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

Abstract

The invention relates to the field of drug development against acute radiation injury caused by exposure to high-energy electromagnetic waves (X-rays, gamma rays) or particles (alpha particles, beta particles, neutrons). To date, there is no effective drug to ameliorate radiation injury after accidental exposure to ionizing irradiation. The invention provides a method of treating radiation injury of a subject in need thereof comprising administering to the subject a peptide, or functional analogue or derivative thereof, of smaller than 30 amino acids. Furthermore, the invention provides use of a peptide, or functional analogue or derivative thereof, of smaller than 30 amino acids for the production of a pharmaceutical composition for the treatment of a subject suffering from or believed to be suffering from radiation injury. In particular, the invention provides anti-radiation peptides having a dose reduction factor (DRF) against acute gamma irradiation of at least 1.10, said DRF determinable by testing which dose of radiation results in 50% mortality at 30 days (LD50/30) after whole body radiation (WBI) in a test group of mice treated with said peptide at 72 hours after WBI and, testing which dose of radiation results in 50% mortality at 30 days (LD50/30) after whole body radiation (WBI) in a control group of mice treated only with the vehicle of said peptide at 72 hours after WBI and wherein the DRF is calculated by dividing the LD5O/3O of the peptide-treated animals by the LD50/30 of the vehicle-treated animals.

Description

Peptide is controlling the purposes in radiation damage

Technical field

The present invention relates to for because being exposed to High energy electromagnetic ripple (X-ray, gamma ray) or particle (alpha particle, beta particle, neutron) and the Field of Drug Discovery of the acute radiation injury caused.There is no effective medicine so far and can alleviate the radiation damage of accidental exposure after ionizing irradiation.

Background technology

Radiation damage is the tissue injury being exposed to radiation and causing.At this, radiation refers to the ionizing radiation produced by High energy electromagnetic ripple (X-ray, gamma ray) or particle (alpha particle, beta particle, neutron).This type of radiation is discharged by radioactive substance (radiosiotope) such as uranium, radon and plutonium.This type of radiation also can be produced by artificial radiation source, such as X-ray and radiotherapy instrument.The measurement of radiation dose uses multiple different unit, but they all relate to the amount of energy deposition.These units comprise roentgen (R), gray(Gy) (gray, Gy) and sievert (Sv) (sievert, Sv).Sievert (Sv) and gray(Gy) similar, difference is that sievert (Sv) take into account the biological effect of different emission types.The radioactive exposure of two kinds of main Types is irradiated and pollutes.Many radiation accidents make people be exposed to this two type simultaneously.

Irradiation is exposed to from the external radiated wave directly penetrating body.Irradiation can cause a disease (acute radiation disease) immediately.In addition, irradiation, particularly high dose radiation, can damage the hereditary material (DNA) of people, cause chronic (Delayed onset) disease, such as cancer and birth defect.But, irradiation can not cause people or its tissue to have radioactivity.Pollution contacts and is detained radioactive substance, the form of the latter dust or liquid typically.Radioactive substance can be stayed on skin, and can drop from skin or wipe, and pollutes other people and object.These materials also can be absorbed by body by lung, digestive tract or skin injury place.The material absorbed is transported to each position of body, such as bone marrow, continues release of radiation at this place.The radiation of this internalization not only can cause acute radiation disease such as internal hemorrhage, and can produce chronic disease as cancer.

People are exposed to low-level natural radiation (background radiation) for a long time.Radiation comes from outer space (cosmic radiation), but its major part is stopped by earth atmosphere.In high radioactivity element condition, the people of life or work is exposed to more cosmic radiation, particularly radon gas, and it is also present in many rocks and minerals.These elements finally appear in various material, comprise food and construction material.In addition, people are also exposed to the radiation from artificial radiation source, comprise the environmental radiation from nuclear weapon test and the radiation from various medical inspection and treatment.Ordinary people is subject to the radiation from natural radiation and artificial radiation source that total amount is about 3 to 4mSv (1mSv=1/1000Sv) every year.The people contacting radioactive substance and x-ray source in work is easily exposed to higher levels of radiation.The people accepting cancer radiation therapy can be subject to the radiation of high level.Nuclear weapon discharge a large amount of radiation.Since 1945, also this type of weapon had not been used to the mankind.But, present many countries have nuclear weapon, and some gang of terrorist are also reaching out for nuclear weapon, which increase the probability that this type of weapon is used again some day.

The damage effect of radiation depends on many factors, comprises amount (dosage) and the persistent period of exposure.Can be lethal to the radiation of single dose fast of whole body, same accumulated dose exposes then effect more than several weeks or several months can be obviously light.For a given dosage, expose fast and more easily cause hereditism to damage.The effect of radiation also depends on the exposure range of body.Such as, if be scattered in whole body more than the radiation of 6Gy, usually death is caused; If but only concentrated on a little region, such as, as cancer radiation, then could use the radiation of 3 to 4 times of these dosage and serious injury is not had to object entirety.The distribution of radiation is also important, because some part of body is more responsive to radiation.Organ and tissue such as intestinal and bone marrow that cell is bred rapidly, the easier infringement being subject to radiation of Organ and tissue such as MT of propagation slower in cell.The hereditary material of sperm and ovum can by radiation destruction.Therefore, in cancer radiation process, these more fragile positions of body to be covered as far as possible and make it to avoid radiation, the radiation of high dose mainly can be applied to cancer.

Radioactive exposure produces two kinds of damages: acute (at once) and chronic (Delayed onset).Acute radiation injury causes vascular leakage by vascular endothelial injury and causes inflammation.Follow by blood vessel response and cell response.Ionizing radiation immunosuppressant also damages gut epithelium, and both all promote that microorganism is shifted from intestinal.

The position that cancer radiation mainly accepts radiation at body produces symptom.Such as, in the radiotherapy of rectal cancer, because radiation is on the impact of small intestinal, abdominal colic is very common with diarrhoea.

Soon just start after World War II to find atoxic radioprotectant, resist radiation damage to protect normal structure.Deep radiobiological studies has found multiple protective agent; if used before radioactive exposure; they watch for animals (mainly rodent) avoids radiation damage [PrasadKN.Handbook of radiobiology.2nd edn.Boca Raton; FL:CRC Press, 1995].These researchs find those scavenging free radicals and/or cause the medicament of hypoxia to have radiation protection to be worth.But, the great majority of these compounds are poisonous at radiation protection dosage to people.Along with the decline of the danger of the nuclear confrontation occurred in cold war process, afterwards the research interest of radioprotectant was significantly declined.Due to the diagnostic instruments based on X-ray increase rapidly and radiologic method is applied in increase in disease early diagnosis, start more to close the somatic cell that day by day increases of frequency of heart and genetic mutation, it can make the danger of current people and the chain disease of offspring's producer raise.Therefore, normal structure must be protected to make it to avoid potential radiation damage, even this damage is very small.

Usually, radioprotectant is defined as such compound, described compound used the damage effect that can reduce radiation before being exposed to ionizing radiation, comprise radiation-induced death [H.B.Stone etal., Models for evaluating agents intended for the prophylaxis, mitigation andtreatment of radiation injuries.Report of an NCI Workshop, December3-4,2003, Radiat Res162:711-728.].They can be used for radiology terrorism, military event, Clinical Oncology, space travel, radiant removes [R.H.Johnson, Dealing with the terror ofnuclear terrorism, Health Phys87:S3-7., F.A.J.Mettler, G.L.Voelz, Majorradiation 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 managementof the acute radiation syndrome:Recommendations of the Strategic NationalStockpile Radiation Working Group, Ann Intern Med140:1037-1051.].Recently, the new radioprotector of exploitation is classified as override research project by American science technical policy office (U.S.Office of Science and Technology Policy) and Department of Homeland Security (Homeland Security Council).Although radioprotector such as the amineothiot class (aminothiols) of synthesis can produce the highest protecting factor, they are larger than naturally occurring protective agent toxicity usually.Usually, best radioprotectant is also considered to cause the highest toxicity.

In military radiation event, effectively alleviate the injures and deaths load that radiation-induced health problem and fighting capacity decline effect can reduce medical science treatment place, more effective action edge is kept after radioactive exposure event, allow commanding officer instruct action at radiation ground environment and be subject to the danger reduction that acute tissue injury causes ability to act to decline, and reduce the negative psychological wound of the personnel that those execute the task in contaminated environment.Desirable radioprotectant should be nontoxic, can not reduce ability to work, and i.e. onset after single administration, particularly when needs enter fast there is external radiation deathtrap particularly like this.

Report (Landauer et al. in NATO HumanFactors and Medicine Panel Research Task Group099 " the Radiation Bioeffects andCountermeasures " conference held at Maryland, USA Bethesda on June 21st, 2005, NATO RTG-0992005) and be published in one section of article of AFRRI CD05-2, proposing genistein (genisteine) can protect mice to avoid the death of gamma radiation induction, and it is at optimal dose (200mg/kg; Within 24 hours, use this dosage at pre-irradiation and can obtain the highest survival rate) time " dose reduction factor " (dose reduction factor, DRF) be 1.16.If within 1 hour, used before total irradiation (WBI), do not observe radiate protective action.In the 51st radiation research association (in April, 2004); other research reports code name is the Study On The Radioprotective of the medicine of ON-01210, points out that this medicine ON-01210 (similar with the other drug for radioactive exposure studied at present) only to use and just has protective effect before radioactive exposure.This medicine has sulfydryl composition (4-carboxystyrl-4-chlorobenzylsulfone), can be used as antioxidant and works, and the free radical scavenging produced is fallen during radiation damage cell.

Equally, as U.S. Department of Defense's conference annual report described in (March2005; Http:// medchembio.amedd.army.mil/docs/CBDP_Report_To_Congress.p df), also do not have at present to be commercially suitable for nontoxic medicine in military operation environment or diagnosis capability.Amifostine (amifostine) is a kind of amineothiot compounds, by the patient of FDA approval for accepting chemotherapy or radiotherapy, but its toxicity untoward reaction reducing performance makes it cannot be used for suitable combat forces, and its intravenous administration approach needs health care professional.The hematopoietic cytokine of other drug as being used for the treatment of bone marrow injury can use the dosage outside mark by each doctor according to the concrete condition of different case, but the restriction of the regulation of this type of purposes makes it be difficult to use in a large amount of casualties processed in military operation.Antibiotic is usually used in the infectious sequela of therapeutic radiology damage, but has to pass through and suitably select effectively to treat exogenous and endogenous systemic infection and affect useful Intestinal Anaerobic Bacteria hardly simultaneously.In order to solve current selectable medical countermeasure this situation limited, Armed Forces RadiobiologyResearch Institute (AFRRI) is at research noval chemical compound 5-androstenediol (5-AED; Whitnall et al., Experimental Biology and Medicine226:625-627 (2001)).Equally, this compound was used and can be produced good radioprotectant effect in mouse model before irradiation is attacked.Before Gamma irradiation mice, 24 hours and 2 hours afterwards subcutaneous administration AED can improve survival rate.The dose reduction factor calculated from the probit survival curve used before WBI is 1.3.All protective effect is observed in inoculation subsequently or the male and female mice of not inoculating lethal dose Klebsiella Pneumoniae (Klebsiella pneumoniae).Other steroid multiple then do not observe protective effect: dehydroepiandros-sterone (DHEA), 5-androstene-3B, 7B, 17B-triol (AET), androstenedione or estradiol.But, in the past year, the further investigation carried out on non-human primate (NHP) model to prepare IND application confirms, far away from effective like that on mouse model when 5-AED uses as radioprotectant, but on NHP model, create good effect with when successive doses therapeutic administration soon after irradiation.

Acute radiation disease.Acute radiation disease comes across systemic exposure usually in the people of radiation.The progress of acute radiation disease has multiple stage, and starting as early symptom (prodrome), is asymptomatic stage (incubation period) subsequently.The amount of the radiation accepted according to people subsequently and occur different symptoms (Symptom Spectrum).Amount of radiation is larger, and symptom is more serious, and it is faster to advance to acute syndrome from early symptom.For specific radioactive exposure amount, the symptom between different people and time course are consistent.Doctor can judge the radioactive exposure situation of people according to the time course of symptom and character.According to the major organ systems got involved, acute radiation syndrome is divided into three groups by doctor, but has overlap between each group.

Hemopoietic syndrome be by radiation on the main portions of bone marrow, spleen and lymph node-generation hemocyte (hemopoietic)-impact cause.Appetite declines (anorexia), drowsiness, nausea and vomiting appears at 2 to 12 hours after being exposed to 2Gy or higher radiation.Within after exposure 24 to 36, these remissions, in one week or longer time, people can feel good.In this asymptomatic stage, the hematopoietic cell in bone marrow, spleen and lymph node starts to exhaust and does not upgrade, and causes leukocyte famine, lacks platelet subsequently, then after be erythrocyte.Lack leukocyte and can cause severe infections.Lacking platelet can cause unmanageable hemorrhage.Lack erythrocyte (anemia) cause weak, weak, pale and when physical exertion dyspnea.After 4 to 5 weeks, if people can survive, then hemocyte starts to regenerate, but people can feel weak weakly reaches the several months.

Gastro-intestinal tract syndrome is caused the effect that digestive tract is coated to cellular layer (cells lining the digestive tract) by radiation.Serious Nausea and vomiting and diarrhoea appear at 2 to 12 hours after being exposed to 4Gy or higher radiation.These symptoms can cause serious dehydration, but these remissions after 2 days.In 4 to 5 days subsequently, people can feel good, but is coated to cellular layer usually used as the digestive tract of protective barrier and comes off in dead disease.After this, again there is serious diarrhoea-normally bloody diarrhea, cause dehydration.From in gastral antibacterial intrusive body, cause severe infections.Also probably there is hemopoietic syndrome in the people accepting so strong radiation, causes hemorrhage and infect, and increasing its mortality risk.

The accumulated dose of radiation is more than there will be cerebrovascular (brain) syndrome during 20 to 30Gy.There is rapidly confusion, Nausea and vomiting, bloody diarrhea and shock in people.Blood pressure drops in a few hours, with tic and stupor.Cerebrovascular syndrome is considered to be all lethal.

The Chronic Effect of radiation.The Chronic Effect of radiation is caused by the damage of hereditary material in somatoblast.These changes can cause cell growth abnormity, such as cancer.Being subject to serious animal of irradiating, have been found that the damage of sexual cell causes defect offspring (birth defect).But, in the offspring of Japan Atomic bullet blast survivor, the deformity of irradiating and causing seldom is observed.Reason may be not enough to produce lower than the radioactive exposure of specific (the unknown) level cause the hereditary material of birth defect to change.

If people is accepting radiotherapy or accidental exposure is sick after radiation, then may be irradiation damage.There is no special test for diagnosing this disease, but some test can be used for detecting infection, low cytometry or organ dysfunction extremely.For determining the order of severity of radioactive exposure, doctor can measure lymphocyte (a kind of leukocyte) quantity of blood.The lymphocyte count exposing latter 48 hours is lower, then radioactive exposure is more serious.

Be different from irradiation, by the radioactive pollution of the Geiger counter measures human body of radiation can be detected.Swab from nose, throat and any wound also can be used for checking radioactivity.

The consequence of radiation damage depends on dosage, dose rates (exposing the how rapid of generation) and the distribution at body, also depends on the health status that people is original.In a word, the overwhelming majority is subject to dying from gastro-intestinal tract syndrome more than the people of the WBI of 6Gy.Because doctor is difficult to the accurate amount of radiation of learning that people is subject to, therefore they generally carry out judged result according to the symptom of people.Cerebrovascular syndrome causes death usually within a few hours extremely several days.Gastro-intestinal tract syndrome caused death at 3 to 10 days usually, but groups of people can viable for weeks.According to their total radiation, many people receiving suitable medical rescue do not die from hemopoietic syndrome; Those people got off of not surviving are normally dead after 8 to 50 days.

Irradiate and there is no emergency treatment at present, but doctor can monitor the various symptoms that people occurs closely, and treat when symptom occurs.Meanwhile, anything is not regrettably almost had to tackle the medical product being attacked the various acute and long term toxicity caused by nuclear attack or radiation at present.Need to remove radioactive substance immediately when running into pollution to be absorbed by body to prevent it.The skin of contaminated by radioactive substances should immediately with a large amount of soap and water or with for this purpose and the solution (if any) of design is cleaned.Little damaged wound should fully clean to remove all radioactive grains, can cause pain although clean.The hair polluted will be cut but not wipe off, and scraping can scratch mark make pollutant enter body.Till scouring will continue to the disappearance of Geiger enumerator display radioactivity.If people has engulfed radioactive substance, induced emesis.Some radioactive substances have special antidote, can prevent the material swallowed from being absorbed.This type of antidote of great majority only for being exposed to remarkable alpha-contamination people, such as, is large reactor accident or nuclear explosion.Potassium iodide can prevent thyroid from absorbing radioiodine and reduce the risk that thyroid carcinoma occurs.Other drug, such as diethylene-triamine pentaacetic acid (diethylene triaminepentaacetic acid, DTPA), ethylenediaminetetraacetic acid (ethylenediamine tetraacetic acid, EDTA) and penicillamine, can intravenous administration to remove some absorbed radioelement.

When there is no suspicious pollution, alleviate nausea and vomiting by using the medicine (antiemetic) of prevention of emesis; These medicines are the routine administrations of the patient carrying out radiotherapy.Dehydration can carry out vein fluid-supplement therapy.

Generation gastrointestinal tract or the syndromic people of hemopoietic should isolate so that they can not contact infection microorganism.Can carry out transfusing blood and injecting stimulating the somatomedin (as erythropoietin and colony stimulating factor) of hemocyte generation to reduce hemorrhage and to increase cytometry.If bone marrow is badly damaged, these somatomedin are invalid, sometimes need to carry out bone marrow transplantation, but success rate are lower.

Occur that gastro-intestinal tract syndrome person needs antiemetic, venous transfusion and tranquilizer.Some can eat light diet.Can administration of antibiotics if neomycin is to kill the antibacterial that may invade body in intestinal.Intravenous administration antibiotic and antifungal and antiviral drugs if desired.The object of cerebrovascular syndrome treatment makes patient comfort by alleviating pain, anxiety and dyspnea.Anti-tic medicine can be used.

What the Chronic Effect of the radiation that appearance radiotherapy causes or the people of disease accepted is symptomatic treatment.Diabrosis or ulcer can be carried out Surgery or reparation and be used hyperbaric oxygen with Promotive union.Radiation-induced leukemia is with chemotherapeutic treatment.Hemocyte supplements by transfusing blood.Infertility cannot reverse, but ovary and the abnormal sex hormone level reduction caused of testicular function are treated by hormone replacement.Researcher adopts cytokine, somatomedin and various other treatment prevent or reduce the approach of radiation-induced normal tissue injury in research and development at present.Have been found that amifostine or pilocarpine hydrochloride can reduce xerostomia (xerostomia) symptom of the incidence cancer patient accepting radiotherapy.

Enhance our knowledge in radiotherapy about the Clinical and experimental study of radiation to the acute of cell and Delayed onset effect, make can optimize now radiation treatment plan and adopt more accurate radiation mode of administration.But, because normal structure has similar reaction with cancerous tissue to radioactive exposure, the damage of radiation-induced normal tissue may therefore be there is after radiation period or radiotherapy complete.Research finds that NSAID class and prostaglandin have some signs of radiological protection really.This two classes medicine all can increase the survival of cell, but mechanism is completely different.Cyto-dynamics research finds, compares in early days with S with the cell of G1/G0 phase, and the mitotic phase (M) and the cell in G2 late period that are in cell cycle are usually responsive to radiation.In addition, radiation causes the mitotic delay of cell cycle.Therefore, in the restrictive cell cycle cell proportion of M and G2 phase chemical agent or strengthen the chemical agent that grows fast of cell and can be used in principle study its potential use as normal structure radioprotector in injection process.Have been found that NSAID passes through to cause cell cycle arrest to make cell trend towards quiescent condition (G0/G1) and play antitumaous effect.Identical mechanism of action is observed in the radioprotection of normal tissue.After being exposed to NSAID, the increase of arachidonic acid concentration also causes producing inducer of apoptosis ceramide.NSAID is the level of superoxide dismutase in elevate cellular also.Improve the survival of cell by the expression of change cytokine after heat shock protein is activated by NSAID.NSAID also may have the effect of antiproliferative effect by angiogenesis inhibitor mechanism.The evidence implicates NSAID that some In vivo study provide can protect normal structure from radiation damage.Prostaglandin uncomfortable ganglion cell's cycle, but they have multiple effect to the growth and differ entiation of cell.PGE2 mediate vascular generates, and increases the supply of oxygen and cell survival and the necessary nutrient of growth.Therefore, PGE2 can strengthen cell survival by suppressing proinflammatory cytokine as TNF-a and IL-1 β in sufficiently high plasma concentration.Therefore, what PGE2 played is the instrumentality of inflammation but not the effect of mediators.Perspective study prompting uses misoprostol (misoprostol), i.e. a kind of PGE1 analog before irradiating, in the untoward reaction that prevention is radiation-induced, have potential use.Illustrate NSAID class and the pharmacological understanding of prostaglandins at present, when preventive use, the untoward reaction of radiation normal tissue likely minimizes by they.

Except instantaneous T suppression cell cycle progression and kill except those cells that can breed, irradiate the homeostasis that also interference affects by the endogenous mediators (tissue responds to the body fluid components of radiation) of cell-cell communication.The change of mediators level is by promoting recover normal (such as by raising H type cell line specificity growth factor) or regulate radiation effect by increasing the weight of infringement.Rear a kind of pattern is by the report about irradiating rear eicosanoid (eicosanoid) level change and illustrate about using the report of the result of anti-inflammatory drug empirical treatment radiation damage.The forerunner of radiation, acute and Chronic Effect are with excessive generation Eicosanoids (prostaglandin, prostacyclin, thromboxane and leukotriene).The endogenous mediator of these inflammatory reactions can cause radioactive exposure after vasodilation, vasoconstriction, microvascular permeability increase, thrombosis and chemotaxis.Glucocorticoid suppresses eicosanoid to synthesize by interference phospholipase A2, and NSAID (non-steroidal anti-inflammatory drug) blocks prostaglandin/thromboxane synthesis by suppressing cyclo-oxygenase.Use the medicine belonging to this two class after irradiation based on experience and all contribute to alleviating the forerunner of radiation, acute and Chronic Effect in humans and animals.

The United States Patent (USP) 5,380,668 (Jan.10,1995) of Herron discloses variously has compound of the antigen-binding activity of hCG etc., by reference its full content is incorporated to the application at this.The wherein general diagnostic uses disclosing described oligopeptide.Multiple patent of Gallo etc. and patent application (as United States Patent (USP) 5,677,275 (corresponding to WO96/04008A1), United States Patent (USP) 5,877,148 (also corresponding to WO96/04008A1), WO97/49721A1, United States Patent (USP) 6,319,504 (corresponding to WO97/49373), U.S. Patent application 2003/0049273A1 (also corresponding to WO97/49373), United States Patent (USP) 5,968,513 (corresponding to WO97/49418), United States Patent (USP) 5,997,871 (corresponding to WO97/49432), United States Patent (USP) 6,620,416, United States Patent (USP) 6,596,688, WO01/11048A2, WO01/10907A2. with United States Patent (USP) 6,583,109) relate to different oligopeptide and uses thereof, as " suppression HIV ", " treatment or pre-preventing HIV infection ", " treatment or prophylaxis of cancer ", " treatment or prevention are characterised in that the disease that somatic cell amount declines ", " treatment or prevention generate relevant disease with pathologic vessels ", " treatment or prevention hematopoietic defect ", " ex vivo gene therapy ", amplification in vitro hemocyte " and/or " hemocyte is supplied to patient ".As PCT International Publication text No.WO03/029292A2 (publication date: April10, 2003), PCT International Publication text No.WO01/72831A2 (publication date: October4, 2001) and U.S. 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, applying date October13, 2005, international application No.PCT/EP2005/003707, applying date April8, 2005, U.S. Patent application No.10/821, 256, applying date April8, 2004, U.S. Patent application No.10/262, 522, applying date September30, 2002, international application No.PCT/NL01/00259 (International Publication text No.WO01/72831A2), applying date March3, 2001, United States Patent (USP) 6, 844, 315 and United States Patent (USP) 6, 921, described in 751, compositions containing some oligopeptide described herein has immunoregulatory activity, can owing to such as treating septicemia and other diseases state or disease, by reference the content of above-mentioned all documents is incorporated to the application.

The present invention relates to the natural route that body regulates important physiological processes, and based on the understanding in PCT International Publication text WO99/59617 and WO01/72831 and PCT International Application Serial No. PCT/NL02/00639, by reference the full content of above-mentioned document is incorporated to the application at this.These applications disclose and be present in gene-regulating peptide in gravid woman's body, that produced as the proteolytic cleavage of hCG by placental gonadotropin.The length of these cleavage products is generally only 2 to 6 aminoacid, has been found that they have superior immunocompetence, and described activity realizes by regulating the gene expression of encoding inflammatory mediators as cytokine.Unexpectedly, find that the fracture of hCG creates a series of peptide contributing to the immunology homeostasis maintaining gravid woman.These peptide balanced immune system are to guarantee that parent keeps immunology to stablize, and its fetus can not be ostracised in the pregnancy period but be bred safely until be born simultaneously.

In addition, the present invention relates to U. S. application 10/821,240, which provide for other little gene-regulating peptides of screening and identification and use the result of screening according to this type of such as from the method for the peptide with reference to peptide.Such as, peptide to be analyzed is from c reactive protein (CRP) (as Human C-reactiveprotein), and this type of peptide comprises LTSL, FVLS, NMWD, LCFL, MWDF, FSYA, FWVD, AFTV and WDFV; From the peptide of beta-catenin (as people 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; From the peptide of β-hCG (as people CG), such as GLLLLLLLS, MGGTWA, TWAS, TLAVE, RVLQ, VCNYRDV, FESI, RLPG, PRGV, NPVVS, YAVALS, LTCDDP, EMFQ, PVVS, VSYA, GVLP, FQGL and AVAL; From the peptide of Bu Ludun type tyrosine kinase (Bruton ' styrosine kinase) (as people BTK), such as 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 the peptide of MMP-2 (as people MM02), such as FKGA, FFGL, GIAQ, LGCL, YWIY, AWNA, ARGA, PFRF, APSP, CLLS, GLPQ, TFWP, AYYL, FWPE, CLLG, FLWC, RIIG, WSDV, PIIK, GLPP, RALC, LNTF, LSHA, ATFW, PSPI, AHEF, WRTV, FVLK, VQYL, KFFG, FPFR, IYSA and FDGI, etc.

Summary of the invention

The present invention relates to for because being exposed to High energy electromagnetic ripple (X-ray/photon and/or natural gamma ray) and/or other high energy ionizing particles (alpha particle, beta particle, neutron, proton, π-meson) and the Field of Drug Discovery of the acute radiation injury caused.There is no so far effective medicine can unexpected be exposed to ionizing irradiation after, or in therapeutic radiation process or radioactivity simulant cause normal tissue injury after alleviate radiation damage; Effective preventive medicine is not had (such as, the first respondent) to use prevent this type of damage or make it to minimize before the event yet.The present inventor observes relatively little non-toxic peptide unexpectedly can be used as antiradiation injury medicine effectively.Importantly, radioprotective peptide of the present invention not only can be used as preventive, also has protective effect when a few hours use after being exposed to radiation.This makes them be particularly suitable for military radiation event, such as, for tackling the terrorist activity of nuclear terrorism.Therefore, the invention provides the method for radiation damage for preventing or treat object in need, comprise using to described object and be less than 30 amino acid whose peptides or its functional homologue.Preferably, use described peptide or its functional homologue, namely after described object is exposed to radiation source to after irradiation described object.In addition, the invention provides and be less than 30 amino acid whose peptides or its functional homologue in the purposes suffering from radiation damage for the preparation for the treatment of or in the pharmaceutical composition of the object of radiation damage of being thought suffering from.Particularly, the invention provides radioprotective peptide, its dose reduction factor (DRF) irradiated for acute systemic is at least 1.10, described DRF can determine as follows: the total irradiation (WBI) testing which kind of dosage causes test group experiment rodent (as mice) to occur the mortality rate (LD50/30) of 50% for 30 days after WBI, described test group processes with described peptide immediately or in maximum 72 hours after WBI, compared with the WBI dosage causing untreated control group to occur the mortality rate (LD50/30) of 50% with after WBI 30 days, wherein by the LD50/30 radiation dose of peptide processed group animal is calculated described DRF divided by the LD50/30 radiation dose of vehicle treated treated animal.

The invention provides treatment and suffer from radiation damage or the method for the object of radiation damage of being thought suffering from, the method comprises provides pharmaceutical composition to described object, and described pharmaceutical composition comprises and is less than 30 amino acid whose radioprotective peptides.Current radiation protective is non-peptide or comprises large protein as cytokine.The invention discloses can be used for protect and treatment radiation damage be less than 30 amino acid whose peptides, such as MTRVLQGVLPALPQVVC.This is the damage effect that Late Cambrian uses that after being exposed to radiation peptide medicament can reduce radiation.Such as, described radioprotective peptide by the most 29, at the most 28, at the most 27, at the most 26, at the most 25, at the most 24, at the most 23, at the most 22, at the most 21, at the most 20, at the most 19, at the most 18, at the most 17, at the most 16 or at the most 15 aminoacid form.

But, described peptide is preferably less than 15 aminoacid.Such as, described radioprotective peptide preferably by the most 14, at the most 13, at the most 12, at the most 11, at the most 10, at the most 9 or at the most 8 aminoacid form.The example of some useful peptides is LPGCPRGVNPVVS, DINGFLPAL and QPLAPLVG.But, if peptide is for self treatment, such as use autoinjector (autoinjector) described herein carries out self treatment, then for security consideration, preferably described peptide is less than 7 aminoacid.Such peptide in conjunction with MHC receptor, can not thus reduce the autoimmune risk occurring to be caused by the immunne response for used peptide usually.

To be less than 7 aminoacid (aa) be particularly preferred Another reason is, find that size is 7 amino acid whose peptides (when comparing the peptide from the proteasome of people and the peptide from pathogen (particularly virus or antibacterial) proteasome (Burroughs et al., Immunogenetics, 2004,56:311-320)) self and non-self between only have 3% overlap.For 6 amino acid whose peptides, overlapping between mankind itself is non-self with pathogen is confirmed as 30%, for 5 amino acid whose peptides, overlapping between the peptide existed in human protein enzyme body with the peptide existed in pathogen protein enzyme body is confirmed as 90%, and for 4 aminoacid and less peptide, this overlap is confirmed as 100%.Based on these data, recognize now when do not exist self-non-self difference time, the dangerous ground of adverse immune response such as anaphylactic shock reduces, and this is useful for not used for any medicine to oneself or other people by the people of medical training.

Therefore, with regard to prevention untoward reaction as with regard to anaphylactic shock, preferably described peptide is most preferably made up of 3 or 4 aminoacid by 2 to 6 aminoacid, more preferably by 3 to 5 aminoacid.With regard to active, based on general viewpoint, as long as can stand proteolysis completely longlyer, then the larger activity of peptide is just more remarkable, and 3 amino acid whose metabolism fragments still have activity thus, and preferably, described peptide is made up of 4 aminoacid.Above and hereafter described compositions is preferably used for treating acute radiation injury.

Prior art has been mentioned and has been used peptide to protect radiation damage.Japanese patent application JP09157291 and JP09157292 discloses 6 concrete aggressiveness and 9 mer peptide sequence, and it has the effect of vitro inhibition active oxygen, scavenging capacity oxygen-derived free radicals and antioxidant activity.These peptides are considered to can be used for suppressing in body to form the relevant untoward reaction comprising the various events of radiation damage to active oxygen.But it does not carry out internal radiation experiment.

JP09176187 teaches the 6 mer peptides analog containing histidine, and it has the activity of Scavenger of ROS.The peptide that 660mg/kg body weight is used in pre-irradiation 20 minutes abdominal cavities makes the survival rate of mice rise to 70% of processed group by 10% of matched group.But it is tested after not carrying out the irradiation in body.

WO2006/032269 discloses a kind of hemocyte homogenate, has therefrom eliminated the composition of molecular weight more than 3kDa.It is reported that this homogenate is suitable for improving the cellullar immunologic response of object.In many different amynologic diseases and pathologic condition, think this homogenate can in the treatment using chemotherapy and/or radiotherapy the preventative patient of being applied to improve the general status of patient.But this research does not relate to any radiation experiments.In addition, although this homogenate may comprise the mixture of protein, the character of active component is unclear, and active component also may right and wrong albuminous.In any case, the document is not separated or identifies any concrete peptide.

EP0572688 discloses the concrete peptide comprising 14 amino acid residues, and this peptide can protect mice to resist total irradiation in 20mg/kg body weight.The protective effect of this peptide is observed when only within 1 hour, using before irradiation.Within latter 1 hour, use this peptide and then do not observe it and matched group data have difference being exposed to irradiate.

Content and the present invention disclosed in these prior aries form significant contrast; Radioprotective peptide of the present invention even after total irradiation a few hours use and still have protective effect.

Benefit the anti-inflammatory property of some little peptides that the object accepting sub-lethal radiation dose can have been identified from the present invention, but unexpectedly, the anti-gastrointestinal tract syndrome that the overwhelming majority benefits from these little peptides is active, particularly from 3 aggressiveness when being more than more than more than 1mg/kg body weight, preferably 5mg/kg body weight, more preferably 10mg/kg body weight when dosage and 4 mer peptides.Consider the reduced immunogenicity feature of little peptide (i.e. 3 to 4 amino acid whose peptides), little peptide dosage can up to 100mg/kg, and consider in some cases and need the situation of object for the treatment of to need emergency treatment, its dosage can up to 200ng/kg, 500mg/kg or even 1g/kg.Therefore, can there is to those object comprising the radiation damage of (lining) infringement of intestinal coating and so-called gastro-intestinal tract syndrome now to treat; Described peptide can make epithelium coating slowly recover.

In high radiation dose situation, there is better activity in order to make peptide, peptide is preferably selected to be placed in pharmaceutical composition of the present invention or autoinjector of the present invention, the dose reduction factor for acute gamma irradiation (DRF) that described peptide has is at least 1.10, described DRF can determine as follows: the radiation testing which kind of dosage causes test group mice to occur the mortality rate (LD50/30) of 50% for latter 30 days in total irradiation (WBI), described test group mice processes with described peptide for 72 hours after WBI, control group mice within latter 30 days, is caused to occur the mortality rate (LD50/30) of 50% with the radiation of which kind of dosage of test in total irradiation (WBI), described control group mice only processes with the carrier of described peptide for 72 hours after WBI, wherein by the LD50/30 of peptide processed group animal is calculated described DRF divided by the LD50/30 of vehicle treated treated animal.

More preferably, the dose reduction factor (DRF) that the peptide used has is at least 1.20, is more preferably at least 1.25, is that the situation of irradiation damage is especially true for described radiation damage.The peptide that the present invention identifies is also referred to as radioprotective peptide.The invention provides the method and pharmaceutical composition that are used for the treatment of irradiation damage, described radiation be sent as uranium, radon and plutonium by radioactive substance (radiosiotope) or by artificial radiation source such as X-ray and radiotherapy instrument produce.

The present invention also provides and is less than 30 amino acid whose peptides in the purposes suffering from radiation damage for the preparation for the treatment of or in the pharmaceutical composition of the object of radiation damage of being thought suffering from.As mentioned above, described peptide is preferably less than 15 aminoacid, and cures for oneself or used by layman, and more preferably, described peptide is less than 7 aminoacid.Some 3 mer peptides that can be used to for the preparation of the pharmaceutical composition for the treatment of radiation damage identified at this are VVC, LAG and AQG.

Similarly, some 4 mer peptides that can be used for treating 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, 5 mer peptides that can be used for treating radiation damage are TLAVE, VEGNL, and LNEAL, 6 mer peptides that can be used for treating radiation damage are VLPALP, MGGTWA, LTCDDP, 7 mer peptides that can be used for treating radiation damage are VLPAPLQ, VCNYRDV, and CPRGVNP, 8 mer peptides that can be used for treating radiation damage are QPLAPLVG, and 9 mer peptides that can be used for treating radiation damage are DINGFLPAL.

Other peptide, particularly 3 or 4 mer peptides is found, such as, by adopting plant growth analysis method described herein by the anti-cell cycle-active of test peptides in proliferation assay.There is provided the peptide be made up of 2 to 6 aminoacid in the purposes suffering from radiation damage for the preparation for the treatment of or in the pharmaceutical composition of the object of radiation damage of being thought suffering from especially at this.Equally, with regard to prevention untoward reaction as with regard to anaphylactic shock, the peptide for the preparation of described pharmaceutical composition is preferably most preferably made up of 3 or 4 aminoacid by 2 to 6 aminoacid, more preferably by 3 to 5 aminoacid.If only with regard to active, based on general viewpoint, as long as (after using) then the larger activity of peptide is just more remarkable can to stand proteolysis completely longlyer, 3 amino acid whose metabolism fragments still have activity thus, preferably, described peptide is made up of 4 aminoacid.

In addition, useful especiallyly be, those need the object for the treatment of radiation damage can be treated by simply subcutaneous or intramuscular injection now, make it possible to thus carry out Heal Thyself with autoinjector, or treated by unbred or non-medical personnel, thus thousands of people need treat in emergency circumstances make rescue organization work very convenient.And if only have intravenous injection or same dangerous intraperitoneal injection to be only effectively, so relative to there is the situation of simple mean for applying as autoinjector provided by the invention, need the object for the treatment of more seldom will arrive relief.

Particularly, the present invention also provides and is less than the purposes of 30 amino acid whose peptides in the pharmaceutical composition for the preparation for the treatment of radiation damage, and wherein said pharmaceutical composition is placed in autoinjector.Autoinjector is a kind of medical treatment device, for using the specific drugs (typically first aid medicine) of single dose, sometimes also referred to as precharging type syringe, injects for oneself injection or by non-medical personnel or layman.In this application, term ＂ autoinjector ＂ does not refer in analytical system, as Husek et al. (J.of Chromatography B:Biomedical Sciences & Applications, Elsevier, Amsterda, Vol.767, no.1, (2002) pg.169-174) described in chromatogram arrangement in for automatization apply biological sample (such as peptide) syringe.

By design, autoinjector is easy to use and can be used to oneself by patient or be used to patient by layman.Injection site is typically in thigh or buttocks, and wherein said treatment comprises peptide described in subcutaneous or intramuscular injection.Because autoinjector can be designed to the medicine automatically and reliably using required dosage, therefore they contribute to quick, easy and drug administration exactly.Specifically, autoinjector is well suited for be used to the object of oneself administering therapeutic material or the medical personnel that must implement injection (situation as emergency treatment) to multiple object within a short period of time by those by those.In addition, the autoinjector with acupuncture injection mechanism (needled injection mechanism) can be designed, so as before injection operation, period, even after all can't see syringe needle, reduce thus or eliminate any anxiety relevant when thrusting object tissue with visible syringe needle.Although the concrete size heterogeneity of acupuncture autoinjector is very large, but they generally include body or housing, acupuncture syringe or similar device and one or more driving mechanism, the latter is used for syringe needle to thrust the tissue of object and the liquid medicine of syringe needle conveying required dosage by thrusting.Driving mechanism in existing acupuncture autoinjector generally includes can provide the energy source of power for driving mechanism.This energy source can be such as mechanical (i.e. spring pressurization formula), pneumatic type, electromechanical or chemistry, see United States Patent (USP) 6,149,626,6,099,504,5,957,897,5,695,472,5,665,071,5,567160,5,527,287,5,354,286,5,300,030,5,102,393,5,092,843,4,894,054,4,678,461 and 3,797,489, by reference the content of above-mentioned each patent is incorporated to the application.International Publication text WO01/17593, WO98/00188, WO95/29720, WO95/31235 and WO94/13342 also disclose the various syringes comprising different driving mechanism.Most of autoinjector is (optionally spring pressurization formula) syringe.

Autoinjector of the present invention, the body particularly directly contacted with peptide or housing, preferably obtained by the material having a minimum affinity with peptide.This at utmost can reduce peptide and adheres to or be attached to autoinjector.Most suitable material is a polypropylene, particularly substantially pure polypropylene.

Autoinjector is designed to overcome with pin to hesitation sense during oneself drug administration at first.The example of this type of autoinjector is Epipen or the Twinject introduced recently , the latter is generally used for the people with anaphylactic reaction danger.Another example of autoinjector is for using interferon beta to treat the Rebiject of multiple sclerosis .Be usually used in ground force during autoinjector and resist chemical operation agent with protection personnel.In AUS, in each biological or chemical weapons reaction bag, all there is autoinjector.This reaction bag is distributed to the soldier of each possibility in the face of biological or chemical weapons.Once start, pin can be injected people automatically, stings any medicated clothing (or even multilamellar medicated clothing) on the person.Autoinjector of the present invention not only comprises above-mentioned injection device (normally spring driven) automatically to carry out skin penetrating and/or drug injection, also comprises precharging type syringe or personal formula injection cartridge case etc.

The invention provides this type of autoinjector that can be used for treating (putting) radiation damage, and no matter radiation is sent by radioactive substance (radiosiotope) such as uranium, radon and plutonium, or produced by artificial radiation source such as X-ray and radiotherapy instrument.The present invention also provides the autoinjector of the pharmaceutical composition of protection, and described pharmaceutical composition is by being less than 30 amino acid whose peptides (referred to here as radioprotective peptide) and suitable excipient forms.Excipient is known in the art, such as, see that (Sarfaraz K Niazi edits Handbook ofPharmaceutical Manufacturing Formulations; ISBN:0849317460, in this not the application by reference).

Excipient consist of such as water, propylene glycol, ethanol, sodium benzoate and benzoic acid as cushion and benzyl alcohol as antiseptic; Or be mannitol, human serum albumin, sodium acetate, acetic acid, sodium hydroxide and water for injection.Other comprise injection solution or suspension for the exemplary composition used by autoinjector parenteral, it contains, such as, the acceptable diluent of suitable non-toxic, parenteral or solvent, such as mannitol, 1,3-butanediol, water, ringer's solution, etc. sodium chloride solution or other suitable dispersants or wetting agent and suspensoid, comprise the monoglyceride of synthesis or diglyceride and fatty acid, comprise oleic acid.

In one embodiment, autoinjector comprises the radioprotective peptide (or its functional homologue) as active component, and when using after being exposed to radiation at object, it can reduce the untoward reaction of radiation.Preferably, if at least 30 minutes after irradiation, more preferably at least 1 hour, most preferably at least a few hours or even a couple of days (as 3 days) uses, described peptide can produce at least part of protective effect for radiation damage.This type of autoinjector, also referred to as ＂ first aid autoinjector ＂, represents it in burst purposes in emergency circumstances.

In one embodiment, the invention provides the autoinjector containing the sterile solution be packaged in syringe-like device, after startup, this syringe-like device can its whole 5mL content of automatic transport.Every mL contains 100mg, preferably 200mg radioprotective peptide and excipient, such as, comprise propylene glycol, ethanol, sodium benzoate and benzoic acid as cushion and the benzyl alcohol excipient as antiseptic.In a preferred embodiment, the autoinjector being used for the treatment of radiation damage carries and is less than 15 amino acid whose radioprotective peptides, is more preferably less than 7 aminoacid.

It is 3 to 4 amino acid whose peptides that the autoinjector being preferably used for the treatment of acute radiation injury carries length, preferably, the dose reduction factor (DRF) of the anti-acute gamma irradiation that peptide has is at least 1.10, described DRF can determine as follows: the radiation testing which kind of dosage causes test group mice to occur the mortality rate (LD50/30) of 50% for latter 30 days in total irradiation (WBI), described test group mice processes with described peptide for 72 hours after WBI, control group mice within latter 30 days, is caused to occur the mortality rate (LD50/30) of 50% with the radiation of which kind of dosage of test in total irradiation (WBI), described control group mice only processes with the carrier of described peptide for 72 hours after WBI, and wherein by the LD50/30 of peptide processed group animal is calculated DRF divided by the LD50/30 of control animals.

The dose reduction factor (DRF) that the peptide that preferred autoinjector carries has is at least 1.20, is more preferably at least 1.25.The anti-suitable peptide be placed in autoinjector also has those peptides plant with anti-cell cycle-active as determined in the present invention.The peptide being suitable for very much autoinjector of the present invention is 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 is also provided for the pharmaceutical composition for the treatment of the object suffered from radiation damage or be thought suffering from radiation damage, described pharmaceutical composition comprises: the radioprotective peptide of medicinal effective dose or its functional homologue, or as this pharmaceutical composition identified and medicinal acceptable diluent.The present invention is provided in the method for the treatment of or pre-antiradiation injury in the object needing or have potential demand at this, described method comprises: to object drug administration compositions, described compositions comprises the means for the treatment of or pre-antiradiation injury and medicinal acceptable excipient, wherein said means are included in this radioprotective peptide identified or pharmaceutical composition, particularly wherein said radiation damage and comprise irradiation damage.

In one embodiment, the invention provides the method being used for the treatment of the object suffering from radiation damage, it comprises uses a kind of compositions to object, and described compositions comprises oligopeptide, described oligopeptide obtain from or derived from peptide MTRVLQGVLPALPQVVC or peptide LPGCPRGVNPVVS.Preferably, described oligopeptide is selected from the group be made up of MTR, MTRV, LQG, LQGV, VLPALP, VLPALPQ, QVVC, VVC, AQG, AQGV, LAG, LAGV and combination in any thereof.In another embodiment, preferably, described oligopeptide is selected from LPGC, CPRGVNP and PGCP.When described radiation damage comprises irradiation damage, this type of oligopeptide is particularly useful.The present invention is also provided for the pharmaceutical composition for the treatment of radiation damage, it comprises oligopeptide, described oligopeptide obtain from or derived from peptide MTRVLQGVLPALPQVVC or peptide LPGCPRGVNPVVS, such as oligopeptide is selected from by MTR, MTRV, LQG, LQGV, VLPALP, VLPALPQ, QVVC, VVC, AQG, AQGV, LAG, LAGV, LPGC, CPRGVNP and PGCP and combination in any thereof, and provides the purposes of described (widow) peptide in the pharmaceutical composition for the preparation for the treatment of radiation damage.

6 mer oligopeptide (VLPALP) that previously we had reported the beta chain of derived from human chorionic-gonadotropin hormone suppress septic shock in mice.Equally, we find, have similar anti-inflammatory activity derived from some other small peptide (from trimer peptide) of the β chain link 2 (residue 41-57) of hCG and some trims of described peptide of obtaining by carrying out single amino acids replacement with alanine.And then we create and filter out some to continue to research and develop the reasoning being used for the treatment of the acutely inflamed therapeutic compound of accidental exposure after ionizing irradiation in these peptides.

Human chorionic gonadotropin (hCG) is a kind of heterodimer Placenta Hominis glycoprotein hormones that anemia of pregnant woman produces.There is various ways in it, comprise cleavage product in the urine of anemia of pregnant woman He in commercial hCG preparation.In During Pregnancy, have effect due to estimating it to prevention fetus allograft refection, therefore some researcheres have studied heterodimer hCG and variant thereof to immune impact.The anti-immunity moderation system of hormone that some report promptings are complete, but this type of effect of cleavage product was not also in the news.Previously our (Khan et al., Hum.Immunol.2002Jan; 63 (1): 1-7) 6 mer oligopeptide (VLPALP) reporting the beta chain of derived from human chorionic-gonadotropin hormone suppress septic shock in mice.Carry out with this six peptides the septic shock that single therapy inhibits mice to after the lipopolysaccharide of injected in mice high dose (LPS).Benner and Khan (Scand.J.Immunol.2005Jul; The immunologic competence that the fragments of peptides that 62Suppl1:62-6) have studied release in body may have, described fragments of peptides is produced by the fracture of the sequence MTRVLQGVLPALPQVVC (residue 41-57) of hCG beta subunit ring 2.---and some peptides being replaced by alanine and derive from it---show significant anti-inflammatory activity (measuring by suppressing the septic shock syndrome of mice) to report at this some 3 to 7 amino acid whose peptides taking from beta subunit ring 2, and exceeded the activity being considered to can be used for treating radiation damage, particularly comprising the radiation damage of gastro-intestinal tract syndrome, have also exceeded and be considered to can be used for for the preparation for the treatment of radiation damage, the activity particularly comprising the pharmaceutical composition of the radiation damage of gastro-intestinal tract syndrome.

The present invention also provides the pharmaceutical composition with anti-cell cycle-active.Cell cycle is one group of orderly event, consequently Growth of Cells be split into two sister cells.The stage of cell cycle is G1-S-G2-M.The G1 phase represents ＂ GAP1 ＂.The S phase represents ＂ and synthesizes (Synthesis) ＂.This is the stage that DNA replication dna occurs.The G2 phase represents ＂ GAP2 ＂.The M phase represents ＂ mitosis (mitosis) ＂, is nucleus (chromatin separation) and Cytoplasm (cytokinesis (cytokinesis)) to occur when dividing.Term " anti-cell cycle-active " referred to herein as described peptide and can change cell cycle kinetics.Such as, it comprises change, namely improves or reduces, fissional frequency.In one embodiment, it refers to antiproliferative activity.

Additionally provide the pharmaceutical composition with anti-cell cycle-active, it comprises PGCP; Have the pharmaceutical composition of anti-cell cycle-active, it comprises VGQL; Have the pharmaceutical composition of anti-cell cycle-active, it comprises RVLQ; Have the pharmaceutical composition of anti-cell cycle-active, it comprises EMFQ; Have the pharmaceutical composition of anti-cell cycle-active, it comprises AVAL; Have the pharmaceutical composition of anti-cell cycle-active, it comprises FVLS; Have the pharmaceutical composition of anti-cell cycle-active, it comprises NMWD; Have the pharmaceutical composition of anti-cell cycle-active, it comprises LCFL; Have the pharmaceutical composition of anti-cell cycle-active, it comprises FSYA; Have the pharmaceutical composition of anti-cell cycle-active, it comprises FWVD; Have the pharmaceutical composition of anti-cell cycle-active, it comprises AFTV; Have the pharmaceutical composition of anti-cell cycle-active, it comprises LGTL; Have the pharmaceutical composition of anti-cell cycle-active, it comprises QLLG; Have the pharmaceutical composition of anti-cell cycle-active, it comprises YAIT; Have the pharmaceutical composition of anti-cell cycle-active, it comprises APSL; Have the pharmaceutical composition of anti-cell cycle-active, it comprises ITTL; Have the pharmaceutical composition of anti-cell cycle-active, it comprises QALG; Have the pharmaceutical composition of anti-cell cycle-active, it comprises GVLC; Have the pharmaceutical composition of anti-cell cycle-active, it comprises NLIN; Have the pharmaceutical composition of anti-cell cycle-active, it comprises SPIE; Have the pharmaceutical composition of anti-cell cycle-active, it comprises LNTI; Have the pharmaceutical composition of anti-cell cycle-active, it comprises LHNL; Have the pharmaceutical composition of anti-cell cycle-active, it comprises CPVQ; Have the pharmaceutical composition of anti-cell cycle-active, it comprises EVVR; Have the pharmaceutical composition of anti-cell cycle-active, it comprises MTEV; Have the pharmaceutical composition of anti-cell cycle-active, it comprises EALE; Have the pharmaceutical composition of anti-cell cycle-active, it comprises EPPE; Have the pharmaceutical composition of anti-cell cycle-active, it comprises LGTL; Have the pharmaceutical composition of anti-cell cycle-active, it comprises VGGI; Have the pharmaceutical composition of anti-cell cycle-active, it comprises RLPG; Have the pharmaceutical composition of anti-cell cycle-active, it comprises LQGA; Have the pharmaceutical composition of anti-cell cycle-active, it comprises LCFL; Have the pharmaceutical composition of anti-cell cycle-active, it comprises TLAVE; Have the pharmaceutical composition of anti-cell cycle-active, it comprises VEGNL; Have the pharmaceutical composition of anti-cell cycle-active, it comprises LNEAL; Have the pharmaceutical composition of anti-cell cycle-active, it comprises MGGTWA; Have the pharmaceutical composition of anti-cell cycle-active, it comprises LTCDDP; Have the pharmaceutical composition of anti-cell cycle-active, it comprises VCNYRDV; Have the pharmaceutical composition of anti-cell cycle-active, it comprises CPRGVNP; With the pharmaceutical composition with anti-cell cycle-active, it comprises DINGFLPAL.

Accompanying drawing explanation

Fig. 1: the mice of the total irradiation processed with AQGV (Ala-Gln-Gly-Val)

＂ WBI ＂ represents total irradiation.Use the C57B1/6 mice of anesthesia, at the later evaluation of WBI (6.5 to 9.8Gy, Philips MG30,81cGy/min) to the Protective effect of radiation damage, the difference of being survived by Kaplan-Meirer analysis to measure.All first the mice of all groups injects peptide or carrier (control animals) in 3 hours after WBI.The group mortality rate of injection placebo is 80%, consistent with to the prediction of this model.The radiation dose of known 8.6 gray(Gy)s (=8.6Gy) can cause the mortality rate of about 80% at these species, be therefore called LD80 (80% fatal dose).Dead beginning at about the 10th day occurs---this was common situation at the WBI of animal or human: at about the 10th day, intestinal coating damages because of radiation and leakage causes antibacterial to enter circulating and cause gastro-intestinal tract syndrome, and the damage of bone marrow causes cannot producing enough leukocyte to resist infection (" bone marrow syndrome "), then there is death.The group that symbol ＂ x ＂ represents accepts intravenous injection first, injects second time subcutaneous injection (SC) after 3 hours first.These animals 100% survive.Do not show them in figure and in fact do not occur any signs of disease at all.For unwitting observer, they look the same with completely normal mice.The group of triangle representative injects peptide first by SC approach, then within every 48 hours, carries out extra SC injection, totally 3 doses (except first dose)---namely at the 3rd, 5 and 7 day.A death is only had in these animals.The group of block symbol representative continues to carry out outside totally 6 doses (except first doses) except 48 hours SC inject, and other are identical with the program of triangle group.Therefore its administration continues to the 13rd day.The treatment of this prolongation brings protective effect completely (none is dead for this group).Any signs of disease is there is not in this treated animal.From these data, we may safely draw the conclusion, if animal accepts the peptide (first dose is intravenous injection) of two multiple doses at first day, can produce protective effect completely to the WBI of fatal dose.If animal accepts the treatment (only SC) of reduced levels, then treatment is extended to second week and also produce protective effect completely.

Fig. 2: the second group of radiation protection experiment carried out with peptide AQGV

Adopt the total irradiation (WBI) of Titration, be single exposure for each group, and group reconditioning subsequently raises gradually.Subcutaneous administration single dose Ala-Gln-Gly-Val (AQGV), but after treatment delay to WBI, 3 days (72hr) carries out.This test is called dose reduction factor (" DRF "), and it is defined as the ratio between the LD50 of processed group and the LD50 of matched group.LD50 representative be the dosage of the test animal death causing 50%.Acceptable DRF value is 1.20.For by this test, after WBI the 30th day, a kind of drug candidate must make LD50 radiation dose than the LD50 dosage height at least 20% (coefficient is the rising of 1.20) of control animal.Such as, if the LD50 of control animal is 8.2Gy, so the LD50 that causes of drug candidate should height at least 20%, and namely this dosage should be 8.2x1.20=10.4Gy in this case.

Fig. 3: oligopeptide acts in arabidopsis (Arabidopsis thaliana) cell cycle analysis.Compound N AK4 (LQGV) and NAK9 (VVC) demonstrates clear and definite impact to the note thing that mapping is tried.For Cell Cycle Markers (pCDG), all observing to have root at two time points clearly affects.The impact of time and/or dose dependent is observed in transition region and cotyledon.For auxin respond flag thing (DR5::GUS), observed the situation identical with Cell Cycle Markers.NAK26 (DINGFLPAL) demonstrates not too consistent time dependence impact.Only observed impact in time at root.Impact is not observed in transition region and cotyledon.

Fig. 4: test the effect of on cell proliferation when rapidly division occurs cell in the Mus mononuclear cell fast growth process of being induced by CD3 of representative oligopeptide.Mice (n=5) lumbar injection PBS, Nak4 (LQGV), Nak47 (LAGV), Nak46 (AQGV) (Ansynth BV, The Netherlands provides) or Nak46* (AQGV that Diosynth BV, The Netherlands provides).With the peptide process mice 1 hour of 0.5mg/kg or 5mg/kg, separating spleen prepares splenocyte suspension subsequently.Collect each group splenocyte suspension and under the condition that there is PBS or anti-cd 3 antibodies In vitro culture (three parts), after incubation 0,12,24 and 48 hour measure propagation.

Detailed Description Of The Invention

In this application, " purification, synthesis or be separated " peptide is to have originated the peptide be purified into from its natural origin or biotechnology, or more preferably, is the peptide synthesized as described herein.

In this application, " compositions " refers to containing oligopeptide or the multiple compounds that is made up of oligopeptide.Preferably, oligopeptide is separated joins in compositions afterwards again.Preferably, oligopeptide is made up of 2 to 6 aminoacid, more preferably, is made up of 3 to 4 aminoacid.

Such as, in one embodiment, preferred compound can be: NT A Q G V CT, and wherein the NT of N-terminal is selected from H--, CH3--, acyl group or general blocking group; And the CT of C-terminal is selected from little peptide (as 1 to 5 aminoacid),--OH,--OR ¹,--NH ₂,--NHR ¹,--NR ¹r ², or--N (CH ₂) _1-6nR ¹r ², wherein R ¹and R ², if present, independently selected from H, alkyl, aryl, (virtue) alkyl, and wherein R ¹and R ²can be connected to each other ring formation.

In this application, " alkyl " is preferably the hydrocarbon of saturated side chain or straight chain, has 1 to 6 carbon atoms, such as methyl, ethyl and isopentyl.

In this application, " aryl " is aryl, preferably has 6 to 10 carbon atoms, such as phenyl or naphthyl.

In this application, " (virtue) alkyl " is aryl (simultaneously having aliphatic portion and aryl moieties), preferably has 7 to 13 carbon atoms, such as benzyl, Ethylbenzyl, n-pro-pyl benzyl and isobutylbenzyl.

In this application, " oligopeptide " be linked together by peptide bond there are 2 to 12 amino acid whose peptides.The equivalent of oligopeptide is the compound with identical with the specific amino acids in oligopeptide or equivalent side chain, and it puts in order identical with the order of described peptide, but linked together by non-peptide bond, such as, by waiting row to connect (isosteric linkages) as ketone isostere, hydroxyl isostere, diketone isostere or ketone-difluoromethyl isostere.

" compositions " also comprises the acceptable salt of such as oligopeptide or the oligopeptide of labelling.In this application, " acceptable salt " refers to the salt of the required activity retaining oligopeptide or isovalent compounds, preferably or other components in the system of described oligopeptide will be used to produce harmful effect to oligopeptide.The example of this type of salt is the acid-addition salts formed by mineral acid example hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, nitric acid nitric acid etc.Salt also can with organic acid as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid etc. be formed.Salt can be with multivalent metal cation as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel etc. or with by N, the organic cation that N '-Dibenzylethylenediamine (N, N '-dibenzylethylenediamine) or ethylenediamine are formed or their combination (as tannic acid zinc) are formed.

Such pharmaceutical composition is by parenteral or by oral administration to object.Such pharmaceutical composition can be made up of oligopeptide and PBS substantially.Preferably, oligopeptide is synthesis.Suitable treatment such as needs by the oligopeptide intravenous administration in pharmaceutical composition in patient, and the amount used is about 0.1 to about 35mg/kg body weight.Pharmaceutical composition can be made up of one to three kind of different oligopeptide substantially.

The chemical entities of such generation can be used systemically, outwardly or partly and introduce in body.Peptide or its trim itself can be used or use as medicinal acceptable acid or base addition salts by its entity, and described acid or base addition salts are by reacting with mineral acid (example hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, Hydrogen thiocyanate, sulphuric acid and phosphoric acid) and formed; Or by reacting with organic acid (as formic acid, acetic acid, propanoic acid, hydroxyacetic acid, lactic acid, acetone acid, oxalic acid, malonic acid, succinic acid, maleic acid and fumaric acid) and formed; Or by reacting with inorganic base (as sodium hydroxide, ammonium hydroxide, potassium hydroxide) and formed; Or by reacting with organic base (ethanolamine as monoamine, diamidogen, triamine and arylamine and replacement) and formed.Selected peptide and any derivative entity also can be coupled to sugar, lipid, other polypeptide, nucleic acid and PNA; And work in position as conjugate or after reaching target tissue or organ local release work.

With regard to each seed amino acid, " replacement " is usually directed to replace with the group of such as alkoxyl, halogen, hydroxyl, nitrogen or low alkyl group the hydrogen that aromatic ring exists.Replace and also can carry out on the alkyl chain connecting aryl moieties and peptide main chain, such as, replace hydrogen with low alkyl group.Other replacement also can be carried out in amino acid whose α position, also uses alkyl.

Preferred replacement uses fluorine or chlorine as halogen and uses methoxyl group as alkoxyl.As for alkyl and low alkyl group, usually, the alkyl with less (1 to 3) carbon atom is preferred.

The conventional method preparation of these compounds of preparation can be adopted to meet the compound of general formula.For this reason, suitable N is held protected (and if when there is reactive side chain, side chain the is protected) amino acid analogue of α or peptide activation and in the solution or solid support is coupled to the protected aminoacid of suitable carboxyl or peptide derivant.The protection of alpha-amido functional group is carried out with urethane functional group usually; as used acidproof tertbutyloxycarbonyl (tertiary-butyloxycarbonyl group; " Boc "), benzyloxycarbonyl group (benzyloxycarbonyl; " Z ") and replace analog or alkaline-resisting 9-fluorenylmethyloxycarbonyl (9-fluoremyl-methyloxycarbonyl, " Fmoc ").Z group is also removed by catalytic hydrogenation.Other suitable protecting groups comprise Nps, Bmv, Bpoc, Aloc, MSC etc.The summary of amido protecting group is shown in The peptides, Analysis, Synthesis, Biology, Vol.3, E.Gross and J.Meienhofer, eds. (Academic Press, New York, 1981).Carboxy protective is undertaken by esterification, such as alkaline-resisting ester as methyl ester or ethyl ester, resistance to acid esters as the tert-butyl ester or replacement, benzene methyl or undertaken by hydrogenolysis.Aforementioned group can be used to carry out the protection of side chain functionalities as the side chain of lysine and glutamic acid or aspartic acid.The protection of sulfydryl (although and not always required), guanidine radicals, alcohol and imidazole group can adopt plurality of reagents to realize; for example, see The Peptides; Analysis; Synthesis; Biology; or see Pure and Applied Chemistry, 59 (3), 331-344 (1987) id..Activation is carried out to the carboxyl of suitable protected aminoacid or peptide and can use azide, mixed acid anhydride, active ester or Carbodiimide method; particularly adopt the compound of the suppression racemization effect adding catalytic as 1-N-N-hydroxybenzotriazole, N-hydroxy-succinamide, 3-hydroxyl-4-oxo-3; 4-dihydro-1; 2; 3;-phentriazine, N-hydroxyl-5 norborene-2,3-dicarboximide.Also the anhydride of phosphorous acid can be used.See, such as, The Peptides, Analysis, Synthesis, Biology, supra andPure and Applied Chemistry, 59 (3), 331-344 (1987).

Solid phase method also by Merrifield prepares described compound.Different solid supports and different strategies are known, such as, see Barany and Merrifield, The Peptides, Analysis, Synthesis, Biology, Vol.2, E.Gross and J.Meienhofer, eds. (Acad.Press, NewYork, 1980); Kneib-Cordonier and Mullen, Int.J.Peptide Protein Res., 30,705-739 (1987); With Fields and Noble, Int.J.Peptide Protein Res., 35,161-214 (1990).Those wherein peptide bond aforesaid blocking group and activation method usually can be adopted to synthesize by the compound that isostere replaces.The method of the isostere of synthetic modification can list of references, such as relevant--CH ₂--NH--isostere and--CO--CH ₂--the document of isostere.

According to the type being connected to the joint of solid support used in the character of blocking group and Solid-phase peptide synthesis, distinct methods can be adopted to remove blocking group and cracking on solid support.Usually protect with carrying out when there is scavenger in acid condition.Such as see volumes3,5and9of the series on The Peptides Analysis, Synthesis, Biology, supra.

Another kind of possible method uses this compounds of enzymatic synthesis.For example, see summary H.D.Jakubke in The Peptides, Analysis, Synthesis, Biology, Vol.9, S.Udenfriend andJ.Meienhofer, eds. (Acad.Press, New York, 1987).

Although may be not too suitable from environment, recombinant DNA method also can be adopted to prepare oligopeptide of the present invention.These class methods relate to the required oligopeptide of method preparation by expressing recombination of polynucleotide in suitable host microorganism, sequential coding one or more oligopeptide interested of described polynucleotide.The method is usually directed to the DNA sequence of one or more concrete oligopeptide of coding to import cloning vehicle (as plasmid, phage DNA or other DNA sequence that can copy in host cell), cloning vehicle is imported suitable eucaryon or prokaryotic host cell, and cultivates the host cell so transformed.If use eukaryotic host cell, compound can contain glycoprotein fraction.

In this application, " functional homologue " of peptide comprises aminoacid sequence or other sequence monomers, and its sequence is changed, and makes the functional characteristic of sequence substantially identical in nature, but need not be identical in amount.

The function of peptide or its functional homologue can adopt in body and/or testing in vitro is determined.Testing in vitro is preferred.In one embodiment, test is compared to functional peptide analog, wherein adopt reference or control peptide, the peptide analogues be such as only made up of L aminoacid.Suitable test comprises determines that candidate peptide affects the ability of cell cycle kinetics.Such as, the impact of plant model system determination cell cycle process can be adopted, such as, in the arabidopsis system that this exemplifies, or adopt (mammal) cell cultivated.It relates to the ability determining candidate peptide apoptosis inhibit on the other hand, such as, by induction (temporary) G2-M cell cycle arrest.

Analog can be produced in many ways, such as, by " conservative amino acid replacement ".In addition, can design peptide mimetic compound, they can functionally or original peptide structure is similar to as starting point, but such as be made up of alpha-non-natural amino acid or polyamide.By " conservative amino acid replacement ", a kind of amino acid residue is replaced by the residue of another kind of character cardinal principle similar (size, hydrophobicity), and general function can not be had a strong impact on substantially thus.But, generally more expect to improve concrete function.Also analog is produced by improving at least one desirable characteristics of aminoacid sequence comprehensively.This can such as be realized by Ala scanning (Ala-scan) and/or replacement web drawing (replacement net mapping) method.Adopt these methods, much different peptide can be produced based on original aminoacid sequence, but each replacement all containing at least one amino acid residue.Amino acid residue can be replaced (Ala-scanning) or replace (replacement web drawing) by any other amino acid residue by alanine.So just, many positional variants (positional variant) of original amino acid have been synthesized.Screen the specific activity of each positional variant.The data produced are for designing the peptide derivant of the improvement of specific amino acid sequence.

Also analog is produced by such as L-amino acid residue being replaced with D-amino acid residue.This replacement causes the peptide producing non-natural existence, can improve the characteristic of aminoacid sequence.The peptide sequence of whole known activity be made up of D aminoacid of reverse reversion (retro inversion) form such as can be provided, it can be kept thus active and increase the half-life.By producing many positional variants of original amino acid and screening its specific activity, the peptide derivant comprising the amino acid whose improvement of this type of D with the characteristic improved further can be designed.The peptide that prior art has been found that at one end or two ends D aminoacid is protected is more stable than the peptide be only made up of L aminoacid.The modification of other types comprises the modification that research and development peptide medicament known in the art makes it to have the useful effect that can be used for pharmaceutical composition.These effects can comprise the reduction that effect improves, pharmacokinetics changes, stability improves and cause Increased Plasma Half-life and require cold chain process stringency.

In an embodiment of the invention, radioprotective peptide comprises the aminoacid sequence that the peptide bond between and carboxyl amino by it links together, and wherein at least one aminoacid is D aminoacid.Such as, radioprotective peptide is selected from as next group: 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 amino acid residue represented by the using single letter code of standard is D aminoacid.

Those skilled in the art can produce the analog compounds of aminoacid sequence.This is undertaken by such as screening peptide library.This analog has the functional characteristic identical with former sequence substantially, but need not be identical in amount.In addition, peptide or analog can by cyclisation, such as, pass through to provide (end) cysteine to them; By dimerization or multimerization, such as, by being connected to lysine or cysteine or other, there is the compound allowing the side chain that connection or multimerization occur; Form series connection or repeating pattern; Coupling or be otherwise connected to carrier known in the art, as long as by reliably connecting of allowing to dissociate.The synthetic of these oligopeptide as above and functional homologue or cleavage product, can be used for treating in the method for radiation damage and follow-up disease.

In this application, " functional homologue " of peptide is preferably less than the peptide deriving it, therefore prepares preferably by disappearance and/or replacement, and its length of non-increasing.In addition, in this application, " functional homologue " of peptide does not relate to those and contains the aminoacid sequence being accredited as radioprotective peptide at this and simultaneously there is larger protein or the peptide of more amino acids in its one or both sides.

Term " pharmaceutical composition " is intended to contain active compound of the present invention itself and the compositions containing compositions of the present invention and medicinal acceptable carrier, diluent or excipient at this simultaneously.Pharmaceutical composition can contain the mixture of at least two kinds of radioprotective peptides described herein or analog.The acceptable diluent of oligopeptide described in detail at this is such as physiology's saline solution or phosphate buffered saline(PBS).In one embodiment, oligopeptide or compositions are applied to animal or human with valid density general, such as, used by vein, intramuscular or intraperitoneal.Another kind of route of administration comprises organ or tissue's perfusion, can in vivo or external, use the infusion liquid comprising oligopeptide of the present invention or compositions to carry out.Can use by single dose, discontinuous multiple dose is used, or continues one period being enough to allow abundant regulator gene to express.Use for continuing, the time continuing to use becomes with many factors, and this is those skilled in the art's easy understand.

The application dosage of bioactive molecule can have sizable scope.The dissolubility restriction of compound when the effect when concentration of the bioactive molecule that can use is subject to low concentration usually and higher concentration.Should and can be determined by relevant doctor or medical expert the optimal dose of concrete patient, its Chinese medicine considers the state of an illness, the body weight and age etc. of the correlative factor such as patient known.

Bioactive molecule directly can be used in suitable carrier, such as, solution in phosphate buffer (" PBS ") or ethanol or DMSO.But, according to the preferred embodiment of the present invention, delivery system is adopted to use bioactive molecule by single dose conveying.Suitable delivery system should be pharmacology's non-activity or at least can tolerate.It preferably neither has immunogenicity and also not to cause inflammation reaction, and the release of bioactive molecule should be made can to maintain its effect level in required time.The possibility being suitable for controlled release object is known in the art and belongs within scope of the present invention.Suitable delivery vehicles includes but not limited to: microcapsule or microsphere; Liposome and other delivery systmes based on lipid; Sticky droplet (viscous instillates); Absorbable rod and/or biodegradable mechanical barrier and implant; And polymer transport material, as polyoxyethylene/polyoxypropylene block copolymers, polyester, cross-linking polyvinyl alcohol, polyanhydride, polymethacrylates and polymethacryladmide hydrogels, anionic carbohydrate polymer etc.Available induction system is well known in the art.

A kind of preparation realizing bioactive molecule release comprises injection microcapsule or microsphere, it is obtained by biodegradable polymers, as poly-(dl-lactide), poly-(dl-lactide-co-glycolide), polycaprolactone, PGA, polylactic acid-altogether-Acetic acid, hydroxy-, bimol. cyclic ester, poly-(hydroxybutyric acid), polyester or acetal resin.Comprising diameter is that the microcapsule of about 50 microns to about 500 microns or the injection system of microsphere have advantage compared with other induction systems.Such as, they usually use less bioactive molecule and can be used by medical assistance personnel.In addition, by selecting the size of microcapsule or microsphere, explosive payload and application dosage, in this type systematic has in the design of the time that different pharmaceutical discharges and speed motility.In addition, its asepticize is made by Gamma irradiation.

The design of microcapsule and microsphere, preparation and use are well known in the art, about the concrete ins and outs of this respect can see document.Also preparation beyond microcapsule and microsphere can be prepared with biodegradable polymers (as lactide, Acetic acid, hydroxy-, bimol. cyclic ester and caprolactone polymers); As the prefabricated membrane containing bioactive molecule and coating film can be used for the present invention.Comprise the filter membrane of bioactive molecule or fiber also within the scope of the invention.

Another kind is highly suitable for single dose and carries the preparation of bioactive molecule of the present invention to be liposome.By bioactive molecule capsulation in liposome or multilamellar vesicle be targeted drug conveying and prolong drug retain know technology.It is that those skilled in the art are known and have a detailed description in the literature that the preparation of the liposome of medicine and use are housed.

Single dose carries the suitable mode of the another kind of bioactive molecule of the present invention to relate to sticky dripping.In the art, high molecular carrier and bioactive molecule used in combination, the structure obtained can produce has full-bodied solution.Suitable high molecular carrier includes but not limited to: glucosan and cyclodextrin; Hydrogel; (being cross-linked) cohesive material, comprises (being cross-linked) viscoelastic material; Carmellose; Hyaluronic acid; And chondroitin sulfate.Preparation and use that sticky of medicine is housed are that those skilled in the art are known.

According to another kind of mode, bioactive molecule can with Absorbable rod mechanical barrier as oxidized regenerated cellulose combined administration.Bioactive molecule can covalently or non-covalently (as passed through ionic bond) be incorporated into this barrier, or be only disperse thereon.

The present invention is explained further by following exemplary embodiment.

Embodiment

The selection of peptide

The upper known preferential cracking site of sequence MTRVLQGVLPALPQVVC (residue 41-57) based on hCG beta subunit ring 2 carries out selecting (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., Endocrinology1991; 129:1541-1550; Cole etal., Endocrinology1991; 129:1559-1567; S.Birken, Y.Maydelman, M.A.Gawinowicz, Methods2000; 21:3-14), and select on the aminoacid sequence from c reactive protein (CRP) (beta-catenin, as people CTNB), Bu Ludun type tyrosine kinase (as people BTK), MMP-2 and p-53.

The synthesis of peptide

Described herein peptide is prepared as solid support by solid-phase synthesis (Ansynth BV) commercialization using 2-chlorine trityl chloride resin (2-chlorotrityl chloride resin) based on the method for 9-fluorenylmethyloxycarbonyl (Fmoc)/tert-butyl group by proprietary method (Diosynth BV) or use.With the side chain of trityl protective group glutamine.These peptides are synthesized by hand.Each coupling comprises the following steps: (i) removes alpha-amino Fmoc with the piperidines in dimethyl formamide (DMF) and protect; (ii) by Fmoc aminoacid (3eq) and the coupling in DMF/N-methylformamide (NMP) of DIC (DIC)/I-hydroxybenzotriazole (HOBt), and (iii) adds cap to remaining amido functional group with acetic anhydride/diisopropylethylamine (DIEA) in DMF/NMP.Synthesize complete, with trifluoroacetic acid (TFA)/H ₂the mixture process peptide resin of O/ tri isopropyl silane (TIS) 95:2.5:2.5.After 30 minutes, add TIS until decolouring.Solution dewaters in a vacuum, with ether sedimentation peptide.Rough peptide is dissolved in water (50-100mg/ml) and passes through RPHPLC (reversed-phase high-performance liquid chromatography) (RP-HPLC) purification.HPLC condition: post: Vydac TP21810C18 (10x250mm); Eluent system: gradient system is that the water-soluble v/v of 0.1%TFA (A) and 0.1%TFA is dissolved in acetonitrile (ACN) v/v (B); Flow velocity 6ml/ minute; Absorption is measured at 190-370nm.Employ different gradient system.Such as peptide LQG and LQGV:10 minute 100%A, succeeded by linear gradient 0-10%B, 50 minutes.Such as peptide VLPALP and VLPALPQ:5 minute 5%B, succeeded by linear gradient 1%B/ minute.By rotating film evaporation at 40 DEG C of lower pressure, the component of collection is concentrated into about 5ml.By eluting twice on anion exchange resin (Merck II) post of acetate form, remaining TFA and acetate are exchanged.Concentrated eluate lyophilizing 28 hours.Subsequently peptide is dissolved in PBS, for subsequent use.

Embodiment 1 and embodiment 2

In first experiment, to single injection PBS (n=9) or peptide (LGQV, VLPALP, LPGCPRGVNPVVS, MTRVLQGVLPALPQVVC in 12-female BAl BIc/c mouse peritoneal in age in week; N=8,10mg/kg).Process latter 1 and a half hours mouse systemic and be exposed to single dose 10Gy ¹³⁷cs-gamma-irradiation.In second experiment, the first systemic exposure of 12-female BAl BIc/c mice in age in week is in single dose 10Gy ¹³⁷cs-gamma-irradiation, then 1.5 hours intraperitoneal single injection PBS (n=9) or peptide (n=8 or 9,10mg/kg) after irradiation.Mortality rate and clinical sign (e.g., eyes are shed tears and represented conjunctivitis, and lose weight) is observed in different time points in experimentation.As seen from Table 2, all test peptides all have the good effect alleviating conjunctivitis in processed group mice, and mortality rate is not affected, this impels us to remove to select the most applicable antagonism of one acutely inflamed peptide, to test it in the lower irradiation of the repeated doses of carrying out in the later stage.

Embodiment 3

6 kinds of oligopeptide (i.e. A:LAGV, B:AQGV, C:LAG are tested in double blinding zoopery, D:AQG, E:MTR, and F:MTRV) and compare with PBS (contrast), test the relative ability that each peptide promotes to recover in the test of mouse kidney ischemia-reperfusion.In experiment, by mouse anesthesia, excision side kidney.Opposite side kidney ligation 25 minutes, serum urea level raises.Use often kind of different peptide (5mg oligopeptide/kg body weight) to before and after ligation 30 different mouse veins, determine mortality rate and the BUN concentration of the mice of various peptide process subsequently 2 hours, 24 hours and 72 hours.The results are shown in Table for 3 (not comprising the result of the peptide A (LAGV (SEQ ID NO:4)) that embodiment 3 obtains).

Under inhalation anesthesia condition, isolate left kidney together with its artery and vein and block 25 minutes with micro-vessel clamps.In operation, animal is placed on heating cushion to keep body temperature at 37 DEG C.To place before vascular forceps 5 minutes and before unclamping vascular forceps 5 minutes, use the peptide of 5mg/kg to animals iv, be dissolved in the Sterile Saline of 0.1mL.Right kidney is excised after left kidney Reperfu-sion.By measuring before folder closes and the blood urea nitrogen evaluate renal function of 2,24 and 72 hours after Reperfu-sion.

Result-Biao 3 mortality rate of 72 hours (after the Reperfu-sion)

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 X 2 test.df＝1

Peptide A (SEQ ID NO:4) is the first peptide used in renal ischemia/reperfusion injury test.The personnel carrying out testing are being familiar with learning curve when using peptide A.Use in this peptide process at postcava, in injection site, some animals occur that moderate is lost blood, but other animals do not have.Send these animals back to cage unintentionally but for the first night after surgery not put drinking water in cage.In addition, these animals should be put to death at 72 hours, but within 48 hours after Reperfu-sion, were condemned to death mistakenly.For peptide B-F, in experimentation, there is not these or other problems.

Can find out, application of oligopeptide MTRV and the particularly animal of AQGV comparatively matched group (PBS) or to use the group of other oligopeptide far better in survival (obviously reducing relative to PBS matched group mortality rate) and BUN lowering of concentration, have more mouse survival and serum urea level is much lower compared with other groups.But, oligopeptide LAG, AQG and MTR do not reduce BUN concentration in this experiment, but significantly reduce mortality rate respectively compared with PBS matched group, and wherein MTR even raises in the 72 little BUN levels of test mice that make constantly.

Embodiment 4

For above-mentioned reason, retest the ability that a kind of oligopeptide (A) reduces mice BUN level.The results are shown in Table 4.Can find out, the mice accepting oligopeptide LAGV is all much better in survival (obviously reducing relative to PBS matched group mortality rate) and reduction BUN concentration relative to matched group (PBS).

Embodiment 5

The ability that other 4 kinds of oligopeptide (G (VLPALPQ), H (VLPALP), I (LQGV) and J (LQG)) reduce BUN level is tested in mouse experiment as above.The results are shown in Table 4.Can find out, the mice receiving oligopeptide LQG demonstrates BUN concentration in early days in experiment and reduces (after Reperfu-sion 24 hours), and the mice receiving VLPALPQ after the test the phase (after Reperfu-sion 72 hours) reduction BUN concentration aspect comparatively matched group (PBS) or other oligopeptide processed group far better, have more mouse survivals and serum urea level is much lower compared with other groups.

Table 4: the renal ischemic mice of 25 minutes is with the BUN after peptide A-J process

the P value that after Reperfu-sion, 2 hours statistical analysis obtain:

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

the P value that after Reperfu-sion, 24 hours statistical analysis obtain:

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

(SPSS for Windows) is checked to calculate P value by Mann Whitney U-.

Embodiment 6

In order to determine dose response relationship, two kinds of peptide (D (AQG are tested in mice renal failure experiment as above, embodiment 3 test mice in good result is revealed to ultimate mortality table) and B (AQGV, embodiment 3 test mice in BUN, there is superior result) dose response mode.Described peptide is tested as described in Example 3 with the dosage of 0.3,1,3,10 and 30mg/kg.Relative to the serum urea level of PBS, PEPD group folder close after 72 hours P value (by MannWhitney U-test (SPSS for Windows) calculating) be: 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.Can find out, relative to peptide B (AQGV), PEPD (AQG) is good unexpectedly in reduction BUN is horizontal at tested comparatively low dosage, and tested also be significant compared with low dosage to the beneficial effect of mortality rate.

Table 6: the urea level in dose response experiment
				24h	72h
PBS	57.8	85.4
			PEPD (AQG) 0.3mg/kg	38.4	30.4
PEPD (AQG) 1.0mg/kg	48.4	38.4
			PEPD (AQG) 3.0mg/kg	39.3	40.3
PEPD (AQG) 10.0mg/kg	46.8	25.8
			PEPD (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
			PEPD (AQG) 0.3mg/kg	8.6	3.5
PEPD (AQG) 1.0mg/kg	7.2	10.2
			PEPD (AQG) 3.0mg/kg	3.5	10.7
PEPD (AQG) 10.0mg/kg	8.0	3.4
			PEPD (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
The moon too 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

Septic shock experiment is set and resists acute inflammation which kind of to determine peptide is the most applicable.

For septicemia or septic shock experiment mice: 8-12 week age female BAl BIc/c mice be used for all experiments.According to Report of European Laboratory Animal ScienceAssociations (FELASA) Working group on Animal Health (Laboratory Animals28:1-24,1994) scheme, by the facility of animal feeding under specific pathogen free concrete conditions in the establishment of a specific crime.

Infusion protocol: for endotoxin model, to BALB/c mouse lumbar injection 150-300 μ g LPS (E.coli026:B6; Difco Lab., Detroit, MI, USA).Matched group is lumbar injection PBS only.In order to the effect of test peptides, peptide is dissolved in PBS and predetermined point of time lumbar injection after PBS process.

Adopt following measurement scheme to mouse disease severity scale:

0 is without exception

1 fur transudate, but the behavior difference that can detect is not had compared with normal mouse

2 fur transudates, placing reflex, responding to stimulation (as patted cage), equally showing active in touching process with healthy mice

3 pairs of beating cage delay of response, passive or docile during touching, but be in alone in new environment still curious

4 lack curious sense, and to stimulation, few or not response, seldom movable

5 breathe effort, motionless or after going over, slowly ajust (dying, put to death)

D is dead

The experiment of first group of septic shock is set to determine the septic shock which kind of peptide LQG, LQGV, VLPALP, VLPALPQ, MTR, MTRV, VVC or QVVC can suppress lipopolysaccharide (LPS) and induces in mice, latter 2 hours of LPS process with single dose peptide process mice.Use the peptide of 5mg/kg body weight to BALB/c mouse lumbar injection, LPS (E.coli026:B6; Difco Lab., Detroit, MI, USA) dosage be cumulative, estimate that LPS caused 80-100% dead at 24 to 72 hours.Matched group is lumbar injection PBS only, without dead.

The experiment of second group of septic shock is set with the septic shock determining which kind of peptide LQG, LQGV, VLPALP, VLPALPQ, MTR, MTRV, VVC or AQG, AQGV, LAG and LAGV can suppress high dose LPS induce in mice, LPS process afterwards 2 and 24 hours with the peptide process mice of doubling dosage.Throughout in reason group, use the peptide of 5mg/kg body weight.To BALB/c mouse lumbar injection high dose LPS (E.coli026:B6; Difco Lab., Detroit, MI, USA), estimate that LPS caused 80-100% dead at 24 to 72 hours.Matched group is lumbar injection PBS only, without dead.

Another group septic shock experiment is set to determine which kind of studied peptide LQG, LQGV, VLPALP, VLPALPQ, MTR, MTRV, VVC or AQGV is adapted at shock most and occurs to use in early stage and/or later stage or whole process.In order to determine the percent of the endotoxin shock survival rate of carrying out later stage or early time treatment with peptide, to BALB/c mouse lumbar injection 300 μ g LPS (E.coli026:B6; Difco Lab., Detroit, MI, USA), estimate without causing 100% death under peptide disposition at 48 hours.Matched group is lumbar injection PBS only, without dead.

Arranging comparative experiments to compare peptide MTR and AQGV, is all commercial source.Comparative experiments has 6 groups, often organize 6 animals, wherein two groups (1A and 1B) accepts placebo (PBS), one group (2) accept peptide MTR (source: Pepscan), one group (3) accept peptide MTR (source: Ansynth), one group (4) accept peptide AQGV and (originate: Pepscan), and another group accept AQGV (source: Ansynth).Peptide/placebo is used for 2 hours after LPS.LPS (source) dosage is 10-11mg/kg.Within 0,2,22,26,42 and 48 hour, disease score is carried out after injection LPS.

Result

The selection of peptide

We select synthetic peptide MTR, MTRV, LQG, LQGV, VLPALP and VLPALPQ, and QVVC and VVC.In the later stage of research, we also select to synthesize the peptide variant replaced derived from the alanine of LQG and LQGV, and be wherein alanine by an aminoacid replacement, to 4 wherein kind of (AQG, AQGV, LAG and LAGV), result is as follows.

Septic shock is tested

In order to test peptides, in shock, early stage effect occurs, within latter 2 hours or 24 hours, giving mouse peritoneal ejection testing peptide with the LPS process of various dose, dosage is 5mg/kg body weight.To the mice without peptide process, the dosage of LPS caused 100% death at 48-72 hour.The results are shown in Table 8.In 7 kinds of peptides of test, peptide VLPALP and LQGV demonstrates the protective effect of the septicemia of significant anti-LPS induction.

In shock, effect that the is early stage or later stage occurs to evaluate peptide process, within latter 2 hours or 24 hours, give mouse peritoneal ejection testing peptide in injection LPS process, dosage is 5mg/kg body weight.Observe 48 hours in mice 84 hours instead of earlier experiments.The results are shown in Table 10.In all peptides of test, AQGV is only had can latter 84 hour to keep 100% survival in LPS process when there is the early stage of shock or later stage administration and not leave over clinical sign.

Compare test MTR and AQGV, two kinds of peptides all have two kinds of sources, all use with 5mg/kg dosage.

Table 11

LPS＝10-11mg/kg

#970=5mg/kg LPs compound

#971=5mg/kg process processes

#Ansynth12

#Ansynth46 disease score

Table 11

LPS＝10-11mg/kg

#970=5mg/kg LPS compound

#971=5mg/kg process processes

#Ansynth12

#Ansynth46 disease score

The hCG immunity-regulating system that some report promptings are complete, but this type of of its cleavage product has no report in scientific and technical literature.Benner and Khan (Scand.J.Immunol.2005Jul; The immunocompetence that in the body that sequence MTRVLQGVLPALPQVVC (residue 41-57) cracking that 62Suppl1:62-6) have studied hCG beta subunit ring 2 produces, release peptide fragment may have, although the fact is small enough to 3 to 7 amino acid whose peptides be usually considered to do not have obvious biologic activity.

We devise the peptide of the septic shock that can to block LPS induction in mice completely, even within 24 hours after injection LPS, start to carry out processing with these peptides still effective in some cases.These peptides can also suppress the generation of MIF.These are found to be the therapeutic use using these peptides to treat the patient suffering from radiation damage and provide probability.

Embodiment 7

Present embodiment shows that the experimental result of peptide AQGV to the mice of 8.6Gy total irradiation (WBI), wherein the mice of all groups all accepts first time injection in 3 hours after TBI.The animal 80% accepting placebo injection group is dead, as estimating this model.The radiation dose of known used radiation (8.6 gray(Gy)s=8.6Gy) can cause the mortality rate of about 80% at these species, be therefore called LD80 (80% fatal dose).Dead beginning at about the 10th day occurs---this was common situation at the WBI of animal or human: at about the 10th day, intestinal coating damages because of radiation and leakage causes antibacterial enter circulation and cause septicemia because of gastro-intestinal tract syndrome, and the damage of bone marrow causes cannot producing enough leukocyte to resist infection (" bone marrow syndrome "), then there is death.

Represent first group of peptide process mice group acceptance intravenous injection AQGV first of (Fig. 1) with symbol ＂ x ＂, this accepts secondary subcutaneous injection (SC) after injecting 3 hours first.Unexpectedly, these animals 100% survive.In addition, there is not any signs of disease in these animals.For unwitting observer, they look the same with completely normal mice, and GI syndrome does not appear in these mices of particularly peptide process.

Second group of mice, by SC approach acceptance peptide injection first, then carries out extra SC injection for every 48 hours, totally 3 doses (except first dose)---namely at the 3rd, 5 and 7 day.Only having a death in these animals, there is not the syndromic symptom of any GI in other.

Except the SC injection of 48 hours continues to carry out outside totally 6 doses (except first doses), the mice of the 3rd group is identical with the program of second group.Therefore its administration continues to the 13rd day.The treatment of this prolongation brings protective effect completely (none is dead for this group).There is not any signs of disease in this treated animal, do not occur the syndromic symptom of any GI equally.

From these data, we may safely draw the conclusion; if animal accepts the peptide (first dose is intravenous injection) of two multiple doses at first day; AQGV can produce protective effect completely to the WBI of fatal dose, and particularly produces protective effect for the GI syndrome that this dosage is adjoint.If animal accepts the treatment (only SC) of reduced levels, then treatment is extended to second week and also produce protective effect completely, and particularly produce protective effect for the GI syndrome that this high dose is adjoint.

Time compared with these results are the research of the Study On The Radioprotective of the medicine of ON-01210 with at upper relevant code name report of the 51st radiation research association (in April, 2004), can find that this medicine ON-01210 (similar with the current other drug for radioactive exposure studied) only uses and just has protective effect before radioactive exposure.This makes this medicine not be very useful in the protection to " dirty bomb ".This medicine has sulfydryl composition (4-carboxystyrl-4-chlorobenzylsulfone), can be used as antioxidant and works, and the free radical scavenging produced is fallen during radiation damage cell.If but in the body at that time of radioactive exposure not this medicine, in any case so it does not have effect.On the contrary, use peptide of the present invention to treat and can work after exposure.

Equally, look back the treatment carried out with other existing medicines, all data (namely about using the treatment of anabolic steroids) about these medicines all show existing non-peptide medicament needs (namely 24 hours) before WBI and use, and use later and do not support effect to the protection of acute radiation injury.

Embodiment 8:DRF studies

In the present embodiment, we report the research of the total irradiation (WBI) to Titration, be single exposure, and each group of reconditioning subsequently raise gradually for each group.Subcutaneous administration single dose peptide AQGV, but after treatment delay to WBI, 3 days (72hr) carries out.This test is called dose reduction factor (" DRF "), and it is defined as the ratio between the LD50 of processed group and the LD50 of matched group.LD50 representative be the dosage of the test animal death causing 50%.

Acceptable DRF ratio is that this coefficient is at least 1.10; But be preferably at least 1.20 or or even at least 1.25.For the test by DRF1.20, after WBI the 30th day, a kind of drug candidate must make LD50 radiation dose than the LD50 dosage height at least 20% (coefficient is the rising of 1.20) of control animal.Such as, if the LD50 of control animal is 8.2Gy, so in order to by this test, the LD50 that drug candidate causes should height at least 20%, and namely this dosage should be 8.2x1.20=10.4Gy in this case.

DRF test in test animal quantity and the results are shown in Table 12.

Table 12

Dosage (Gy)	“n”	The absolute number of 30 days surviving animals after WB1	30 days percent survival	30 days percent mortality
					7.4	50	45	90％	10％
8	100	60	60％	40％
					8.6	120	24	20％	80％
9.2	30	0	0％	100％
					8.6+AQGV, the 3rd day	20	20	100％	0％
9.2+AQGV, the 3rd day	10	10	100％	0％
					9.8+AQGV, the 3rd day	10	10	100％	0％
10.4+AQGV, the 3rd day	10	4	40％	60％
					11.0+AQGV, the 3rd day	10	0	0％	100％

It is very important for discussing the reason for the treatment of delay this decision in 72 hours: when some radiation exposure (as be exposed to the nuclear trigger device of transport steamer, or aircraft impact is to nuclear reactor being close to key city etc.), its destructive power big may causing needs a couple of days all the wounded could be delivered to therapeutic community.Therefore military science man (it pays close attention to the initial responder of protection) and folk scientist (it pays close attention to a large amount of casualty for the treatment of) can need to determine whether a kind of drug candidate can eliminate toxicity (the GI syndrome of acute radiation by any way naturally; bone marrow syndrome), otherwise it breaks out suddenly afterwards by so long delay.

The DFR test result of AQGV.The radiation dose killing 50% control animals is ~ 8.2Gy.The protective effect of peptide AQGV is so good, to such an extent as to radiation dose must be improved 25% (coefficient is 1.25) until reach ~ 10.4Gy, just can kill the animal of 50%, and this is the result obtained when delay treatment in 3 days.If this treatment can be used sooner, such as, when 24hr or 48hr, the radiation of so killing the animal of 50% will be higher.

Embodiment 9

In order to study the anti-cell cycle-active of different oligopeptide further, in Arabidopsis thaliana Seedlings, carry out proliferation experiment.Object to test the impact of oligopeptide on the gene expression of plant label thing in the cell fast growth process that division rapidly occurs of one group of 140 kinds of different length.Two kinds of marker gene all relate to cell cycle progression, and protrude mark thing activity represents high cell cycle activity, and the representative of marker free activity does not have cell cycle activity, therefore also just do not breed.The example of the effect of oligopeptide in arabidopsis cell cycle analysis is shown in Fig. 3.

Method

Peptide is resuspended in 1x phosphate buffer (PBS) pH8, and final concentration is 5mg/ml.Gained solution is assigned to (Coming Incorporated) in 96-hole circle base plate, every hole 40 microlitre.Plate-20 DEG C deposit 4 days for subsequent use.The seed of arabidopsis ecotype Ws-0 is placed in 2% commercialization bleach (Glorix) and within 10 minutes, carries out surface sterilization, with aseptic MQ water washing 5 times.Then seed to be resuspended in the agar of 0.1% and to be layered on MS20 plate, wherein supplementing the kanamycin of 80mg/l.

Plate placed for two nights at 4 DEG C, was then transferred to climatic chamber 210 DEG C, periodicity of illumination 16/8 hour.Grow after 4 days, seedling is transferred to (every hole 4 strain seedling) in the 96-orifice plate containing peptide solution, hatches 4 to 8 hours.

In order to this experiment, employ the arabidopsis homozygote seedling of carrying the two kinds of reporter genes being blended in GUS.First reporter gene used is Cell Cycle Markers, pCDG (Carmona et al., The Plant Journal, 1999,20 (4), 503-508), second is auxin respond flag thing, DR5::GUS (Ulmasov et al., The Plant Cell, Vol.9,1963-1971).After compound incubation, for GUS, seedling is dyeed.Staining reaction 100mM sodium phosphate buffer (pH7.0) (containing 10mM EDTA, 10%DMSO, 0.1%Triton X-100,2mM X-Gluc, 0.5mM K ₃fe (CN) ₆with 0.5mM K ₄fe (CN) ₆) in carry out 16 hours in 37 DEG C.React to stop GUS and remove chlorophyll, seedling with 96% Ethanol Treatment 1 hour, is then deposited in 70% ethanol subsequently.The seedling of stereomicroscopy Microscopic observation dyeing, there is the seedling preparation section of compound treatment effect.Fix in chloral hydrate solution and clean seedling to examine under microscope, and taking pictures under the microscope being equipped with DIC eyeglass.

Result

Test the impact that peptide is expressed the Arabidopsis thaliana Seedlings marker gene of growth fast.The change distributed in Different Organs by GUS is monitored this: root, root-plumular axis transition region and cotyledon.

In tested 140 kinds of compounds, have 43 kinds and demonstrate and have clear and definite impact to the expression of two kinds of labels of test.Fig. 3 show in more detail the example of the significant change that test compounds causes, the change of the electronmicroscopic level that peptide LQGV, VVC and DINGFLPAL cause.Unexpectedly, this effect is obviously correlated with from the length of different test peptides.Can find out from table 13, anti-cell cycle-active shows extremely strong in small peptide, and length more than 9 amino acid whose peptides, none can reduce cell cycle activity.Length is in 5 to 9 amino acid whose peptides, and about 22% demonstrates reduction, but demonstrates reduction cell cycle activity more than 50% in 3 aggressiveness and 4 aggressiveness of test.

Table 13: the frequency distribution of the positive test peptides/peptide length found in arabidopsis cell period measuring.The length (aminoacid) of #AA=peptide; The quantity of #=test; The quantity of the #+=positive; The positive percent of %+=

Embodiment 10

In order to study the anti-cell cycle-active of different oligopeptide further, the mouse peripheral blood cell stimulated through anti-cd 3 antibodies is used to carry out experiment in vitro.Object tests the effect of some representative oligopeptide on cell proliferation when division rapidly occurs cell in the Mus mononuclear cell fast growth process of CD3 induction.Mice (n=5) lumbar injection PBS, Nak4 (LQGV), Nak47 (LAGV), Nak46 (AQGV) (Ansynth BV, The Netherlands provides) or Nak46 ^*(AQGV that Diosynth BV, TheNetherlands provide).With the peptide process mice 1 hour of 0.5mg/kg or 5mg/kg, separating spleen prepares splenocyte suspension subsequently.Collect the splenocyte suspension often organized and under the condition that there is PBS or anti-cd 3 antibodies In vitro culture (three parts), within 0,12,24 and 48 hour, measure propagation after incubation.All test peptides all demonstrate and can reduce propagation (see Fig. 4).

The result of embodiment 9 and 10

According to the research that the cell cycle research in plant and external reduction peripheral blood cells are bred, 3 mer peptides that can be used for treating radiation damage through qualification are VVC, LAG, AQG.Similarly, 4 mer peptides that can be used for treating 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 for treating radiation damage are TLAVE, VEGNL, LNEAL, 6 mer peptides that can be used for treating radiation damage are VLPALP, MGGTWA, LTCDDP, 7 mer peptides that can be used for treating radiation damage are VLPAPLQ, VCNYRDV, CPRGVNP, 8 mer peptides that can be used for treating radiation damage are QPLAPLVG, and 9 mer peptides that can be used for treating radiation damage are DINGFLPAL.

List of references

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Claims (6)

1. peptide is in the purposes suffering from radiation damage for the preparation for the treatment of or in the pharmaceutical composition of the object of radiation damage of being thought suffering from, and described peptide is selected from following group: LQGV, LAGV and AQGV, and described radiation damage is gastro-intestinal tract syndrome.

2. the purposes of claim 1, whole aminoacid of wherein said peptide are L-aminoacid.

3. the purposes of claim 1 or 2, wherein said peptide is AQGV.

4. the purposes of claim 1 or 2, wherein said treatment comprises peptide described in subcutaneous or intramuscular injection.

5. the purposes of claim 1 or 2, wherein said pharmaceutical composition is placed in autoinjector.

6. the purposes of claim 1 or 2, wherein said treatment comprise after irradiation at least 30 minutes time use described peptide.