue until the Geiger counter shows radioactivity disappears. Vomiting is induced if a person swallows radioactive material. Some radioactive materials have special antidotes that prevent swallowed material from being absorbed. Most of these antidotes are only used for people exposed to significant radioactive contamination, such as a major reactor accident or nuclear explosion. Potassium iodide prevents the thyroid gland from absorbing radioactive iodine and reduces the risk of developing thyroid cancer. Other drugs, such as diethylene triaminepentaacetic acid (DTPA), ethylenediamine tetraacetic acid (EDTA), and penicillamine, are given intravenously to remove certain absorbed radioactive elements .

When contamination is not suspected, nausea and vomiting can be reduced with drugs to prevent vomiting (antiemetics); these drugs are routinely prescribed in patients undergoing radiation therapy. Dehydration can be treated with intravenous fluids.

People who develop gastrointestinal or hematopoietic syndrome should be isolated so that they are not exposed to infectious organisms. Blood transfusions and injections of growth factors that stimulate blood cell production (such as erythropoietin and colony-stimulating factor) may be given to reduce bleeding and increase blood counts. If the bone marrow is severely damaged, these growth factors are ineffective, and a bone marrow transplant is sometimes required, although the success rate is low.

Those who develop gastrointestinal syndrome require antiemetics, intravenous fluids, and sedatives. Some people can eat bland diets. Antibiotics such as neomycin are given to kill bacteria in the gut that might invade the body. Antibiotics and antifungal and antiviral drugs are administered intravenously as necessary. The goal of cerebrovascular syndrome treatment is to make the patient comfortable by relieving pain, anxiety, and dyspnea. Anticonvulsants may be administered.

People who develop chronic effects of radiation or disease caused by radiation therapy receive symptomatic treatment. Ulcers or ulcers may be surgically removed or repaired with hyperbaric oxygen to promote healing. Radiation-induced leukemia is treated with chemotherapy. Blood cells are replenished through blood transfusions. Infertility is irreversible, but low levels of sex hormones due to abnormal ovarian and testicular function can be treated with hormone replacement. Investigators are currently developing ways to prevent or reduce radiation-induced normal tissue damage using cytokines, growth factors, and various other treatments. Amifostine or pilocarpine hydrochloride have been found to reduce symptoms of dry mouth (xerostomia) in patients with head and neck cancer receiving radiation therapy.

Clinical and experimental studies of the acute and delayed effects of radiation on cells have enhanced our knowledge in radiation therapy, making it now possible to optimize radiation therapy regimens and employ more precise patterns of radiation administration. However, because normal and cancerous tissue respond similarly to radiation exposure, radiation-induced damage to normal tissue may occur during or after radiation therapy has been completed. Studies did find some signs of radioprotection from NSAIDs and prostaglandins. Both classes of drugs increase cell survival, but through completely different mechanisms. Cell dynamics studies have found that cells in the mitotic (M) and late G2 phases of the cell cycle are generally the most sensitive to radiation compared to cells in the early S and G1/G0 phases. Furthermore, radiation causes mitotic delay of the cell cycle. Thus, chemicals 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 investigated for their potential use as normal tissue radioprotective drugs during injection. NSAIDs have been found to exert anti-cancer effects by causing cell cycle arrest so that cells tend towards a quiescent state (G0/G1). The same mechanism of action was observed in the radioprotection of normal tissues. Increased concentrations of arachidonic acid after exposure to NSAIDs also lead to the production of the apoptosis inducer ceramide. NSAIDs also raise intracellular levels of superoxide dismutase. Activation of heat shock proteins by NSAID can improve cell survival by changing the expression of cytokines. NSAIDs may also have the effect of inhibiting cell proliferation through anti-angiogenic mechanisms. Several in vivo studies provide evidence that NSAIDs protect normal tissues from radiation damage. Prostaglandins do not regulate the cell cycle, but they have various effects on the growth and differentiation of cells. PGE2 mediates angiogenesis, increasing the supply of oxygen and nutrients necessary for cell survival and growth. Therefore, PGE2 at sufficiently high plasma concentrations can enhance cell survival by inhibiting pro-inflammatory cytokines such as TNF-α and IL-1β. Therefore, PGE2 acts as a modulator rather than a mediator of inflammation. Prospective studies suggest the potential use of misoprostol, a PGE1 analog, administered before irradiation to prevent radiation-induced adverse effects. Current understanding of the pharmacology of NSAIDs and prostaglandins suggests that, when used prophylactically, they have the potential to minimize adverse effects of radiation on normal tissues.

In addition to transiently inhibiting cell cycle progression and killing those cells capable of proliferating, irradiation also disturbs homeostasis influenced by endogenous mediators of intercellular communication (the humoral components of tissue in response to radiation). Changes in the levels of mediators can modulate the effects of radiation by promoting reversion to normal (for example, by raising H-type cell line-specific growth factors) or by exacerbating damage. The latter pattern is illustrated by reports of changes in eicosanoid levels after irradiation and by reports of the results of empirical treatment of radiation injury with anti-inflammatory drugs. The precursor, acute, and chronic effects of radiation are accompanied by excess production of eicosanoids (prostaglandins, prostacyclins, thromboxanes, and leukotrienes). Endogenous mediators of these inflammatory responses can cause vasodilation, vasoconstriction, increased microvascular permeability, thrombosis, and chemotaxis following radiation exposure. Glucocorticoids inhibit eicosanoid synthesis by interfering with phospholipase A2, while NSAIDs block prostaglandin/thromboxane synthesis by inhibiting cyclooxygenase. Drugs belonging to both classes are administered empirically after irradiation to help mitigate the prodromal, acute and chronic effects of radiation in humans and animals.

US Patent 5,380,668 (Jan. 10, 1995) to Herron discloses various compounds having antigen binding activity of hCG, etc., the entire contents of which are hereby incorporated by reference. The diagnostic use of said oligopeptides is generally disclosed therein. Various patents and patent applications of Gallo et al. (such as US Patent 5,677,275 (corresponding to WO96/04008A1), US Patent 5,877,148 (also corresponding to WO96/04008A1), WO97/49721A1, US Patent 6,319,504 (corresponding to WO97/49373), U.S. Patent Patent Application 2003/0049273A1 (also corresponding to WO97/49373), US Patent 5,968,513 (corresponding to WO97/49418), US Patent 5,997,871 (corresponding to WO97/49432), US Patent 6,620,416, US Patent 6,596,688, WO01/11048A2, WO01/ 10907A2. and U.S. Patent 6,583,109) relate to different oligopeptides and uses thereof, such as "inhibiting HIV infection", "treating or preventing HIV infection", "treating or preventing cancer", "treating or preventing disorders characterized by decreased somatic cell mass ", "treatment or prevention of conditions associated with pathological angiogenesis", "treatment or prevention of hematopoietic defects", "ex vivo gene therapy", in vitro expansion of blood cells" and/or "providing blood cells to patients". Such as PCT International Publication No.WO03/029292A2 (publication date: April10, 2003), PCT International Publication No.WO01/72831A2 (publication date: October4, 2001) and US Patent Application Publication 20020064501A1 (publication date: May30, 2002), 20030119720A1 (publication date: June26, 2003), 20030113733A1 (publication date: June19, 2003) and 20030166556A1 (publication date: September4, 2003), U.S. Patent Application No.11/249,541, application date October13, 2005, International Application No.PCT/ EP2005/003707, application date April8, 2005, U.S. patent application No.10/821,256, application date April8, 2004, U.S. patent application No.10/262,522, application date September30, 2002, international application No.PCT/NL01/00259 ( International Publication No.WO01/72831A2), application date March3, 2001, U.S. Patent 6,844,315 and U.S. Patent 6,921,751, compositions containing some of the oligopeptides described herein have immunomodulatory activity, which can be used, for example, to treat sepsis and other Disease states or disorders, the contents of all of the above documents are incorporated into this application by reference.

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

In addition, the present invention relates to US application 10/821,240, which provides methods for screening and identifying other small gene-modulating peptides and using peptides based on the results of such screening, eg, from reference peptides. For example, the peptides to be analyzed are from C-reactive protein (CRP) (such as human CRP), such peptides include LTSL, FVLS, NMWD, LCFL, MWDF, FSYA, FWVD, AFTV, and WDFV; from β-catenin (such as 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 (such as 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), e.g., LSNI, YVFS , LYGV, YVVC, FIVR, NILD, TIMY, LESI, FLLT, VFSP, FILE, TFLK, FWID, MWEI, QLLE, PCFW, VHKL, LYGV, LESI, LSNI, YVFS, IYSL, and NILD; and from matrix metalloproteinases- 2 (such as human MM02) peptides 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, among others.

Contents of the invention

The present invention is directed against acute radiation 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, pions) Damaged drug development field. To date there are no effective drugs to reduce radiation damage after accidental exposure to ionizing radiation, or following normal tissue damage during therapeutic radiation or by radiosimulants; responder) to prevent or minimize such damage. The present inventors have unexpectedly observed that a relatively small non-toxic peptide can be effectively used as an anti-radiation damage drug. Importantly, the anti-radiopeptides of the present invention are not only useful as prophylactic agents, they are also protective when administered hours after exposure to radiation. This makes them particularly suitable for use in military radiation events, such as terrorism used to counter 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, said peptide or functional analogue thereof is administered to said subject after radiation, ie after exposure of said 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 be suffering from radiation damage. In particular, the present invention provides radioresistant peptides that have a dose reduction factor (DRF) for acute total body irradiation (DRF) of at least 1.10, which can be determined by testing which dose of whole body irradiation (WBI) results in a test 30 days after WBI. A 50% mortality (LD50/30) occurred in groups of experimental rodents (such as mice) that were treated with the peptide immediately or within a maximum of 72 hours after WBI, compared to 30 days after WBI resulting in untreated controls. The group was compared to the WBI dose at which 50% mortality occurred (LD50/30), where the DRF was calculated by dividing the LD50/30 radiation dose of the peptide-treated animals by the LD50/30 radiation dose of the vehicle-treated animals.

The present invention provides a method of treating a subject suffering from, or believed to be suffering from, radiation injury comprising providing to said subject a pharmaceutical composition comprising a radioresistant peptide of less than 30 amino acids. Current radioprotectants are non-peptidic or include large proteins such as cytokines. The present invention discloses peptides less than 30 amino acids, such as MTRVLQGVLPALPQVVC, which can be used for protection and treatment of radiation damage. This is the first time a peptide drug has been found to reduce the damaging effects of radiation when administered after it has already been exposed. 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 Composed of up to 15 amino acids.

However, the peptides are 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. Some examples of useful peptides are LPGCPRGVNPVVS, DINGFLPAL and QPLAPLVG. However, if the peptide is to be used for self-treatment, eg, using an autoinjector as described herein, it is preferred that the peptide is less than 7 amino acids for safety reasons. Such peptides generally do not bind MHC receptors, thereby reducing the risk of autoimmunity induced by an immune response to the administered peptide.

Another reason why less than 7 amino acids (aa) is particularly preferred is that peptides are found to be 7 amino acids in size (when comparing peptides from human proteasomes and peptides from pathogenic (especially viral or bacterial) proteasomes ( Burroughs et al., Immunogenetics, 2004, 56: 311-320)) have only 3% overlap between self and non-self. The overlap between human self and pathogen non-self was determined to be 30% for 6 amino acid peptides and 30% for 5 amino acid peptides between the peptides present in the human proteasome and those present in the pathogen proteasome by The overlap was determined to be 90%, while for peptides of 4 amino acids and smaller 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 self-nonself differences are absent, which would be beneficial for a person with no medical training to administer any drug to self 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, based on a general point of view, as long as it can withstand complete proteolysis for a longer period of time, the larger the peptide, the more active it will be. Thus, the metabolic fragment of 3 amino acids still has activity. Preferably, the peptide consists of Composed of 4 amino acids. The compositions described above and below are preferably used in the treatment of acute radiation injury.

The prior art has mentioned the use of peptides for protection against radiation damage. Japanese patent applications JP09157291 and JP09157292 disclose specific 6-mer and 9-mer peptide sequences, which have the effect of inhibiting active oxygen, scavenging active oxygen free radicals and antioxidant activity in vitro. These peptides are thought to be useful in vivo to inhibit the adverse effects of various events, including radiation injury, associated with the formation of reactive oxygen species. But it did not conduct in vivo radiation experiments.

JP09176187 teaches histidine-containing 6-mer peptide analogues having active oxygen scavenging activity. Intraperitoneal administration of 660 mg/kg of peptide 20 minutes before irradiation increased the survival rate of mice from 10% in the control group to 70% in the treated group. However, post-irradiation experiments in vivo were not performed.

WO2006/032269 discloses a homogenate of blood cells from which components with a molecular weight above 3 kDa have been removed. The homogenate is reported to be suitable for improving the cellular immune response of a subject. In many different immunological diseases and pathological conditions, it is believed that the homogenate can be administered prophylactically to patients during treatment with chemotherapy and/or radiotherapy to improve the general condition of the patient. But the study did not involve any radiation experiments. Furthermore, although the homogenate may include a mixture of proteins, the nature of the active ingredient is not at all known, and the active ingredient may also be non-proteinaceous. Regardless, the document does not isolate or identify any specific peptide.

EP0572688 discloses a specific peptide comprising 14 amino acid residues which is able to protect mice against whole body irradiation at 20 mg/kg body weight. The protective effect of this peptide was only observed when administered 1 hour before irradiation. No difference from the control data was observed when the peptide was administered 1 hour after exposure to radiation.

These prior art disclosures are in marked contrast to the present invention; the antiradiogenic peptides of the present invention are protective even when administered several hours after whole body irradiation.

Subjects receiving sublethal radiation doses have been able to benefit from the anti-inflammatory properties of some of the small peptides identified in this invention, but surprisingly, the majority of the benefit comes from the anti-gastrointestinal syndrome activity of these small peptides , especially from 3- and 4-mer peptides when the dose is above 1 mg/kg body weight, preferably above 5 mg/kg body weight, more preferably above 10 mg/kg body weight. Given the low immunogenic profile of small peptides (i.e., peptides of 3 to 4 amino acids), small peptide doses can be as high as 100 mg/kg, and in some cases given the need for emergency treatment given the condition of the subject in need, the dose can be up to 100 mg/kg. Up to 200ng/kg, 500mg/kg, or even 1g/kg. Thus, it is now possible to treat subjects with radiation damage including damage to the intestinal lining, the so-called gastrointestinal syndrome; said peptides allow a slow recovery of the epithelial lining.

In order to make the peptide have better activity in the case of high radiation dose, it is preferable to select the peptide to be placed in the pharmaceutical composition of the present invention or the self-use injector of the present invention, and the peptide has a dose for acute gamma irradiation A reduction factor (DRF) of at least 1.10 can be determined as follows: test which dose of radiation causes 50% mortality (LD50/30) in the test group of mice 30 days after whole body irradiation (WBI), the Test group mice were treated with the peptide 72 hours after WBI, and it was tested which dose of radiation caused 50% mortality (LD50/30) in control group mice 30 days after whole body irradiation (WBI), so The control group mice were treated with the vehicle of the peptide only 72 hours after WBI, and wherein the DRF was calculated by dividing the LD50/30 of the peptide-treated animals by the LD50/30 of the vehicle-treated animals.

Even more preferably, the peptide used has a dose reduction factor (DRF) of at least 1.20, even more preferably at least 1.25, especially where said radiation damage is radiation 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 emitted by radioactive substances (radioisotopes) such as uranium, radon, and plutonium or produced by artificial sources of radiation such as X-rays and radiotherapy equipment .

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

Similarly, some 4-mer peptides useful in the treatment of radiation damage are 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, CPVQ, EVVR, MTEV, EALE, EPPE, LGTL, VGGI, RLPG, LQGA, and LCFL, which can be used to treat radiation-damaged 5-mer The body peptides are TLAVE, VEGNL, and LNEAL, the 6-mer peptides that can be used to treat radiation damage are VLPALP, MGGTWA, LTCDDP, and the 7-mer peptides that can be used to treat radiation damage are VLPAPLQ, VCNYRDV, and CPRGVNP, which can be used to treat radiation damage The 8-mer peptide is QPLAPLVG, and the 9-mer peptide that can be used to treat radiation damage is DINGFLPAL.

Additional peptides, particularly 3- or 4-mer peptides, can be found by testing the anti-cell cycle activity of the peptides in a proliferation assay, for example by employing the plant growth assays described herein. In particular provided herein is the use of a peptide consisting of 2 to 6 amino acids for the manufacture of a pharmaceutical composition for the treatment of a subject suffering from or believed to be suffering from radiation damage. Also, in terms of preventing adverse reactions such as anaphylactic shock, the peptide used for the preparation of 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. amino acid composition. If only in terms of activity, based on a general point of view, as long as (after administration) it can withstand complete proteolysis for a longer period of time, the larger the peptide, the more active it is, thus the metabolic fragment of 3 amino acids is still active, preferably Typically, the peptide consists of 4 amino acids.

Furthermore, and of particular interest, those subjects requiring treatment for radiation damage can now be treated by simple subcutaneous or intramuscular injections, thereby enabling self-treatment with self-administered syringes, or by untrained or non-medical personnel treatment, thus facilitating the work of ambulance organizations in emergency situations where thousands of people need treatment. And if only intravenous injection or equally dangerous intraperitoneal injection is effective, it will be more difficult for the subject in need of treatment to be rescued than if there are simple administration tools such as the self-use syringe provided by the present invention.

Specifically, the present invention also provides the use of a peptide less than 30 amino acids in the preparation of a pharmaceutical composition for treating radiation damage, wherein the pharmaceutical composition is placed in a self-use syringe. A self-administered syringe is a medical device used to administer a single dose of a specialty drug (typically emergency medicine), sometimes called a prefilled syringe, for self-injection or injection by a non-medical or layperson. In the present application, the term "self-contained syringe" does not mean that in the analytical system, such as Husek et al. (J.of Chromatography B: Biomedical Sciences & Applications, Elsevier, Amsterdam, Vol.767, no.1, (2002) pg. 169-174) Injectors for automated application of biological samples (e.g. peptides) in the chromatographic apparatus described.

By design, self-administered syringes are easy to use and can be self-administered by the patient or administered to the patient by a layperson. The injection site is typically within the thigh or buttock, wherein the treatment comprises subcutaneous or intramuscular injection of the peptide. Since self-injectors can be designed to automatically and reliably administer the required dose of a drug, they facilitate quick, easy and accurate drug administration. In particular, self-administered syringes are well suited for use by those subjects who must administer a therapeutic substance to themselves, or by medical personnel who must administer injections to multiple subjects within a relatively short period of time, as in the case of an emergency department. In addition, self-injectors with a needled injection mechanism can be designed so that the needle is not visible before, during, or even after the injection procedure, thereby reducing or eliminating complications associated with penetration of the visible needle into the subject's tissue. any anxiety. Although the exact specifications of needling self-use syringes vary widely, they generally include a body or housing, a needling syringe or similar device, and one or more drive mechanisms for penetrating the needle into a subject's tissue and The required dose of liquid medicine is delivered through a pierced needle. The drive mechanism in existing needle-punching self-use syringes usually 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. Pat. , 5,300,030, 5,102,393, 5,092,843, 4,894,054, 4,678,461, and 3,797,489, the contents of each of which are incorporated herein by reference. International publications WO01/17593, WO98/00188, WO95/29720, WO95/31235, and WO94/13342 also disclose various syringes including different drive mechanisms. Most self-contained syringes are (optionally spring loaded) syringes.

The self-contained syringe of the present invention, especially the body or housing that is in direct contact with the peptide, is preferably made of a material that has minimal affinity for the peptide. This minimizes peptide sticking or sticking to the self-administering syringe. A very suitable material is polypropylene, especially substantially pure polypropylene.

Self-administered syringes were originally designed to overcome the hesitation of administering medication to oneself with a needle. An example of such a self-contained syringe is the Epipen or the recently introduced Twinject , the latter is usually used in people at risk of anaphylaxis. Another example of a self-administering syringe is Rebiject for the administration of interferon beta for the treatment of multiple sclerosis . Self-use syringes are often used by the Army to protect personnel from chemical warfare agents. In the U.S. Army, self-use syringes are included with each biological or chemical weapons response kit. This response kit is distributed to every soldier who may be confronted with a biological or chemical weapon. Once activated, the needle automatically injects the person, piercing any clothing (even layers of clothing) on the person. Self-injectors of the present invention include not only injection devices as described above (usually spring-driven) for automatic skin puncture and/or drug injection, but also prefilled syringes or self-injection cartridges and the like.

The present invention provides such self-contained syringes that can be used to treat (radio)radiation damage, whether the radiation is emitted by radioactive substances (radioisotopes) such as uranium, radon, and plutonium, or by artificial sources of radiation such as X-rays and radiotherapy equipment produced. The present invention also provides a self-container syringe of the protected pharmaceutical composition consisting of a peptide of less than 30 amino acids (referred to herein as a radioantipeptide) 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, hereby incorporated by reference and not the present application).

Excipients consist 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 injectable use water. Other exemplary compositions for parenteral administration via a self-administering syringe include injection solutions or suspensions containing, for example, a suitable non-toxic, parenterally acceptable diluent or solvent, such as mannitol, 1 , 3-butanediol, water, Ringer's solution, isotonic sodium chloride solution, or other suitable dispersing or wetting agents and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.

In one embodiment, the self-administering syringe includes as an active ingredient a radioantipeptide (or a functional analog thereof), which, when administered after a subject has been exposed to radiation, is capable of reducing adverse effects of radiation. Preferably, the peptide is capable of at least partial protection against radiation damage if administered at least 30 minutes, more preferably at least 1 hour, most preferably at least hours or even days (eg 3 days) after irradiation. Such self-container syringes are also known as "first aid self-container syringes", representing their use in sudden emergency situations.

In one embodiment, the present invention provides a self-contained syringe containing a sterile solution packaged in a syringe-like device capable of automatically delivering its entire 5 mL contents upon activation. Each mL contains 100 mg, preferably 200 mg, of the anti-radiopeptide and excipients such as those comprising propylene glycol, ethanol, sodium benzoate and benzoic acid as buffers, and benzyl alcohol as preservatives. In a preferred embodiment, the self-administered syringe for treating radiation damage carries a radioresistant peptide of less than 15 amino acids, more preferably less than 7 amino acids.

Preferred self-contained syringes for the treatment of acute radiation injury carry a peptide of 3 to 4 amino acids in length, preferably the peptide has a Dose Reduction Factor (DRF) against acute gamma irradiation of at least 1.10, said DRF may be as follows Determination: Test which dose of radiation causes 50% mortality (LD50/30) 30 days after whole body irradiation (WBI) in the test group of mice treated with the peptide 72 hours after WBI treatment, and to test which dose of radiation resulted in 50% mortality (LD50/30) 30 days after whole body irradiation (WBI) in control mice treated with only the The vehicle of the peptide was treated and the DRF was calculated by dividing the LD50/30 of the peptide-treated animals by the LD50/30 of the control animals.

More preferred self-syringe carried peptides have a dose reduction factor (DRF) of at least 1.20, more preferably at least 1.25. Suitable peptides for anti-placement in self-administering syringes are also those peptides which have anti-cell cycle activity in plants as determined according to the present invention. Peptides well suited for use in the self-injector of the present 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, QALG, GVLC, NLIN, SPIE, LNTI, LHNL, CPVQ, EVVR, MTEV, 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 radiation damage or considered to be suffering from radiation damage, said pharmaceutical composition comprising: a pharmaceutically effective amount of a radioresistant peptide or a functional analog thereof, or as described in The identified pharmaceutical composition and pharmaceutically acceptable diluent. The present invention herein provides a method of treating or preventing radiation damage in a subject in need thereof or potentially in need thereof, the method comprising: administering to the subject a pharmaceutical composition comprising a means for treating or preventing radiation damage and a pharmaceutically acceptable An acceptable excipient, wherein said means comprises a radioresistant peptide or pharmaceutical composition identified herein, particularly wherein said radiation damage comprises radiation damage.

In one embodiment, the invention provides a method for treating a subject suffering from radiation damage comprising administering to the subject a composition comprising an oligopeptide obtained or derived from the peptide MTRVLQGVLPALPQVVC or the 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 treating radiation damage comprising an oligopeptide obtained or derived from the peptide MTRVLQGVLPALPQVVC or the peptide LPGCPRGVNPVVS, for example 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 provide the use of the (oligo)peptide in the preparation of a pharmaceutical composition for treating radiation damage.

We previously reported that a 6-mer oligopeptide (VLPALP) derived from the β-chain of human chorionic gonadotropin suppressed septic shock in mice. Likewise, we found that several other short peptides derived from beta-chain loop 2 (residues 41-57) of hCG (starting from the trimeric peptide) as well as those obtained by single amino acid substitutions with alanine Some modifications have similar anti-inflammatory activity. In turn, we generated the rationale to screen some of these peptides to continue the development of therapeutic compounds for the treatment of acute inflammation following accidental exposure to ionizing radiation.

Human chorionic gonadotropin (hCG) is a heterodimeric placental glycoprotein hormone produced by pregnant women. It occurs in various forms, including fragmentation products, in the urine of pregnant women and in commercial hCG preparations. Because of its putative role in preventing fetal allograft rejection during pregnancy, several investigators have investigated the effects of heterodimeric hCG and its variants on the immune system. Some reports suggest that intact hormones counter-regulate the immune system, but such effects of fragmentation products have not been reported. We previously (Khan et al., Hum. Immunol. 2002 Jan; 63(1): 1-7) reported that a 6-mer oligopeptide (VLPALP) derived from the β-chain of human chorionic gonadotropin was Suppresses septic shock. A single treatment with this hexapeptide after injecting mice with high doses of lipopolysaccharide (LPS) suppressed septic shock in mice. Benner and Khan (Scand. J. Immunol. 2005 Jul; 62 Suppl 1:62-6) investigated the possible immunological activity of peptide fragments released in vivo via the sequence MTRVLQGVLPALPQVVC of hCG β-subunit loop 2 (residues 41-57) fracture. It is reported here that some 3 to 7 amino acid peptides taken from loop 2 of the β-subunit—and some peptides derived therefrom by alanine substitution—exhibit significant anti-inflammatory activity (by inhibiting septic shock syndrome), and exceeds the activity considered to be useful for the treatment of radiation injury, especially radiation injury including gastrointestinal syndrome, and also exceeds the activity considered useful for the preparation of radiation injury, especially for the treatment of radiation injury, especially Is the activity of the pharmaceutical composition in radiation injury including gastrointestinal syndrome.

The present invention also provides pharmaceutical compositions having anti-cell cycle activity. The cell cycle is an ordered set of events that results in a cell growing and dividing into two sister cells. The phases of the cell cycle are G1-S-G2-M. G1 phase stands for "GAP1". The S phase stands for "Synthesis". This is the stage where DNA replication occurs. The G2 phase stands for "GAP2". M phase stands for "mitosis" and is when the division of the nucleus (chromatin separation) and cytoplasm (cytokinesis) occurs. The term "anti-cell cycle activity" herein means that the peptide is capable of altering cell cycle kinetics. For example, it includes altering, ie increasing or decreasing, the frequency of cell division. In one embodiment, it refers to antiproliferative activity.

Also provided is a pharmaceutical composition with anti-cell cycle activity comprising PGCP; a pharmaceutical composition with anti-cell cycle activity comprising VGQL; a pharmaceutical composition with anti-cell cycle activity comprising RVLQ; A pharmaceutical composition comprising EMFQ; a pharmaceutical composition with anti-cell cycle activity comprising AVAL; a pharmaceutical composition with anti-cell cycle activity comprising FVLS; a pharmaceutical composition with anti-cell cycle activity comprising NMWD A pharmaceutical composition with anti-cell cycle activity comprising LCFL; A pharmaceutical composition with anti-cell cycle activity comprising FSYA; A pharmaceutical composition with anti-cell cycle activity comprising FWVD; A drug with anti-cell cycle activity A composition comprising AFTV; a pharmaceutical composition with anti-cell cycle activity comprising LGTL; a pharmaceutical composition with anti-cell cycle activity comprising QLLG; a pharmaceutical composition with anti-cell cycle activity comprising YAIT; Pharmaceutical composition with anti-cell cycle activity comprising APSL; pharmaceutical composition with anti-cell cycle activity comprising ITTL; pharmaceutical composition with anti-cell cycle activity comprising QALG; pharmaceutical composition with anti-cell cycle activity , which comprises GVLC; a pharmaceutical composition with anti-cell cycle activity, which comprises NLIN; a pharmaceutical composition with anti-cell cycle activity, which comprises SPIE; a pharmaceutical composition with anti-cell cycle activity, which comprises LNTI; A pharmaceutical composition with anti-cell cycle activity comprising LHNL; a pharmaceutical composition with anti-cell cycle activity comprising CPVQ; a pharmaceutical composition with anti-cell cycle activity comprising EVVR; a pharmaceutical composition with anti-cell cycle activity comprising Comprising MTEV; a pharmaceutical composition having anti-cell cycle activity comprising EALE; a pharmaceutical composition having anti-cell cycle activity comprising EPPE; a pharmaceutical composition having anti-cell cycle activity comprising LGTL; having an anti-cell cycle activity A pharmaceutical composition comprising VGGI; a pharmaceutical composition with anti-cell cycle activity comprising RLPG; a pharmaceutical composition with anti-cell cycle activity comprising LQGA; a pharmaceutical composition with anti-cell cycle activity comprising LCFL A pharmaceutical composition with anti-cell cycle activity comprising TLAVE; A pharmaceutical composition with anti-cell cycle activity comprising VEGNL; A pharmaceutical composition with anti-cell cycle activity comprising LNEAL; A drug with anti-cell cycle activity A composition comprising MGGTWA; a pharmaceutical composition having anti-cell cycle activity comprising LTCDDP; a pharmaceutical composition having anti-cell cycle activity comprising VCNYRDV; a pharmaceutical composition having anti-cell cycle activity comprising CPRGVNP; A pharmaceutical composition having anti-cell cycle activity comprising DINGFLPAL.

Description of drawings

Figure 1: Whole body irradiated mice treated with AQGV (peptide EA-230)

"WBI" stands for Whole Body Irradiation. In vivo protection against radiation injury was assessed following WBI (6.5 to 9.8 Gy, Philips MG30, 81 cGy/min) using anesthetized C57B1/6 mice, with differences in survival measured by Kaplan-Meirer analysis. All groups of mice were first injected with peptide or vehicle (control animals) 3 hours after WBI. The placebo-injected group had an 80% mortality rate, as predicted by the model. A radiation dose of 8.6 Gray (=8.6Gy) is known to cause approximately 80% mortality in this species, hence the name LD80 (80% lethal dose). Mortality begins around day 10 - as is usual in animals or humans with WBI: around day 10, the intestinal lining is damaged and leaks from radiation allowing bacteria to enter the circulation and cause gastrointestinal syndrome, Damage to the bone marrow leads to an inability to produce enough white blood cells to fight infection ("myeloid syndrome") and death. The group represented by the symbol "x" received the first intravenous injection, and the second subcutaneous injection (SC) 3 hours after the first injection. 100% of these animals survived. What’s not shown in the picture is that they didn’t show any signs of disease at all. To an unsuspecting observer, they looked just like perfectly normal mice. The group represented by the triangle symbol received an initial 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 initial dose) - ie on days 3, 5, and 7. Only one of these animals died. The group represented by the square symbol had the same procedure as the triangle symbol group, except that the 48-hour SC injection was continued for a total of 6 doses (except the first dose). Its administration was therefore continued until day 13. This prolonged treatment resulted in complete protection (no deaths in this group). There were no signs of disease in this group of animals. From these data we can conclude that complete protection against a lethal dose of WBI is conferred if animals receive double doses of the peptide on the first day (the first dose is given intravenously). If the animals received a lower level of treatment (SC only), extending the treatment to the second week also produced complete protection.

Figure 2: Second set of radioprotection experiments with peptide AQGV

Whole-body irradiation (WBI) with increasing doses was used, with a single exposure for each group and gradually increasing doses for subsequent groups. A single dose of peptide EA-230 (AQGV) was administered subcutaneously, but treatment was delayed until 3 days (72 hr) 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 which causes death in 50% of the tested animals. An acceptable DRF value is 1.20. To pass this test, a drug candidate must result in an LD50 radiation dose at least 20% higher than the LD50 dose in control animals (an increase by a factor of 1.20) at day 30 after WBI. For example, if the LD50 of the control animal is 8.2Gy, the candidate drug should result in an LD50 that is at least 20% higher, ie the dose in this case should be 8.2x1.20=10.4Gy.

Figure 3: Function of oligopeptides in Arabidopsis thaliana cell cycle analysis. The compounds NAK4(LQGV) and NAK9(VVC) showed a clear effect on the markers tested. For the cell cycle marker (pCDG), a clear effect on 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 auxin response marker (DR5::GUS) as for the cell cycle marker. NAK26(DINGFLPAL) showed less consistent time-dependent effects. The effects were observed in time only at the root. No effects were observed in the transition zone and cotyledons.

Figure 4: Representative oligopeptides 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 intraperitoneally injected with PBS, Nak4(LQGV), Nak47(LAGV), Nak46(AQGV) (Ansynth BV, provided by The Netherlands), or Nak46* (Diosynth BV, AQGV provided by The Netherlands). Mice were treated with 0.5 mg/kg or 5 mg/kg of the peptide for 1 hour, and then the spleen was isolated to prepare a splenocyte suspension. Splenocyte suspensions from each group were pooled and cultured in vitro (triplicate) in the presence of PBS or anti-CD3 antibody, and proliferation was measured at 0, 12, 24 and 48 hours after culture.

Detailed description of the invention

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

In this application, "composition" refers to various compounds containing or consisting of oligopeptides. Preferably, the oligopeptide is isolated before being 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 the NT at the N-terminal is selected from H--, CH3--, acyl, or general protecting groups; and the CT at the C-terminus is selected from small Peptide (eg, 1 to 5 amino acids), --OH, ^--OR1 , _--NH2 , ^{--NHR1 , --NR1R2 ,} or --N( _CH2 ) _1-6NR1R ² , wherein R ¹ and R ² , if present, are independently selected from H, alkyl, aryl, (aryl)alkyl, and wherein R ¹ and R ² may be connected to each other to form a ring.

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

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

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

In this 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 the same or equivalent side chains as the specified amino acids in the oligopeptide, arranged in the same order as the peptide in question, but linked together by non-peptide bonds, for example by isosteric linkages ) such as ketone isosteres, hydroxyl isosteres, diketone isosteres, or ketone-difluoromethyl isosteres.

A "composition" also includes, for example, an acceptable salt of an oligopeptide or a labeled oligopeptide. In this application, "acceptable salt" refers to a salt that retains the desired activity of the oligopeptide or equivalent compound, and preferably does not adversely affect the oligopeptide or other components of the system in which it is used. Influence. Examples of such salts are the acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like. Salts can also be combined 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, pamoate acid, alginic acid, polyglutamic acid, etc. Salts can be with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, etc., or with N,N'-dibenzylethylenediamine (N,N'-dibenzylethylenediamine ) or organic cations formed from ethylenediamine, or a combination thereof (such as zinc tannate).

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

The chemical entities so produced can be administered and introduced into the body systemically, topically or locally. The peptide or a modification thereof can be administered as such or as a pharmaceutically acceptable acid or base addition salt obtained by reacting 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 acid and fumaric acid); or by reaction with inorganic bases (such as sodium hydroxide, ammonium hydroxide, potassium hydroxide); or by reaction with organic bases (such as monoamines, diamines, triamines , and aromatic amines and substituted ethanolamines) formed by the reaction. Selected peptides and any derived entities may also be conjugated to sugars, lipids, other polypeptides, nucleic acids, and PNAs; and act as conjugates in situ or for local release upon reaching the target tissue or organ.

For various amino acids, "substitution" generally involves replacing hydrogen present on an aromatic ring with a group such as alkoxy, halo, hydroxy, nitrogen or lower alkyl. Substitutions can also be made on the alkyl chain linking the aromatic moiety to the peptide backbone, eg substitution of a lower alkyl for a hydrogen. Other substitutions can also be made alpha to amino acids, also using alkyl groups.

Preferred substitutions use fluorine or chlorine as halogen and methoxy as alkoxy. As for the alkyl and lower alkyl groups, generally, those having fewer (1 to 3) carbon atoms are preferred.

Compounds conforming to the general formula can be prepared using conventional methods for preparing such compounds. For this, a suitable N-terminally 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 carboxyl group in solution or on a solid support. Protected amino acid or peptide derivatives. The protection of the α-amino functional group is usually carried out with the urethane functional group, such as the use of acid-resistant tertiary-butyloxycarbonyl (tertiary-butyloxycarbonyl group, "Boc"), benzyloxycarbonyl (benzyloxycarbonyl, "Z") and substituted analogs, or resistant 9-fluoromyl-methyloxycarbonyl ("Fmoc") as a base. The Z group can also be removed by catalytic hydrogenation. Suitable other protecting groups include Nps, Bmv, Bpoc, Aloc, MSC and the like. Amino-protecting groups are reviewed in The peptides, Analysis, Synthesis, Biology, Vol. 3, E. Gross and J. Meienhofer, eds. (Academic Press, New York, 1981). Carboxyl protection can be carried out by esterification, for example base-resistant esters such as methyl or ethyl esters, acid-resistant esters such as tert-butyl esters or substituted, benzyl esters or by hydrogenolysis. Protection of side chain functional groups such as lysine and glutamic acid or aspartic acid can be carried out using the aforementioned groups. Protection of thiol (although not always required), guanidine, alcohol and imidazole groups can be achieved using a variety of reagents, see for example The Peptides, Analysis, Synthesis, Biology, id. or see Pure and Applied Chemistry, 59(3 ), 331-344 (1987). Activation of the carboxyl group of a suitable protected amino acid or peptide can be accomplished using the azide, mixed anhydride, active ester, or carbodiimide approach, particularly by the addition of catalytic racemization-inhibiting compounds such as 1- N-N-hydroxybenzotriazole, N-hydroxysuccinimide, 3-hydroxy-4-oxo-3,4-dihydro-1,2,3,-benzotriazine, N-hydroxy-5-nor Bornene-2,3-dicarboxyimine. Anhydrides of phosphorus-containing acids may also be used. See, e.g., The Peptides, Analysis, Synthesis, Biology, supra and Pure and Applied Chemistry, 59(3), 331-344 (1987).

The compounds can also be prepared by Merrifield's solid phase method. Different solid supports and different strategies are known, see for example Barany and Merrifield, The Peptides, Analysis, Synthesis, Biology, Vol. 2, E. 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 replaced by an isostere can generally be synthesized using the aforementioned protecting groups and activation methods. For methods of synthesizing modified isosteres, reference can be made to literature, for example, literature on --CH ₂ --NH--isosteres and --CO--CH ₂ --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 method, different methods can be used to remove the protecting group and cleavage from the solid support. Deprotection is usually carried out under acidic conditions and in the presence of scavengers. See for example volumes3, 5 and 9 of the series on The Peptides Analysis, Synthesis, Biology, supra.

Another possible approach is to use enzymes to synthesize such compounds. See for example the review by H.D. Jakubke in The Peptides, Analysis, Synthesis, Biology, Vol. 9, S. Udenfriend and J. Meienhofer, eds. (Acad. Press, New York, 1987).

The oligopeptides of the invention can also be prepared using recombinant DNA methods, although it may be less appropriate from the circumstances. 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. This method generally involves introducing a DNA sequence encoding one or more specific oligopeptides into a cloning vector (such as a plasmid, phage DNA, or other DNA sequence capable of replicating in a host cell), introducing the cloning vector into a suitable eukaryotic or prokaryotic host cells, and culturing the thus transformed host cells. If eukaryotic host cells are used, the compound will contain a glycoprotein moiety.

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

The function of a peptide or a 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 a functional peptide analogue using a reference or control peptide, eg a peptide analogue consisting only of L amino acids. Suitable tests include determining the ability of candidate peptides to affect cell cycle kinetics. For example, effects on cell cycle progression can be determined using plant model systems, such as the Arabidopsis system exemplified here, or using cultured (mammalian) cells. In another aspect it involves determining the ability of candidate peptides to inhibit apoptosis, eg by inducing (transient) G2-M cell cycle arrest.

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

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

In one embodiment of the present invention, the anti-radiation peptide comprises an amino acid sequence linked together by a peptide bond between its amino and carboxyl groups, wherein at least one amino acid is a D amino acid. For example, the radioresistant 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, CPVQ, EVVR, MTEV, EALE, EPPE, LGTL, VGGI, RLPG, LQGA, LCFL, TLAVE, VEGNL, LNEAL, VLPALP , MGGTWA, LTCDDP, VLPAPLQ, VCNYRDV, CPRGVNP, QPLAPLVG, and DINGFLPAL, wherein at least one of the amino acid residues represented by the standard one-letter code is a D amino acid.

One skilled in the art is able to generate analog compounds of amino acid sequences. This can be done, for example, by screening peptide libraries. Such analogs have substantially the same functional properties as the original sequence, but need not be identical in quantity. Furthermore, peptides or analogs can be cyclized, for example by providing them with a (terminal) cysteine; be dimerized or multimerized, for example by attachment to lysine or cysteine or other Compounds with linked or multimerized side chains; formed in tandem or repeat configurations; coupled or otherwise linked to carriers known in the art, provided by a secure linkage that allows dissociation. Syntheses and functional analogs or fragmentation products of these oligopeptides as described above are useful in methods of treating radiation damage and subsequent disease.

In this application, a "functional analog" of a peptide is preferably smaller than the peptide from which it is derived, and thus is preferably prepared by deletion and/or substitution rather than increasing its length. Furthermore, in this application, "functional analogs" of peptides do not refer to those larger proteins or peptides that contain the amino acid sequence identified herein as a radioresistant peptide with more amino acids on one or both sides of it.

The term "pharmaceutical composition" herein is intended to cover both the active composition of the present invention per se and compositions containing the composition of the present invention together with pharmaceutically acceptable carriers, diluents or excipients. The pharmaceutical composition may contain a mixture of at least two radioresistant peptides or analogs described herein. Acceptable diluents for the oligopeptides detailed herein are, for example, physiological saline or phosphate buffered saline. In one embodiment, the oligopeptide or composition is administered systemically to an animal or human at an effective concentration, eg, intravenously, intramuscularly or intraperitoneally. Another route of administration involves organ or tissue perfusion, which can be performed in vivo or in vitro, using a perfusate comprising an oligopeptide or composition of the invention. Administration may be in a single dose, in discrete multiple doses, or for a period of time sufficient to allow sufficient modulation of gene expression. For sustained administration, the duration of sustained administration will vary with a variety of factors, as will be readily understood by those skilled in the art.

The dose of active molecule administered can vary considerably. The concentration of active molecule that can be administered is generally limited by the efficacy at lower concentrations and the 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, and the like.

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

One formulation to achieve release of active molecules includes injectable microcapsules or microspheres made from biodegradable polymers such as poly(dl-lactide), poly(dl-lactide-co-glycolide ), polycaprolactone, polyglycolide, polylactic-co-glycolide, poly(hydroxybutyric acid), polyester or acetal resin. 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 fewer active molecules and can be administered by paramedical personnel. Furthermore, such systems have inherent flexibility in designing the timing and rate of release of different drugs by choosing the size of the microcapsules or microspheres, the amount of drug loaded, and the dose administered. In addition, it can be sterilized by gamma irradiation.

The design, preparation and use of microcapsules and microspheres are well known in the art, and specific technical details in this regard can be found in the literature. Formulations other than microcapsules and microspheres can also be prepared using biodegradable polymers such as lactide, glycolide, and caprolactone polymers; for example, prefabricated and sprayed films containing active molecules can be used in the present invention. Filters or fibers comprising active molecules are also within the scope of the invention.

Another formulation that is highly suitable for single dose delivery of the active molecules of the invention is liposomes. 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 drug-loaded liposomes is known to those skilled in the art and is described in detail in the literature.

Another suitable means of single-dose delivery of the active molecules of the invention involves sticky droplets. In this technique, high molecular weight carriers are used in combination with active molecules, resulting in structures that produce solutions with high viscosity. Suitable high molecular weight carriers include, but are not limited to: dextran and cyclodextrin; hydrogels; (cross-linked) viscous materials, including (cross-linked) viscoelastic materials; carmellose; hyaluronic acid; white. The preparation and use of drug-loaded sticky droplets is known to those skilled in the art.

According to another approach, the active molecule can be administered in combination with an absorbable mechanical barrier such as oxidized regenerated cellulose. Active molecules can be bound to this barrier covalently or non-covalently (eg, via ionic bonds), or simply dispersed thereon.

The invention is further explained by the following illustrative examples.

Example

Peptide selection

Selection was based on known preferential cleavage sites on the sequence MTRVLQGVLPALPQVVC (residues 41-57) of hCG β-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; 129:1559-1567; S. Birken, Y.Maydelman, M.A.Gawinowicz, Methods2000; 21:3-14), and in C-reactive protein (CRP) (β-catenin, such as human CTNB), Bruton-type tyrosine kinase (such as human BTK ), matrix metalloproteinase-2 and p-53 amino acid sequences were selected.

peptide synthesis

By a proprietary method (Diosynth BV) or using a 9-fluorenylmethoxycarbonyl (Fmoc)/tert-butyl-based method with 2-chlorotrityl chloride resin (2-chlorotrityl chloride resin) The peptides described herein were prepared commercially by Phase Synthesis (Ansynth BV). The side chain of glutamine is protected with a trityl functional group. The peptides were synthesized manually. Each coupling consists of the following steps: (i) removal of the Fmoc protection of the α-amino group with piperidine in dimethylformamide (DMF), (ii) addition of the Fmoc amino acid (3eq) with diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) was coupled in DMF/N-methylformamide (NMP), and (iii) was coupled with acetic anhydride/diisopropylethylamine (DIEA) in DMF/ The remaining amino functions were capped in NMP. After the synthesis, the peptide resin was treated with a mixture of trifluoroacetic acid (TFA)/H ₂ O/triisopropylsilane (TIS) 95:2.5:2.5. After 30 minutes, TIS was added until decolorization. The solution was dehydrated in vacuo and the peptide was precipitated with ether. The crude peptide was dissolved in water (50-100 mg/ml) and purified by reverse phase high performance liquid chromatography (RP-HPLC). HPLC conditions: column: Vydac TP21810C18 (10x250mm); elution system: gradient system is 0.1% TFA dissolved in water v/v (A) and 0.1% TFA dissolved in acetonitrile (ACN) v/v (B); flow rate 6ml/ min; absorbance measured at 190-370 nm. Different gradient systems were used. For example for peptides LQG and LQGV: 100% A for 10 minutes, followed by a linear gradient of 0-10% B for 50 minutes. For example for peptides VLPALP and VLPALPQ: 5% B for 5 minutes followed by a linear gradient of 1% B/minute. The collected fractions were concentrated to about 5 ml by rotary film evaporation at 40°C under low pressure conditions. Residual TFA was exchanged for acetate by eluting twice on a column of anion exchange resin (Merck II) in the acetate form. The eluate was concentrated and lyophilized for 28 hours. Peptides were then dissolved in PBS for later use.

Example 1 and Example 2

In the first experiment, 12-week-old female BALB/c mice were given a single intraperitoneal injection of PBS (n=9) or peptides (LGQV, VLPALP, LPGCPRGVNPVVS, MTRVLQGVLPALPQVVC; n=8, 10 mg/kg). Mice were systemically exposed to a single dose of 10 Gy ¹³⁷ Cs-γ-irradiation 1.5 hours after treatment. In the second experiment, 12-week-old female BALB/c mice were systemically exposed to a single dose of 10Gy ¹³⁷ Cs-γ-irradiation followed by a single intraperitoneal injection of PBS (n=9) or peptide (n=8 or 9, 10 mg/kg). Mortality and clinical signs (eg, watery eyes indicating conjunctivitis, and weight loss) were observed at various time points during the experiment. It can be seen from Table 2 that all the tested peptides have a good effect on reducing conjunctivitis in the treated mice, but has no effect on the mortality rate, which prompted us to choose a peptide that is most suitable for fighting acute inflammation, so that in the later stage It was tested in repeated doses of lower irradiation.

Example 3

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

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

Results - Table 3 (mortality at 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=1

Peptide A (SEQ ID NO: 4) was the first peptide administered in the renal ischemia-reperfusion assay. The person performing the experiment is familiar with the learning curve when using peptide A. During inferior vena cava administration of the peptide, some animals experienced moderate blood loss at the injection site, but others did not. The animals were inadvertently returned to their cages but were not given drinking water in the cages during the first postoperative night. In addition, the animals should have been sacrificed at 72 hours but were mistakenly sacrificed at 48 hours after reperfusion. For peptides B-F, these or other problems did not arise during the experiment.

It can be seen that the animals administered with oligopeptide MTRV and especially AQGV are much better in terms of survival (significantly reduced mortality relative to the PBS control group) and decreased BUN concentration than the control group (PBS) or the groups administered with 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 the BUN concentration in this experiment, but significantly reduced the mortality compared with the PBS control group, and MTR even increased the BUN level of the test mice at 72 hours.

Example 4

For the reasons mentioned previously, one 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 the mice receiving the oligopeptide LAGV were much better than the control group (PBS) in terms of survival (significantly lower mortality compared to the PBS control group) and lower BUN concentration.

Example 5

Four additional 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. As can be seen, mice that received the oligopeptide LQG showed reduced BUN concentrations early in the experiment (24 hours after reperfusion), whereas mice that received VLPALPQ showed reduced BUN concentrations later in the experiment (72 hours after reperfusion) Much better than control (PBS) or other oligopeptide-treated groups, with more mice surviving and serum urea levels much lower than other groups.

Table 4: BUN after 25 minutes of renal ischemia in mice treated with peptides A-J

P values obtained by statistical analysis 2 hours 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 values obtained from 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 Mann Whitney U-test (SPSS for Windows).

Example 6

To determine the dose-response relationship, two peptides (D(AQG, which showed good results for mortality in the mice tested in Example 3) and B(AQGV) were tested in the mouse renal failure experiment as described above. , had superior results to BUN in the mice tested in Example 3). The peptide was tested as described in Example 3 at doses of 0.3, 1, 3, 10 and 30 mg/kg. Relative to PBS The serum urea level of the peptide D group at 72 hours after clipping was calculated by MannWhitney U-test (SPSS for Windows) as: 0.3mg/kg0.001, 1mg/kg0.009, 3mg/kg0.02 , 10mg/kg0.000, and 30mg/kg0.23, for peptide B group, these P values are 0.88, 0.054, 0.000, 0.001 and 0.003. It can be seen that relative to peptide B (AQGV), peptide D (AQGV) It was unexpectedly good at reducing BUN levels at the lower doses tested, and the beneficial effect on mortality was also significant at the lower doses tested.

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 (AQGV30.0mg/kg 47.8 38.0

standard error 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 Yuetai B (AQGV) 3.0mg/kg 7.6 2.6 Peptide B (AQGV) 10.0mg/kg 8.5 6.9 Peptide B (AQGV) 30.0mg/kg 5.8 7.8

Set up a septic shock experiment to determine which peptides work best against acute inflammation.

Mice used for sepsis or septic shock experiments: 8-12 week old female BALB/c mice were used for all experiments. Animals were housed in facilities under specific pathogen-free conditions following the protocol of the Report of European Laboratory Animal Science Associations (FELASA) Working group on Animal Health (Laboratory Animals 28: 1-24, 1994).

Injection protocol: For the endotoxin model, BALB/c mice were injected intraperitoneally with 150-300 μg LPS (E. coli026:B6; Difco Lab., Detroit, MI, USA). The control group was only intraperitoneally injected with PBS. To test the effect of the peptides, the peptides were dissolved in PBS and injected intraperitoneally at predetermined time points after PBS treatment.

The severity of disease in mice was scored using the following measurement scheme:

0 no exception

1 Fur exudates, but no detectable behavioral differences compared to normal mice

2 Fur exudate, curling reflex, responding to stimuli (eg, slapping cage), as active as healthy mice during touch

3 Slow to slap on cage, passive or docile to touch, but still curious in new surroundings alone

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

5 labored breathing, motionless or slowly straightens up after being overturned (dying, execution)

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, after LPS treatment 2 Mice were treated with a single dose of the peptide for 2 hours. BALB/c mice were injected intraperitoneally with peptides at 5 mg/kg body weight, and the dose of LPS (E.coli026:B6; Difco Lab., Detroit, MI, USA) was gradually increased. LPS was expected to cause 80 -100% death. In the control group, only PBS was injected intraperitoneally, and no death 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 inhibit high-dose LPS-induced septic shock in mice, at Mice were treated with double doses of peptides 2 and 24 hours after LPS treatment. In each treatment group, 5 mg/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), and LPS was expected to cause 80-100% death within 24 to 72 hours. In the control group, only PBS was injected intraperitoneally, and no death occurred.

Set up another set of septic shock experiments to determine which of the investigated peptides LQG, LQGV, VLPALP, VLPALPQ, MTR, MTRV, VVC or AQGV is most appropriate to use early and/or late in the onset of shock or throughout. To determine the percentage of endotoxic shock survival with late or early peptide treatment, BALB/c mice were injected intraperitoneally with 300 μg of LPS (E.coli026:B6; Difco Lab., Detroit, MI, USA), anticipating no peptide treatment Cases resulted in 100% death within 48 hours. In the control group, only PBS was injected intraperitoneally, and no death occurred.

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

result

Peptide selection

We chose the synthetic peptides MTR, MTRV, LQG, LQGV, VLPALP and VLPALPQ, and QVVC and VVC. Later in the study, we also chose to synthesize alanine-substituted peptide variants derived from LQG and LQGV, in which one amino acid was replaced by alanine, and for four of them (AQG, AQGV, LAG, and LAGV), The result is as follows.

Septic shock experiment

In order to test the role of the peptide in the early stage of shock, mice were injected intraperitoneally with the test peptide at a dose of 5 mg/kg body weight 2 hours or 24 hours after treatment with different doses of LPS. Doses of LPS resulted in 100% death within 48-72 hours of no peptide treated mice. The results are shown in Table 8. Among the 7 peptides tested, the peptides VLPALP and LQGV showed significant protection against LPS-induced sepsis.

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

Comparative testing of MTR and AQGV, both peptides from two sources, both administered at a dose of 5 mg/kg.

Table 11

LPS＝10-11mg/kg

#970＝5mg/kg LPs compound

#971＝5mg/kg Treatment Treatment

#Ansynth12

#Ansynth46 disease score

Table 11

LPS＝10-11mg/kg

#970＝5mg/kg LPS compound

#971＝5mg/kg Treatment Treatment

#Ansynth12

#Ansynth46 Disease Scoring

Some reports suggest that intact hCG can modulate the immune system, but such fragmentation products have not been reported in the scientific literature. Benner and Khan (Scand.J.Immunol.2005Jul; 62Suppl1:62-6) investigated the possible immunological activity of in vivo-released peptide fragments generated by cleavage of the sequence MTRVLQGVLPALPQVVC (residues 41-57) of hCGβ-subunit loop 2, although The fact is that peptides as small as 3 to 7 amino acids are generally considered to have no appreciable biological activity.

We designed peptides that completely blocked LPS-induced septic shock in mice, and in some cases treatment with these peptides was effective even starting 24 hours after LPS injection. These peptides are also able to inhibit the production of MIF. These findings open up the possibility of therapeutic use of these peptides to treat patients suffering from radiation damage.

Example 7

This example shows the experimental results of peptide AQGV on mice subjected to 8.6Gy whole body irradiation (WBI), wherein all groups of mice received the first injection 3 hours after TBI. Eighty percent of the animals receiving placebo injections died, as expected for this model. The dose of radiation administered (8.6 Gy = 8.6 Gy) is known to cause approximately 80% mortality in this species, hence the name LD80 (80% lethal dose). Mortality begins around day 10 - as is usual in WBI in animals or humans: around day 10, the intestinal lining is damaged and leaks from radiation allowing bacteria to enter the circulation and die due to gastrointestinal syndrome Sepsis is caused, and damage to the bone marrow prevents the production of enough white blood cells to fight infection ("myeloid syndrome"), followed by death.

The first group of peptide-treated mice represented by the symbol "x" (Fig. 1) received the first intravenous injection of AQGV, which received the second subcutaneous (SC) injection 3 hours after the first injection. Unexpectedly, 100% of these animals survived. Furthermore, the animals did not show any signs of disease. To an unsuspecting observer, they looked just like perfectly normal mice, especially those treated with the peptide that did not develop GI syndrome.

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

The mice in the third group followed the same procedure as the second group, except that the 48-hour SC injection was continued for a total of 6 doses (except for the first dose). Its administration was therefore continued until day 13. This prolonged treatment resulted in complete protection (no deaths in this group). There were no signs of disease in this group of animals, nor were there any symptoms of GI syndrome.

From these data we can conclude that AQGV confers complete protection against a lethal dose of WBI if animals receive double the dose of peptide on day 1 (the first dose is given intravenously), and in particular against the Concomitant GI syndrome produces a protective effect. If the animals received a lower level of treatment (SC only), extending the treatment to the second week also produced complete protection, and in particular protection against the GI syndrome associated with this high dose.

When comparing these results with the study on the radioprotective effect of the drug ON-01210 reported at the 51st Radiation Research Society (April 2004), it can be found that the drug ON-01210 (which is currently Similar to other drugs studied for radiation exposure) the protective effects were only present if administered before radiation exposure. This makes the drug not very useful in protection against "dirty bombs". The drug has a sulfhydryl component (4-carboxystyrl-4-chlorobenzylsulfone) that acts as an antioxidant to scavenge free radicals produced when cells are damaged by radiation. But if the drug isn't in your body at the time of radiation exposure, it won't work anyway. In contrast, treatment with the peptides of the invention may work after exposure.

Likewise, reviewing treatment with other existing agents, all data on these agents (i.e., on treatment with anabolic steroids) indicate that existing non-peptidic agents need to be administered prior to WBI (i.e., 24 hours) Administration has no supportive effect on protection against acute radiation injury.

Example 8: DRF Study

In this example, we report a study of dose-escalating whole-body irradiation (WBI), with a single exposure for each group followed by escalating doses for each group. A single dose of peptide AQGV was administered subcutaneously, but treatment was delayed until 3 days (72 hr) 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 which causes death in 50% of the tested animals.

An acceptable DRF ratio is a coefficient of at least 1.10; but preferably at least 1.20 or even at least 1.25. To pass the DRF1.20 test, a drug candidate must result in an LD50 radiation dose at least 20% higher than the LD50 dose in control animals (an increase by a factor of 1.20) at day 30 after WBI. For example, if the LD50 of the control animals is 8.2Gy, then in order to pass the test, the drug candidate should result in an LD50 that is at least 20% higher, ie in this case the dose should be 8.2x1.20=10.4Gy.

See Table 12 for the number and results of the test animals in the DRF test.

Table 12

Dose (Gy) "n" Absolute number of animals surviving 30 days after WB1 30 day survival percentage 30-day percent mortality 7.4 50 45 90% 10% 8 100 60 60% 40% 8.6 120 twenty four 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 rationale for the decision to delay treatment for 72 hours: in some cases of radioactive exposure (such as exposure to a nuclear fission device on a transport ship, or an aircraft hitting a nuclear reactor near a central city, etc.), the destructive power of So large that it may take days to get all the injured to a treatment center. Thus military scientists (who are concerned with protecting first responders) and civilian scientists (who are concerned with treating mass casualties) will naturally need to determine whether a candidate drug can in any way abrogate acute radiation toxicity (GI syndrome, myeloid syndrome ), otherwise it would burst after such a long delay.

AQGV's DFR test results. The radiation dose to kill 50% of the control animals was -8.2 Gy. Peptide AQGV was so protective that the radiation dose had to be increased by 25% (by a factor of 1.25) until ~10.4Gy was reached to kill 50% of the animals, which was obtained with a 3-day treatment delay result. If the treatment could be administered sooner, eg at 24hr or 48hr, the radiation to kill 50% of the animals would be higher.

Example 9

To further investigate the anti-cell cycle activity of different oligopeptides, proliferation experiments were performed in Arabidopsis seedlings. The aim was to test the effect of a panel of 140 oligopeptides of different lengths on the expression of plant marker genes during rapid growth of rapidly dividing cells. Both marker genes are involved in cell cycle progression, with high marker activity indicating high cell cycle activity and no marker activity indicating no cell cycle activity and thus no proliferation. An example of the role of oligopeptides in Arabidopsis cell cycle analysis is shown in Figure 3.

method

Peptides were resuspended in 1x phosphate buffered saline (PBS) pH 8 at a final concentration of 5 mg/ml. The resulting solution was dispensed into 96-well round bottom plates (Coming Incorporated) at 40 microliters per well. Plates were stored at -20°C for 4 days until use. Seeds of Arabidopsis ecovarian 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 supplemented with 80 mg/l kanamycin.

Plates were left overnight at 4°C and then transferred to a climate chamber at 210°C with a 16/8 hour photoperiod. After 4 days of growth, the seedlings were transferred to 96-well plates (4 seedlings per well) containing the peptide solution and incubated for 4 to 8 hours.

For this experiment, Arabidopsis homozygous seedlings carrying the two reporter genes fused to GUS were used. The first reporter gene used was a cell cycle marker, pCDG (Carmona et al., The Plant Journal, 1999, 20(4), 503-508), and the second was an auxin response marker, DR5:: GUS (Ulmasov et al., The Plant Cell, Vol. 9, 1963-1971). 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 ₃ Fe(CN) ₆ and 0.5mM K ₄ Fe( CN) ₆ ) at 37°C for 16 hours. To terminate the GUS reaction and remove chlorophyll, the seedlings were subsequently treated with 96% ethanol for 1 h and then stored in 70% ethanol. The stained seedlings were observed under a stereomicroscope, and sections were prepared from seedlings showing compound-treated effects. Seedlings were fixed and cleaned in chloral hydrate solution for careful observation under a microscope and photographed under a microscope equipped with DIC optics.

result

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

A total of 43 of the 140 compounds tested showed a clear effect on the expression of the two markers tested. Figure 3 details examples of apparent changes caused by the test compounds, changes at the microscopic level caused by the peptides LQGV, VVC and DINGFLPAL. Unexpectedly, this effect was clearly related to the length of the different tested peptides. It can be seen from Table 13 that the anti-cell cycle activity is extremely strong in the short peptides, and none of the peptides longer than 9 amino acids can reduce the cell cycle activity. About 22% of peptides 5 to 9 amino acids in length showed a reduction, but more than 50% of the 3-mers and 4-mers tested showed reduced cell cycle activity.

Table 13: Frequency distribution of positive test peptides/peptide lengths found in Arabidopsis cell cycle assays. #AA = length of peptide (amino acids); # = number tested; #+ = number of positives; %+ = percent positive

Example 10

In order to further study the anti-cell cycle activity of different oligopeptides, mouse peripheral blood cells stimulated with anti-CD3 antibody were used for in vitro experiments. The purpose is to test the effect of some representative oligopeptides on cell proliferation during the rapid growth of mouse monocytes induced by CD3 when the cells undergo rapid division. Mice (n=5) were intraperitoneally injected with PBS, Nak4(LQGV), Nak47(LAGV), Nak46(AQGV) (Ansynth BV, provided by The Netherlands), or Nak46 ^* (Diosynth BV, AQGV provided by The Netherlands). Mice were treated with 0.5 mg/kg or 5 mg/kg of the peptide for 1 hour, and then the spleen was isolated to prepare a splenocyte suspension. Splenocyte suspensions from each group were pooled and cultured in vitro in the presence of PBS or anti-CD3 antibody (triplicate), and proliferation was measured at 0, 12, 24 and 48 hours after culture. All tested peptides were shown to reduce proliferation (see Figure 4).

Results of Examples 9 and 10

Based on cell cycle studies in plants and in vitro studies of reduced proliferation of peripheral blood cells, 3-meric peptides identified as useful in the treatment of radiation injury are VVC, LAG, AQG. Similarly, 4-mer peptides useful in the treatment of radiation damage are 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, CPVQ, EVVR, MTEV, EALE, EPPE, LGTL, VGGI, RLPG, LQGA, LCFL, 5-mer peptides that can be used to treat radiation damage It is TLAVE, VEGNL, LNEAL, the 6-mer peptides that can be used to treat radiation damage are VLPALP, MGGTWA, LTCDDP, the 7-mer peptides that can be used to treat radiation damage are VLPAPLQ, VCNYRDV, CPRGVNP, and the 8-mer peptides that can be used to treat radiation damage The peptide is QPLAPLVG, and a 9-mer peptide useful in the treatment of radiation damage is DINGFLPAL.

references

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 and molecules. Scand. J. Immunol. 2005 Jul; 62 Suppl1: 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.

Alfthan H., U.H. Stenman. Pathophysiological importance of various molecular forms of human choriogonadotropin. Mol. Cell Endocrinol. 1996; 125: 107-120.

Kardana A., M.M. Elliott, M.A. Gawinowicz, S. Birken, L.A. Cole. The heterogeneity of human chorionic gonadotropin (hCG). I. Characterization of peptide heterogeneity in 13 individual preparations of hCG. Endocrinology 1991;129-1550.1

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 chorionic gonadotropin (hCG). III. The occurrence and biological and immunological activities of nick hCG. Endocrinology 1991;129:1559-1567

Birken S., Y. Maydelman, M.A. Gawinowicz. Preparation and analysis of the common urinary forms of human chorionic gonadotropin. Methods 2000; 21: 3-14

Claims (6)

1. Use of a peptide selected from the group consisting of LQGV, LAGV and AQGV for the preparation of a pharmaceutical composition for the treatment of a subject suffering from or considered to be suffering from radiation damage, the radiation damage being gastric Bowel syndrome. 2. Use according to claim 1, wherein all amino acids of the peptide are L-amino acids. 3. The use according to claim 1 or 2, wherein the peptide is AQGV. 4. The use according to claim 1 or 2, wherein said treatment comprises subcutaneous or intramuscular injection of said peptide. 5. The use according to claim 1 or 2, wherein the pharmaceutical composition is placed in a self-administered syringe. 6. The use according to claim 1 or 2, wherein said treatment comprises administering said peptide at least 30 minutes after irradiation.