CN1310674C

CN1310674C - Medicinal composition for treating serious acute respiratory syndrome

Info

Publication number: CN1310674C
Application number: CNB031312276A
Authority: CN
Inventors: 尼萨尔·艾哈麦德·卡恩; 罗伯特·本纳; 格特·文斯福尔特; 杰弗里·埃利奥特·加尔平
Original assignee: BIOTENPUT Co Ltd
Current assignee: BIOTENPUT Co Ltd
Priority date: 2003-04-30
Filing date: 2003-04-30
Publication date: 2007-04-18
Anticipated expiration: 2023-04-30
Also published as: HK1070835A1; CN1541707A

Abstract

The present invention relates to the production field of a medical composition for treating patients with severe acute respiratory syndrome (SARS) or suspected patients with severe acute respiratory syndrome (SARS). Especially, when the severe acute respiratory syndrome is caused by SARS virus infection and the patients and the suspected patients are in late phase and in the SARS danger at the lethal stage, the present invention provides an application of at least one gene regulatory peptide or an object with similar functions to produce the medical composition for treating patients with severe acute respiratory syndrome (SARS) or suspected patients with severe acute respiratory syndrome (SARS).

Description

The pharmaceutical composition of treatment severe acute respiratory syndrome

Technical field

The present invention relates to treatment suffers from or the doubtful production field of suffering from a kind of compositions of severe acute respiratory syndrome (SARS) object.

Background technology

At the beginning of the year ends 2002 and 2003, many countries are vigilance to the report of the viral pneumonia of rapid diffusion with high mortality rate and extremely.This disease causes sometimes that according to it dead outstanding feature obtains a title---severe acute respiratory syndrome (SARS) rapidly.This disease causes hyperpyrexia, myalgia and shiver with cold, dry cough and dyspnea subsequently.The another kind of stage of patient near 10% is dry cough, dyspnea and histanoxia subsequently.The mortality rate of report is 5-7% at present.Many countries just actively make great efforts to save life now and the health care system makes it to avoid collapsing.Body just is enough to close a hospital sometimes one by one; And infect medical and nursing work person rapidly and threaten other patient and kinsfolk.

SARS has been confirmed to be the droplet transmission that produces by sneeze and cough, and its symptom comprises hyperpyrexia, shiver with cold, dry cough, nasal obstruction, myalgia and dyspnea.According to World Health Organization's report, since November, worldwide reported had 300 people to die from SARS at present, there is people more than 5000 to suffer from the pneumonia of this mystery.First known outburst is in the Guangdong Province of the SOUTHERN CHINA of adjoining with Hong Kong, but has reported that at present this disease all has outburst from Vietnam to Canada, but most of confirmed cases are in China, Hong Kong and Singapore.

Think that at present SARS is caused by virus, described virus is also referred to as SARS virus at this, thinks that it is a kind of variant of coronavirus, has about 50% homology with known human body coronavirus up to now.Shorter usually incubation period in SARS infection back, headache appears in 1-3 days (but also seeing near 16 days) subsequently, throat pain, and myalgia is shivered with cold and is had a stuffy nose.Usually a large amount of thin nasal discharge of stream then, stiff and become mucopurulently gradually, amount reduces.In some cases, infect and in an about week, disappear, yet in other situation, the common persistent fever of this disease surpasses 100.4 degrees Fahrenheits.Heating is had a headache sometimes also with shiver with cold, uncomfortable and whole body headache.Some patients also experience slight dyspnea symptom when onset.After about 3-7 days, dry cough can appear in patient, follows or be further development of hypoxemia.In this stage, in X-ray or cat scan detection, see the performance of lamellar pneumonia usually.

Most patient begins to occur the symptom of common similar influenza in the phase I, and rehabilitation is gone up on the surface then, some patients of second stage in addition the back that takes a turn for the better from the teeth outwards worsen suddenly.The clinical sign and the lab testing of expression generation serious problems are as follows unusually: LDH raises, and creatine kinase raises, neutrophilic leukocytosis, and platelet is suppressed usually, and prolonged prothrombin and whole body blood clotting quicken, and can reflect with the d dimer assay.Other indicative clinical sign comprises animal migration pneumonia (confirming by X-ray or CT scan), and many patients show histanoxia.In the SARS situation that starts of 10%-20%, to such an extent as to problem especially severe patient need carry out mechanical ventilation.The histopathology of this overpowering pneumonia illustrates a small amount of or does not have viral inclusion body, and depicts a kind of usually and other ADRS or adult respiratory distress syndrome and BOOP or bronchioles disappearance machine pneumonia pathology figure much at one.These two kinds of syndromes are caused by the infringement of proinflammatory cytokine and the generation of other inflammatory mediator usually.

Participate in the diagnostic test that the scientist in the WHO laboratory cooperation net has disclosed some SARS.Comprise so-called " PCR " test, it can detect the particular inheritance information of virus, and can determine the copy number of described virus in any particular sample.For PCR crucial be primer, its can by network laboratories's open acquisition on the WHO website of opening on April 4 ( Http:// www.who.int/csr/sars/primers/en/).Described primer is utilized by many countries in the world.Scientist WHO is optimistic further now, because can carry out meticulous rapidly adjustment (fine-tune) to the PCR diagnostic test of SARS at present.Need meticulous adjustment to be because there is great limitation in existing the test on the ability that has other coronavirus in getting rid of SARS.

The ability that detects early infection SARS virus crowd is a key measure.Existing P CR tests very specificity, but can't detect the patient that all drain coronavirus.Early stage and reliable detection SARS virus helps medical and nursing work person to determine to exist the patient of heating and other doubtful symptom to isolate immediately and control according to the infectious strict measure of control very much in sample.This program obviously reduces the possibility of spread of infection to other people thereupon.

As if yet diagnostic techniques is carried out rapidly, but the method that does not have specific treatment SARS to infect is at present just suited the medicine to the illness or careless treatment at the second stage of SARS.The complication that the second stage situation is further worsened is varied, for example comprises the bacterial infection or the chlamydia infection that need antibiotic therapy.Other complication comprises adult respiratory distress syndrome (ARDS), many organ imbalance syndromes (MODS) or multiple organ failure, MOF (MOF), systemic inflammatory response syndrome (SIRS), this is the direct Secondary cases effect of the viral immunoregulatory abnormality that causes immune activation and take place thus among the host, and pyemia, septic shock and death, dead part is because primary infection or combination infection.The doctor attempts to conclude which type of processing is best.CDC recommends to be suffered from " the unclear pneumonia of cause of disease that colony obtains " and just should be treated as long as patient is doubtful.They should be isolated in the negative pressure chamber, give antibiotic and other nursing.The globulin goods that also give their some standard antiviral drugs such as virazole or tamiflu and derive from the SARS infected patient of rehabilitation gradually or survival.About 90% patient rehabilitation after 6 days.There is half to need respirator in remaining 10%.Yet, the specific medicament of also not treating SARS.

Summary of the invention

The invention provides at least a gene regulatory peptides or its functional analogue production for treating suffer from or the doubtful object of suffering from severe acute respiratory syndrome (SARS) or relevant disease, preferred people's pharmaceutical composition in purposes.The present invention provide especially at least a gene regulatory peptides or its functional analogue production for treating suffer from or the pharmaceutical composition of the doubtful object of suffering from severe acute respiratory syndrome (SARS) or relevant disease in purposes, especially cause by SARS virus when severe acute respiratory syndrome, and especially when described object be in suffer from or the doubtful danger of suffering from SARS late period and that may be the deadly stage in the time.

The invention provides a kind of unexpected treatment pattern that never discloses, comprise at the immunocompetent cytokine gene of SARS patient and regulating, suffer from or the doubtful SARS of suffering from relevant disease patient's treatment in obtain remarkable break-throughs.

The present invention relates to the congenital adjusting approach of body of important immunization.In a preferred embodiment, the invention provides at least a gene regulatory peptides or its functional analogue production for treating suffer from or the pharmaceutical composition of the doubtful object of suffering from severe acute respiratory syndrome (SARS) or relevant disease in purposes, wherein said syndrome is by due to the viral infection, due to the pathogen that causes serious respiratory disorder by influenza virus or other, described disease has ARDS immunology pathogeny sign, there is or do not have DIC, SIRS (systemic inflammatory response syndrome) or MODS sign.

The present invention provide especially use at least a gene regulatory peptides or its functional analogue, production for treating suffer from or doubtful suffer from SARS virus infect due to the medicine of object of SARS.Yet remarkably, in the second stage of SARS, SARS virus does not find in pulmonary system it is active usually, and the principal element that patient status worsens is significant inflammatory reaction, is most preferred at this situation with medicine composite for curing of the present invention.We provide such understanding: most patient can be controlled this virus through immunization, and ARDS immunologic dysfunction complication may take place minority, has some DIC and MODS sign; In described a few patients, do not regulate the ability of the short inflammation infringement of host immune system, this is ARDS, the crucial pathogenesis of DIC and MODS.This immunological response is unusually by being regulated with Drug therapy of the present invention, alleviates or eliminates short inflammation infringement, and the host disease human disease is optimum process thereupon, patients ' recovery and by self control viral infection.

The described therein patient of purposes of the present invention is in and suffers from or doubtful that suffer from this secondary or late period and may be effective especially in those situations of danger of SARS in the stage that causes death, SARS described late period and that may be the stage that causes death is characterised in that one or more expression exists this immunoreactive clinical sign or chemical examination finding, for example be characterised in that and be selected from following clinical sign or chemical examination finding: the lactic dehydrogenase enzyme level improves, hormone kinases level improves, neutrophilic leukocytosis, prolonged prothrombin, platelet suppresses, the d-dimer increases, animal migration pneumonia and gradual histanoxia, progress has or does not have DIC for ARDS, SIRS or MODS.

In the present patent application, set forth the little gene regulatory peptides or the purposes of its functional analogue, its natural being present in gravid woman's body, and derived from the proteolysis of placental gonadotropin as the human chorionic gonadotropin (hCG) that produces at pregnancy duration.These peptides (its activated state has only about 4-6 amino acid long usually) illustrate has unsurpassed immunocompetence, plays a role by the gene expression of regulating coding inflammatory mediator such as cytokine.Astoundingly, the decomposition of finding hCG provides a series of peptides of the immune stable state that helps to keep the anemia of pregnant woman.These peptides or its functional analogue are natural self materials, be not ostracised too early at pregnancy duration by its fetus to guarantee mother to keep the stable of immune state for its balance immune system, but in parent, spend safely up to birth, these materials suffer among the patient of SARS second stage particularly effective especially in treatment, described SARS second stage is characterised in that to have above-mentioned severe complication.Described peptide or its functional analogue for example can derive from urine or other organic product source, as comprise serum, milk surum, intacellin, plant, the cell or tissue of promoting sexual gland hormone or its function equivalence chemical compound.At this, can obtain to be meant from described source, directly or indirectly to obtain described peptide or its functional analogue that peptide or its functional analogue for example also can obtain by chemosynthesis, or obtain from occurring in nature animal or plant raw material.Peptide or its functional analogue can for example be found in deriving from gravid woman's urine fraction or in the article of commerce of hCG or other promoting sexual gland hormone; At least contain the catabolite of capacity hCG or other promoting sexual gland hormone and have Gene regulation and especially in the goods of immunoregulatory activity at those.

In one embodiment, the invention provides at least a gene regulatory peptides or its functional analogue purposes in producing medicine, described medicine is that treatment suffers from or doubtfully suffers from severe acute respiratory syndrome (SARS) or relevant disease, wherein said peptide or its analog can derive from gravid woman's urine, and particularly wherein said women is in its pregnant preceding 3 months.This medicine can be easily from being used for separating and other urine residue that abandons of chemical preparation human gonadotropin prepares.

In another embodiment, the invention provides at least a gene regulatory peptides or its functional analogue purposes in producing medicine, described medicine is that treatment suffers from or doubtfully suffers from severe acute respiratory syndrome (SARS) or relevant disease, wherein said peptide or its analog especially can prepare from the promoting sexual gland hormone goods by proteolysis, and especially wherein said promoting sexual gland hormone or its fraction comprise human chorionic gonadotropin (hCG) or its fraction natural or reorganization.

In another embodiment, the invention provides at least a gene regulatory peptides or its functional analogue purposes in producing medicine, described medicine is that treatment suffers from or doubtfully suffers from severe acute respiratory syndrome (SARS) or relevant disease, wherein said peptide or its analog obtain by chemosynthesis, and especially wherein said peptide or its analog are selected from LQG, AQG, LQGV, AQGV, LQGA, VLPALPQVVC, VLPALP, ALPALP, VAPALP, ALPALPQ, VLPAAPQ, VLPALAQ, LAGV, VLAALP, VLPALA, VLPALPQ, VLAALPQ, VLPALPA, GVLPALP, GVLPALPQ, LQGVLPALPQVVC, VCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL, RPRCRPINATLAVEK, EGCPVCITVNTTICAGYCPT, SKAPPPSLPSPSRLPGPS, SIRLPGCPRGVNPVVS, LPGCPRGVNPVVS, LPGC, MTRV, MTR, VVC, the QVVC or derivatives thereof.

The most preferred purposes of the present invention is the mixture that wherein said compositions comprises peptide LQGV, AQGV and VLPALP.In addition, the invention provides any purposes, wherein said compositions or its representative sample are tested its immunoregulatory activity in the bioanalysis in a kind of body, as describing in detail in the present invention in the zoopery that provides in the part, perhaps wherein said compositions in a kind of external biological analysis, test its immunoregulatory activity or be used for the treatment of according to the guilding principle that is provided SARS immunomodulatory compounds existence whether, as in NF-κ B provided by the invention transposition analysis or gene array.In the sort of mode, preparation is found in during production process at the guidance of suitable pharmaceutical composition of treatment SARS or one or more stage afterwards, test the required immunomodulating or the Gene regulation functional activity of described compositions or one or more its chemical compound or representative sample, for example in carrying out zooperal body in the bioanalysis, in external biological is analyzed, determining under the control of cytokine gene to regulate the functional activity of immunologic process, or whether the physiology of required gene regulatory peptides or its functional analogue exists in compositions by determining test or the chemical compound.Certainly, when from Organic Ingredients such as urine, have for example leukocyte elastase active (reorganization) promoting sexual gland hormone goods from carrying out proteolysis, or from original synthetic compositions or chemical compound as in batches or the peptide of chemosynthesis during purification, purge process can be handled the immunoregulatory activity fraction that selection thus needs most easily, to be further purified or to forgive in the pharmaceutical composition of treatment SARS.

Description of drawings

Fig. 1: the figure shows the fraction that has anti-inflammatory activity in the shock that LPS causes in the disease commitment is III and V, and the fraction that has the inflammation elimination activity in stage terminal stage of a disease is IV and VI.

Fig. 2: test the

fraction

3,4 and 5 of fraction 1,2 and set in vivo in the bioanalysis, prove that the

fraction

3,4 and 5 that has only set has anti-inflammatory activity in the shock that LPS causes.

Fig. 3: Pregnyl on the G25 post (206nm) separation graph.

Fig. 4: Pregnyl elution profile on the GPC 300A post.

Fig. 5: the elution profile that gets the fraction 3 of isolating GPC 300A post on the comfortable GPC 60A post.

Fig. 6: the GPC 60A tomographic map of fraction 3 that derives from the GPC 300A post of 3 months women's urines of gestation with anti-inflammatory activity.This ratio that illustrates between fraction 3.2 and 3.3 is about 1: 2.2.

Fig. 7: the GPC 60A tomographic map of fraction 3 that derives from the GPC 300A post of the Pregnyl that does not have anti-inflammatory activity.This ratio that illustrates between fraction 3.2 and 3.3 is about 1: 3.4.

Fig. 8: the GPC 60A tomographic map of fraction 3 that derives from the GPC 300A post of the Pregnyl with anti-inflammatory activity.This ratio that illustrates between fraction 3.2 and 3.3 is about 1: 1.

The GPC 60A tomographic map of Fig. 9: Pregnyl.

Figure 10: Bio-gel P2 (preceding 3 months urines of gestation) tomographic map (206nm).

Figure 11: the tomographic map of Bio-gel P2 (pregnyl) (206).

Figure 12: the result who originally illustrates BEAS 2B cell line: dexamethasone (DX) can suppress inductive IL-6 of TNF-α and RANTES generation in the BEAS 2B cell line.Active fraction also can suppress the generation of the inductive inflammatory cytokine of TNF-α.In addition, dexamethasone can recover the downward modulation by the inductive antiinflammatory TGF-of TNF-α β cytokine, and active fraction can not only be recovered the generation of TNF-β, and this anti-inflammatory cytokines also further raises.In addition, dexamethasone and active fraction all can suppress the inductive RANTES generation of IFN-γ.Flow cytometry analysis BEAS 2B cell line illustrates, and dexamethasone and active fraction all can be born and regulate inductive HLA-DR of TNF-α and ICAM-1 expression.

The specific embodiment

It is generally acknowledged that proteinic minimal decomposition product is to himself no specific biological function (except as the immune system antigen), but show at present body in fact routine utilize proteinic normal protein enzymolysis process to produce important function of gene to regulate chemical compound, this is the small peptide that control body autogene is expressed.Obviously, body exactly uses the Gene Handling system by the proteinic little catabolite control of himself gene code.

Known to for a long time pregnancy duration, parent produces a kind of temporary transient immunoregulatory status, causes parent to reply inhibition at the repulsion of fetus.Contradictory ground in the infection resistance raising of the common parent of pregnancy duration, and finds that its protective to the clinical symptoms of various autoimmune systemic diseases such as rheumatism and multiple sclerosis is better.Therefore the protection of fetus not only is interpreted as the immunosuppressant result, and should be considered as the result of immunologic balance between parent protection and the fetus protection.

(promptly demonstration can be easy to by chemistry or recombination method synthetic the little catabolite of some of known hCG if desired, and as the little peptide of pharmaceutical composition), performance is to the short inflammation of important transcription factor family control or the main adjusting activity of anti-inflammatory cytokines cascade, and NF-κ B family plays an important role in the gene expression of regulating the setting immune response.

The most of hCG that produce at pregnancy duration are produced by placenta cells, and Placenta Hominis is the cell and the close position that contacts of tissue of parent and fetus, and needs most immunomodulating herein to fight off repulsion.The local gene regulatory peptides that decomposes from Placenta Hominis hCG that produces is equilibrated at short inflammation or the anti-inflammatory cytokines cascade found in the no-man's-land between parent and the fetus (no-man ' s land) immediately.By the trophoderm that typical placenta cells produces, described peptide passes it to born of the same parents' external space; Enter immune system cell and bring into play its immunoregulatory activity, thereby in Placenta Hominis, suppress immunne response by the cytokine gene expression of regulating NF-κ B-and AP-1 mediation.

The invention provides at the deutero-peptide of hCG generally excessive enter body and produce to the generation of anemia of pregnant woman's autoimmune disease and the beneficial effect of the order of severity thereof; Yet, can not overestimation to these effects because be easy to recognize from Placenta Hominis far away more, at preventing that the immunoregulatory activity that fetus repels is more little, for no other reason than that the peptide that produces of Placenta Hominis dilutes when spreading all over body generally.Yet the immunomodulating of described peptide and Gene regulation activity never should only take place at pregnancy duration with in Placenta Hominis; The men and women produces hCG with the same manner, and for example in its hypophysis, and nature utilizes the Gene regulation activity of peptide to a great extent really.

Therefore, the invention provides a kind of new treatment measure, use gene regulatory peptides and derivant thereof with materia medica potentiality.Really, with described gene regulatory peptides the extracorporeal treatment immunocyte and handle laboratory animal in vivo after, by expression and distribution figure research, find that in pneumosilicosis gene array reaching separately of NF-κ B or AP-1 driving is consistent with the short inflammation of immune response or the specificity plus or minus adjusting sign of anti-inflammatory cytokines cascade.Think that also NF-κ B (and AP-1) is a disease main effects device, use the deutero-gene regulatory peptides of hCG to have the important potentiality of treatment SARS, thereby investigate thoroughly and help the balance mother's immune system so that keep the medicine potentiality of the accurate material of its pregnant state safely.

The invention provides and suffer from or the doubtful treatment of suffering from the object of SARS virus infection associated diseases, it is by using a kind of pharmaceutical composition for object, described pharmaceutical composition comprises a kind of gene regulatory peptides or its functional analogue and the suitable diluent of a kind of materia medica of materia medica effective dose.Useful especially materia medica diluent be sterilized water or etc. ooze saline solution as 0.9% saline or phosphate buffered solution (PBS).In a preferred embodiment, the invention provides and suffer from or the doubtful treatment of suffering from the object of SARS virus infection associated diseases, it is by using a kind of pharmaceutical composition for object, described pharmaceutical composition comprises two or several genes of materia medica effective dose and regulates peptide or its functional analogue and the suitable diluent of a kind of materia medica.

The invention provides the purposes of regulating peptide medicine composite,, undertaken by the gene expression that general is regulated in the described object systemic cell to be used to suffer from or doubtfully to suffer from the object that SARS virus infects.This gene regulatory peptides or its functional analogue for example are selected from oligopeptide LQG, AQG, LQGV, AQGV, LQGA, VLPALPQVVC, VLPALP, ALPALP, VAPALP, ALPALPQ, VLPAAPQ, VLPALAQ, LAGV, VLAALP, VLPALA, VLPALPQ, VLAALPQ, VLPALPA, GVLPALP, GVLPALPQ, LQGVLPALPQVVC, VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL, RPRCRPINATLAVEK, EGCPVCITVNTTICAGYCPT, SKAPPPSLPSPSRLPGPS, SIRLPGCPRGVNPVVS, LPGCPRGVNPVVS, LPGC, MTRV, MTR, VVC, QVVC and functional analogue thereof or derivant.Functional analogue for example is found in the article of commerce of the urine fraction that derives from the anemia of pregnant woman or hCG or other promoting sexual gland hormone; At least containing capacity hCG or other promoting sexual gland hormone catabolite and having in active those goods of adjusting.

The preferred size that having of forgiving in the pharmaceutical composition of the present invention regulated peptide is 15 aminoacid at the most, although littler molecule also illustrates special effectiveness.Astoundingly, the invention provides such experience, i.e. gene expression in the SARS infected patient can be regulated or control by little peptide and functional analogue system thereof.Preferred peptide is the catabolite of big polypeptide, described big polypeptide is chorionic-gonadotropin hormone (CG) and growth hormone or somatomedin such as fibroblast growth factor, EGF, VEGF, RNA 3 ' terminal phosphate salt cyclase and CAP18 in this way, or its synthetic.In principle, this adjusting peptide sequence can be derived from any peptide molecule albumen of protokaryon and/or eukaryotic cell generation, and the invention provides such experience, the catabolite of the preferred oligopeptide of polypeptide that the size that provides promptly can be provided is to produce general and beneficial effect, and described catabolite participates in the adjusting of gene expression naturally as gene regulatory peptides.Especially, the invention provides a kind of (synthetic) gene regulatory peptides, it can derive from or (β-hCG) preferably derives from or derived from nicked β-hCG derived from β-human chorionic gonadotropin.Thought in the past that the catabolite of β-hCG participated in immunomodulating (WO99/59671 by the regulator gene expression, WO01/72831, PCT/NL02/00639), or participate in the syndromic treatment of expendable (WO97/49721), but the system that do not embody in these publications regulates the relation of cytokine gene expression in the object that SARS infects.Certainly, this gene regulatory peptides or its function equivalent or derivant can derive from or derived from decomposing or proteolysis and near other protein of Gene regulation cascade.Preferably, described peptide molecule derives from has at least 10 amino acid whose peptides, as has the peptide of following aminoacid sequence: MTRVLQGVLPALPQVVC, SIRLPGCPRGVNPVVS, VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL, RPRCRPINATLAVEKEGCPVCITVNTTICAGYCPT, CALCRRSTTDCGGPKDHPLTC, SKAPPPSLPSPSRLPGPS, CRRSTTDCGGPKDHPLTC, TCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ.

The non-theory that is limited to fetters, and the present invention has disclosed a kind of unexpected Gene regulation pattern, reaches the purpose that treatment suffers from the object of SARS relevant disease.With polypeptide such as endogenous CG, EGF etc., but also comprise pathogen polypeptide such as virus, antibacterial or protozoacide polypeptide are decomposed into unique oligopeptide, are for example undertaken by the intracellular protein enzymolysis.Unique proteolysis enzyme can extensively obtain in cell, for example in eukaryotic lysosome or proteasome system.Some gained catabolites are that length is 3-15, preferred 4-9,4-6 amino acid whose oligopeptide most preferably, its pair cell is not without any function or effect astoundingly, show that at this it may be by participating in the adjusting of gene expression by feedback mechanism as signaling molecule in the situation of endogenous polypeptide decomposition, activity or transposition by for example following peptide regulator gene transcription factor such as NF-κ B prove that described peptide is LQGV, VLPALPQVVC, LQGVLPALPQ, LQG, GVLPALPQ, VLPALP, VLPALPQ, GVLPALP, VVC, MTRV and MTR.The invention provides the synthetic of above-mentioned these peptides, and the functional analogue or the derivant of these catabolites, to regulate in the cell gene expression and to be used for the treatment of in the method for SARS relevant disease.Oligopeptide such as LQG, AQG, LQGV, AQGV, LQGA, VLPALP, ALPALP, VAPALP, ALPALPQ, VLPAAPQ, VLPALAQ, LAGV, VLAALP, VLPALA, VLPALPQ, VLAALPQ, VLPALPA, GVLPALP, GVLPALPQ, LQGVLPALPQVVC, SIRLPGCPRGVNPVVS, SKAPPPSLPSPSRLPGPS, LPGCPRGVNPVVS, LPGC, MTRV, MTR, VVC, or the functional analogue of its longer sequence or derivant (comprising catabolite), be effective especially.

In a preferred embodiment, the invention provides suffer from or doubtful suffer from SARS virus infect due to the treatment of object of inflammation disease, treat by use a kind of pharmaceutical composition for it, described pharmaceutical composition comprises a kind of gene regulatory peptides of materia medica effective dose, and described Gene regulation Toplink is regulated the gene expression of coding inflammatory mediator such as cytokine.Be used for the treatment of the SARS relevant disease and especially infect the effective gene adjusting peptide of forgiving in the medicine of deadly inflammation disease common in the second stage at SARS, be those peptides or its functional analogue, its natural proteolysis product that reaches in anemia of pregnant woman's body derived from placental gonadotropin that is present in, the human chorionic gonadotropin (hCG) of described placental gonadotropin as producing at pregnancy duration, yet, those skilled in the art can synthesize same or similar active synthetic (for example reorganization produces) variant and trim on the function of these peptides easily, and use for example zoopery to test its activity, experimentize with mice as described herein.In another embodiment, the invention provides and be used for the treatment of a kind of pharmaceutical composition of SARS relevant disease, described pharmaceutical composition comprises a kind of gene regulatory peptides or its functional analogue, and gene regulatory peptides or the purposes of its functional analogue in the pharmaceutical composition of production for treating SARS relevant disease are provided.Term " pharmaceutical composition " had both referred to independent active function chemical compound, referred to contain described chemical compound and the suitable carrier of materia medica again, the compositions of diluent or excipient.The suitable diluent of described pharmaceutical composition for example is normal saline solution or phosphate buffered solution.In one embodiment of the invention, described pharmaceutical composition is applied to animal or human's body with the valid density general, and for example by intravenous, intramuscular or intraperitoneal are used.

Embodiment

Summary

Be in SARS immunologic dysfunction in late period syndrome outbreak or during patient, preferably use the medicine composite for curing of treatment SARS provided by the invention.There is gradual histanoxia sign usually in this patient, and is lower and can show by oximetry by partial pressure of oxygen and oxygen saturation in its arterial blood.There is converging of previously described peculiar sign of SARS sample disease and symptom in these patient typical cases, and with some chemical examination findings, it comprises following: lymphopenia, thrombocytopenia, the d-dimer positive, partial prothrombin time prolongs, LDH raises, and CK raises.These patients are starved of delivery of supplemental oxygen or even need the support of artificial ventilation.These patients can be according to the therapeutic scheme antiviral treatment of determining, oral as virazole 400mg day three times, or by the intravenous administration of antibiotics, as cephalo thiophene trowel used for plastering and clarithromycin (or levofloxacin), cause the common pathogens of colony's acquired pneumonia with antagonism, with corticosteroid hormone (meticortelone, 1mg/kg body weight/day), or use other anti-inflammatory drug, or special medicine (as vaccine) at SARS virus.

The suitable patient of treatment has the progressivity pneumonia of ARDS form usually, by X-ray or CT susceptible of proof as a result.They were taken a kind of pill in per 8 hours or continue to drip with whole therapeutic doses no longer needs to treat until thinking.By survival rate, histanoxia is eliminated the time, and the elimination time and the process measurement result of pneumonia and other immunologic dysfunction, comprises clotting time, CK, LDH, and the normalization of TH1 and TH2 cytokine/chemotactic factor cascade.

Embodiment 1

In one embodiment, the invention provides a kind of pharmaceutical composition, it is set forth in the detailed Description Of The Invention part, wherein said active component obviously presents less than the elutriated fraction of 15kD with molecular weight, determine by gel permeation chromatography, for example wherein said active component obviously presents less than the elutriated fraction of 2kD with molecular weight, determines by gel permeation chromatography.Although described pharmaceutical composition of the present invention is easy to obtain from urine, for example wherein said mammal chorionic-gonadotropin hormone goods derive from urine, or derive from wherein the urine fraction that only has active hormones fiddling, as the urine fraction that during commercial promoting sexual gland hormone production process, abandons, but can also use other raw material as comprising the serum of promoting sexual gland hormone or its catabolite, cell or tissue.The present invention also provides a kind of pharmaceutical composition that obtains from described raw material, it can for example regulate Th1 and/or Th2 cytoactive, and/or can regulate dendritic cell differentiation.

Preferably, pharmaceutical composition provided by the invention derives from the mammal of gestation, preferred people, for example preparing a kind of pharmaceutical preparation makes it contain the pregnant mare serum promoting sexual gland hormone (PMSG) of (Placenta Hominis) promoting sexual gland hormone as finding in pregnant mare serum, or the pregnant mouse uterus extract (PMUE) that from the pregnant mouse uterus, extracts, or in pregnant women placental, the human chorionic gonadotropin of finding in blood or the urine (hCG or HCG).Pharmaceutical composition provided by the invention can have or not have promoting sexual gland hormone, for example is present in 3 months the pregnant woman urine of gestation or the promoting sexual gland hormone in the commercial hCG goods.

The invention provides a kind of method for the treatment of immunologically mediated disease, comprise with deriving from the mammiferous at least a pharmaceutical composition of gestation animal is treated.Described treatment can be directly, for example is included as described individuality a kind of pharmaceutical composition is provided, and as hCG or PMSG goods, it comprises a kind of immunoregulatory factor provided by the invention.Also can provide and have the described pharmaceutical composition that derives from the animal pregnancy fraction, for example to urine or serum or Placenta Hominis (coming from parent or fetus) or other tissue or cell sampling, and from described urine or serum or tissue or cell, prepare the described immunoregulatory factor that comprises described active component, and its active component of test by fractionation method known in the art (example gel permeation chromatography).

Embodiment 1

The pharmaceutical composition of from the pregnant woman urine fraction, treating SARS by the purification preparation

Urine purification (method 1): the pregnant woman urine (2L) that will collect from 3 months healthy volunteer of gestation places bottle, and cooling is until be transported to laboratory in two days.In transit, add the 1g/L Hydrazoic acid,sodium salt and be 7.2-7.4 with pH regulator with sodium hydroxide, and room temperature (RT) precipitation 1 hour.Decant about 75% supernatant, will centrifugal near sedimentary residue (10 minutes, 25000rpm, 4 ℃) with disgorging, and add in remaining supernatant.Supernatant is concentrated by one of following two kinds of methods.In first method, supernatant is laterally filtered in the filter plant at 0.45 μ m Minitan (Millipore).Subsequently, will filter thing (2L) concentrates in the Amicon ultrafiltration apparatus of the YM Dipole film in equipment 10kD aperture.Final volume (250ml) is dialysed twice with the Milli Q water of 10L.Be 3ml by 10kD aperture ultrafiltration to final volume in Amicon then with sample.In the second approach, (Millipore concentrates in Pellicon ultrafiltration apparatus cat.No.PXC010C50) with the Pellicon XL filter membrane of supernatant (2L) in equipment 5kD aperture.Final volume is 150ml.

Utilize one of following method, described raw material is further used in the gel filtration to obtain the fraction of treatment SARS.

1. desalting column G25: the Sephadex G-25 Fine (Pharmacia) that uses equipment HPLC.Post is of a size of 5 * 60cm, and the application of sample sample volume is 6-10ml, and flow velocity is 3ml/ minute.The running buffer that uses is that the 50mM ammonium bicarbonate adds 5% methanol.Sephadex G-25 is the desalting column that is used to separate little peptide and small protein matter.The fractionated scope of this post is 100Dalton-5000Dalton.Collect fraction (being generally 50ml) and lyophilizing (, seeing Fig. 1) at separating diagram.The lyophilizing fraction that molecular weight is lower than 1000Dalton is dissolved in (pH 7.4) among the 2-4ml PBS, and tests to determine that pharmaceutical composition is at the adaptability that is being used for the SARS treatment in the bioanalysis in vivo.This bioanalysis shows that the fraction that has anti-inflammatory activity in the inductive shock disease of LPS commitment is III and V, and has diminish inflammation active IV of being and VI (Fig. 1) in stage terminal stage of a disease.

2. solvent resistant column Superdex 75: Superdex 75 (Pharmacia) post that uses equipment 10 * 300mm post bed FPLC.The application of sample sample is 50 μ l, and flow velocity is 1ml/ minute, totally 52 minutes.The running buffer that uses is that the 50mM ammonium bicarbonate adds 5% methanol.Superdex 75 posts are the splendid equipment of separating monomer and dimeric forms lower molecular weight recombinant protein and peptide.Fraction scope at this post of globular protein is 3,000Dalton-70,000Dalton.Collect fraction (being generally 1ml), and be integrated into molecular weight and be lower than 15, the fraction of 000Dalton eluting (

fraction

3,4 and 5 according to separating the diagram (see figure 2); Fig. 2).The fraction lyophilizing of at least 20 different running set will be derived from.Freeze dried fraction is dissolved among the 10mM phosphate buffered saline (PBS) pH7.4 of 1-2ml, to determine the adaptability of pharmaceutical composition in being used for the treatment of SARS in the bioanalysis test in vivo, perhaps be dissolved in the 50mM ammonium bicarbonate of 2-4ml, and as further fractionated as described in the 1st.

The

fraction

3,4 and 5 of fraction 1,2 and set is tested in the bioanalysis in vivo, shows that the

fraction

3,4 and 5 of set has anti-inflammatory activity in the inductive shock disease of LPS commitment.

3. gel permeation column (GPC) 300A: we use equipment Alltech megalosphericus size exclusion (GPC) post 300 , and (250 * 4.6mm or 300 * 7.5mm) Shimadzu HPLC system use 50mM ammonium bicarbonate running buffer.The separating ranges of this post is 1,200,000-7,500Dalton.The sample pipetting volume volume is spissated pregnant 3 months pregnant woman urines of 10-50 μ l.Flow velocity is 0.5ml/ minute, turns round 45 minutes.Use objective molecular weight standard with calibration post eluting position.Collect 50 μ l fraction and gather (see figure 3) according to the eluting diagram.To get comfortable molecular weight and be lower than 7, the fraction set lyophilizing that the 000Dalton eluting is at least 20 times, and be dissolved among the 10mM phosphate buffered saline (PBS) pH7.4, and determine the adaptability of pharmaceutical composition in being used for the treatment of SARS in the bioanalysis test in vivo, perhaps be dissolved in the 50mM ammonium bicarbonate of 1-2ml, and as the 1st and the 4th as described in further fractionated.

All fraction are tested in the bioanalysis in vivo, and shown at molecular weight and be lower than 7, the fraction 3 of 000Dalton eluting has anti-inflammatory activity.

4. gel permeation column (GPC) 60A: in another experiment, the molecular weight that derives from the GPC300A post is lower than 7, the lyophilizing set fraction (seeing the 3rd point) of 000Dalton further separates on the GPC60A post.We use equipment Alltech megalosphericus size exclusion (GPC) post 60 , and (250 * 4.6mm or 300 * 7.5mm) Shimadzu HPLC system use the running of 50mM ammonium bicarbonate.The separating ranges of this post is 28,000-250Dalton.The sample pipetting volume volume is that the spissated molecular weight of 10-50 μ l is lower than 7, and the set fraction of 000Dalton derives from the GPC300A post.Flow velocity is 0.3ml/ minute, turns round 25 minutes.Also use objective molecular weight standard calibration post eluting position.The spissated set fraction 3 that derives from the GPC300A post separates on the GPC60A post, selects 3 zonally-gradeds to separate, and is called fraction 3.1, it uses molecular weight＞2000Da eluting, fraction 3.2, and it uses molecular weight at the 2000-300Da eluting, and fraction 3.3, it is lower than 300Da eluting (Fig. 3 .) at molecular weight.To derive from all these fraction lyophilizing of at least 20 different runnings, and be dissolved among the 1-2ml PBS (pH7.4), and determine the adaptability of pharmaceutical composition in being used for the treatment of SARS in the bioanalysis test in vivo.

All fraction are all tested in the bioanalysis in vivo, shown that the fraction 3.2 at molecular weight eluting between 2000-300Da has anti-inflammatory activity, the fraction 3.3 that is lower than the 300Da eluting at molecular weight has the activity of diminishing inflammation.3 months pregnant woman urines of spissated 10-50ml gestation are directly separated on the GPC60A post, select 3 zones to carry out fractionated, be called 1,2 and 3 (see figure 4)s, and collection from least 20 runnings (at separating diagram, seeing Fig. 4).To gather

fraction

1,2 and 3 lyophilizing and be dissolved among the 1-2ml PBS.All fraction are all tested in the bioanalysis in vivo, show that fraction 2 has anti-inflammatory activity, and fraction 3 is in the inductive shock of the LPS activity that diminishes inflammation late period.These two kinds of fraction all are to be lower than 1 at molecular weight, the 000Dalton eluting.

5.Bio-Gel P-2: will be lower than 5 at molecular weight from superdex G25 post, the lyophilizing of 000Dalton eluting set fraction and derive from the fraction 3 of GPC60A post, (96 * 1.5cm) further separate by gel permeation chromatography on Bio-Gel P-2 post.Material (13-17mg) is suspended in the DDW (8-12ml).Described material is dissolving fully not.(Sigma201,10 minutes 3000rpm) separate precipitate (8-11mg) with supernatant by centrifugal.Supernatant is passed through the gel filtration fractionated on Bio-Gel P-2 post.With this post water with 15ml/ minute flow velocity eluting.With LKB 2142 differential refractometers and LKB 2238 Uvicord SII (206nm) monitoring eluting situation.Collect certain fraction and the lyophilizing of fraction (20 minutes) (, seeing Figure 10) set by Pharmacia Frac 100 fraction catchers at separating diagram.Every kind of fraction is dissolved among the 1ml PBS, and tests its anti-inflammatory activity.These experiments show that molecular weight is lower than 1, and the fraction of 000Dalton has anti-inflammatory activity and (sees Figure 10; Arrow is represented anti-inflammatory activity eluting position).

Embodiment 2

From commercial promoting sexual gland hormone goods, the pharmaceutical composition of preparation treatment SARS in recombinant gonadotropins goods or its less fraction

In following all separation methods, all with 30, the commercial hCG goods of 000IU (Pregnyl, Profasi etc.) are dissolved in the 2ml 50mM ammonium bicarbonate.

1. desalting column G25: the Sephadex G-25Fine (Pharmacia) that uses equipment HPLC.Post is of a size of 5 * 60cm, and the application of sample sample volume is that (15,000IU/ml), flow velocity is 3ml/ minute to 2-4ml.The running buffer that uses is that the 50mM ammonium bicarbonate adds 5% methanol.Sephadex G-25 is the desalting column that is used to separate little peptide and small protein matter.The fractionated scope of this post is 100Dalton-5000Dalton.Collect fraction (being generally 50ml) and lyophilizing (, seeing Fig. 3) at separating diagram.The lyophilizing fraction that molecular weight is lower than 1000Dalton is dissolved in (pH 7.4) among the 2-4ml PBS, and tests to determine that pharmaceutical composition is at the adaptability that is being used for the SARS treatment in the bioanalysis in vivo.This bioanalysis shows that the fraction that has anti-inflammatory activity in the inductive shock disease of LPS commitment is III and V, and has diminish inflammation active IV of being and VI (Fig. 3) in stage terminal stage of a disease.

2. solvent resistant column Superdex 75: Superdex 75 (Pharmacia) post that uses equipment 10 * 300mm post bed FPLC.The application of sample sample is that (15,000IU/ml), flow velocity is 1ml/ minute to 50 μ l, totally 52 minutes.The running buffer that uses is that the 50mM ammonium bicarbonate adds 5% methanol.Superdex 75 posts are the splendid equipment of separating monomer and dimeric forms lower molecular weight recombinant protein and peptide.Fraction scope at this post of globular protein is 3,000Dalton-70,000Dalton.Collect fraction (being generally 1ml), and be integrated into molecular weight and be lower than 15, the fraction of 000Dalton eluting (

fraction

3,4 and 5 according to separating the diagram (see figure 2); Fig. 2).The set fraction lyophilizing of at least 20 different runnings will be derived from.The lyophilizing fraction is dissolved among the 10mM phosphate buffered saline (PBS) pH7.4 of 1-2ml, to determine the adaptability of pharmaceutical composition in being used for the treatment of SARS in the bioanalysis test in vivo, perhaps be dissolved in the 50mM ammonium bicarbonate of 2-4ml, and as further fractionated as described in the 1st.

The

fraction

3. gel permeation column (GPC) 300A: we use equipment Alltech megalosphericus size exclusion (GPC) post 300 , and (250 * 4.6mm or 300 * 7.5mm) ShimadzuHPLC system use 50mM ammonium bicarbonate running buffer.The separating ranges of this post is 1,200,000-7,500Dalton.The sample pipetting volume volume be the commercial hCG goods of 10-50 μ l (30,000IU/ml).Flow velocity is 0.5ml/ minute, turns round 45 minutes.Use objective molecular weight standard with calibration post eluting position.The label that uses is: press down the enzyme peptide (6,500Da), cytochrome C (12,400), carbonic anhydrase (29,000), albumin (66,000) and blue glucosan (2,000,000).Collect 50 μ l fraction and gather (see figure 4) according to the eluting diagram.To get comfortable molecular weight and be lower than 7, the fraction set lyophilizing that the 000Dalton eluting is at least 20 times, be dissolved among the 10mM phosphate buffered saline (PBS) pH7.4, and determine the adaptability of pharmaceutical composition in being used for the treatment of SARS in the bioanalysis test in vivo, perhaps be dissolved in the 50mM ammonium bicarbonate of 1-2ml, and as the 1st and the 4th as described in further fractionated.

4. gel permeation column (GPC) 60A: in another experiment, the molecular weight that derives from the GPC300A post is lower than 7, the lyophilizing set fraction (seeing the 3rd point) of 000Dalton further separates on the GPC60A post.We use equipment Alltech megalosphericus size exclusion (GPC) post 60 , and (250 * 4.6mm or 300 * 7.5mm) Shimadzu HPLC system use the running of 50mM ammonium bicarbonate.The separating ranges of this post is 28,000-250Dalton.The sample pipetting volume volume is that the spissated molecular weight of 10-50 μ l is lower than 7, and the set fraction of 000Dalton derives from the GPC300A post.Flow velocity is 0.3ml/ minute, turns round 25 minutes.Also use objective molecular weight standard calibration post eluting position.The spissated set fraction 3 that derives from the GPC300A post separates on the GPC60A post, selects 3 zonally-gradeds to separate, and is called fraction 3.1, it uses molecular weight＞2000Da eluting, fraction 3.2, and it uses molecular weight at the 2000-300Da eluting, and fraction 3.3, it is lower than 300Da eluting (Fig. 5) at molecular weight.To derive from all these fraction lyophilizing of at least 20 different runnings, and be dissolved among the 1-2ml PBS (pH7.4), and determine the adaptability of pharmaceutical composition in being used for the treatment of SARS in the bioanalysis test in vivo.

Fraction 3.2 and 3.3 ratio: then, we on the GPC60A post from early stage non-activity of described disease and activated hCG goods, being further purified the fraction 3 that derives from the GPC300A post.3 months pregnant woman urines of gestation are carried out same analysis, and the ratio of definite fraction 3.2 and 3.3.We find to have in early days in described disease, and ratio is about 1: 2.2 (fraction 3.2: fraction 3.3) (Fig. 5) in 3 months pregnant woman urines of gestation of anti-inflammatory activity, do not have in early days in described disease that ratio is 1: 3.4 (Fig. 7) in the active hCG goods of antiinflammatory (Pregnyl), ratio is 1: 1 (Fig. 8) in the hCG goods of anti-inflammatory activity and have in early days in described disease.

With the commercial hCG goods of 10-50 μ l (15,000IU/ml) directly on GPC 60A post, separate, selects 3 zones to carry out fractionated, be called 1,2 and 3 (see figure 9)s, and from least 20 runnings, collect fraction (illustrate at separating, see Fig. 9).To gather

fraction

1,2 and 3 lyophilizing and be dissolved among the 1-2ml PBS.All fraction are all tested in the bioanalysis in vivo, show that fraction 2 has anti-inflammatory activity in early days in described disease, and fraction 3 has the activity of diminishing inflammation in the described terminal stage of a disease.These two kinds of fraction all are lower than 1 at molecular weight, the 000Dalton eluting.

5.Bio-Gel P-2: will be lower than 5 at molecular weight from superdex G25 post, the lyophilizing of 000Dalton eluting set fraction and derive from the fraction 3 of GPC60A post, (96 * 1.5cm) further separate by gel permeation chromatography on Bio-Gel P-2 post.Material (13-17mg) is suspended in the DDW (8-12ml).Described material is dissolving fully not.(Sigma201,10 minutes 3000rpm) separate precipitate (8-11mg) with supernatant by centrifugal.Supernatant is passed through the gel filtration fractionated on Bio-Gel P-2 post.With this post water with 15ml/ minute flow velocity eluting.With LKB 2142 differential refractometers and LKB 2238Uvicord SII (206nm) monitoring eluting situation.Collect certain fraction and the lyophilizing of fraction (20 minutes) (, seeing Figure 11) set by Pharmacia Frac 100 fraction catchers at separating diagram.Every kind of fraction is dissolved among the 1ml PBS, and tests its anti-inflammatory activity.These experiments show that molecular weight is lower than 1, and the fraction of 000Dalton has anti-inflammatory activity and (sees Figure 11; Arrow is represented anti-inflammatory activity eluting position).

Embodiment 3

The pharmaceutical composition of preparation treatment SARS from the synthetic peptide of commerce

In another embodiment, gene regulatory peptides wherein provided by the invention is used for the treatment of disease as the regulatory factor of NF-κ B, peptide of the present invention or its functional deriv or analog are used to produce a kind of pharmaceutical composition, to treat or to alleviate inflammatory cytokine (the TNF-α of the various levels of in the metainfective patient of SARS virus, seeing usually, IL-1 isotype, IL-6, IL-8, I1-12, IL-23) reply and chemical compound such as nitric oxide (NO) and arachidonic acid metabolite are replied.The negative adjusting of the effective NF-κ B that comprises in this pharmaceutical composition peptide for example is VLPALPQVVC, LQGVLPALPQ, LQG, LQGV, GVLPALPQ, VLPALP, VVC, MTR and annular LQGVLPALPQVVC, in the treatment that its IL-6 that for example is effective to see in severe acute respiratory syndrome (SARS) replys excessively by force.Preferably include and follow antiviral agent such as ribavirine and gene regulatory peptides treatment, to reduce for example IL-6 activity of NF-κ B mediation.

In another embodiment, gene regulatory peptides wherein provided by the invention is used as NF-κ B regulatory factor to be used for the treatment of disease, the invention provides NF κ B and regulate the purposes of peptide or derivatives thereof in the pharmaceutical composition of production for treating primate inflammation disease, and a kind of method for the treatment of the struvite SARS disease of primate is provided.Preferred described treatment is included as the patient and uses a kind of pharmaceutical composition, and it comprises negative peptide or its functional analogue of regulating of NF-κ B.Effectively the negative adjusting of NF-κ B peptide is VLPALPQVVC, LQGVLPALPQ, LQG, LQGV, GVLPALPQ, VLPALP, VVC, MTR and annular LQGVLPALPQVVC.Use method provided by the invention can find more negative peptide and the functional analogue thereof of regulating.

Peptide is synthetic: use the method (Atherton, 1985) based on fluorenylmethoxycarbonyl (Fmoc)/tert-butyl, as solid support, (Merrifield, 1963) prepares described peptide by solid phase synthesis with 2-chlorotrityl chloride resin (Barlos, 1991).The side chain of glutamine triphenylmethyl function and protecting.The described peptide of synthetic.Each coupling may further comprise the steps: (i) remove the α amido protecting by piperidines in dimethyl formamide (DMF); (ii) in DMF/N-methylformamide (NMP) with DIC (DIC)/l-hydrozybenzotriazole (HOBt) and Fmoc aminoacid (3eq.) coupling, and (iii) use acetic anhydride/diisopropylethylamine (DIEA) capping residue amino functional in DMF/NMP.On synthetic basis of finishing, with described peptide resin trifluoroacetic acid (TFA)/mixture process of 95: 2.5: 2.5 of H2O/ tri isopropyl silane (TIS).After 30 minutes, add TIS, until decolouring.Precipitate described peptide with the solution for vacuum evaporation and with diethyl ethyl phosphonate.Rough peptide is dissolved in the water (50-100mg/ml) also by reversed-phase high-performance chromatography (RP-HPLC) purification.The HPLC condition is as follows: chromatographic column: Vydac TP21810C18 (10 * 250mm) (selection of chromatographic column is very crucial, because peptide has short retention time); Elution system: 0.08-1%TFA v/v (B) gradient system in 0.1%TFA v/v (A) and the acetonitrile (ACN) in the water; Flow velocity 6ml/ minute; Detect absorbance at 190-370nm.Can use different gradient systems.For example at peptide LQGV:10 minute 100%A, linear gradient 0-10%B carried out 50 minutes subsequently.At peptide VLPALP:5 minute 5%A, linear gradient 1%B/ minute subsequently.Is about 5ml by rotation film evaporation and concentration with the fraction of collecting under reduced pressure at 40 ℃.Remaining TFA exchanges with the acetate form by twice eluting through the post with anion exchange resin (Merck II) with acetic acid.Eluate is concentrated and lyophilizing in 28 hours.Then freeze dried peptide is tested to determine the adaptability of pharmaceutical composition in being used for the treatment of SARS in the bioanalysis in vivo, undertaken by testing its effect in the BALB/c mouse model to the inductive inflammation of LPS.

Table 1

ID	Sequence	Early activity	Late period activity
ID	Sequence	Early activity	Late period activity	Peptide-1 peptide-2 peptide-3 peptide-4 peptide-5 peptide-6 peptide-7 peptide-8 peptide-9 peptide-10 peptide-11 peptide-12 peptide-13 peptide-14 peptide-43 peptide-44 peptide-45 peptide-46 peptide-47 peptide-48 peptide-49 peptide-50 peptide-51 peptide-52 peptide-53 peptide-54 peptide-55 peptide-56 peptide-57 peptide-58 peptide-59 peptide-60 peptide-61 peptide-62 peptide-63 peptide-64 peptide-65 peptide-66 peptide-67 peptide-68 peptide-69 peptide-70 peptide-71 peptide-74 peptide-75 peptide-76 peptide-78	VLPALPQVVC LQGVLPALPQ LQG LQGV GVLPALPQ VLPALP VLPALPQ GVLPALP VVC QVVC MTRV MTR LQGVLPALPQVVC ring-LQGVLPALPQVVC AQG LAG LQA AQGV LAGV LQAV LQGA ALPALP VAPALP VLAALP VLPAAP VLPALA ALPALPQ VAPALPQ VLAALPQ VLPAAPQ VLPALAQ VLPALPA VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQ SIRLPGCPRGVNPVVS LPGCPRGVNPVVS CPRGVNPVVS LPGC CPRGVNP PGCP RPRCRPINATLAVEKEGCPVCITVNTTICAGYCPT MTRVLQGVLPALPQ MTRVLPGVLPALPQVVC CALCRRSTTDCGGPKDHPLTC SKAPPPSLPSPSRLPGPS TCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ CRRSTTDCGGPKDHPLTC	+ + + + + + + + + + + + + + + + + + + + + + + -+ -+ + + + -+ -+ + -+	+ + + + + + + + + + + + + + + + + + + + + + + + -+ + -+ -+ -+ + + +

Table 1: useful peptide activity.From ± to+be illustrated in LPS to induce small activity the shock to pure activity

Embodiment 4

Therapeutic scheme

Treat for example to be included in pharmaceutical composition of the present invention and injected Ringer ' s lactic acid in preceding 24 hours, has a kind of pharmaceutical composition provided by the invention in described Ringer ' the s lactic acid, preferred 1-1000mg/l peptide, as VLPALPQ, GVLPALP or MTRV, or two or the mixture of three kind of these peptide.During severe infections patient's treatment, for example by under the mechanical respiration, importantly the maintenance capacity is stable usually, and if desired, in addition more provide described pharmaceutical composition (for example peptide or its functional analogue) in the hypisotonic solution, as in 0.3-0.6% saline.

Gene regulatory peptides can give at identical injection liquid, and the preferred simmer down to of described peptide (or analog) is from about 1-1000mg/l, but described peptide also can give in the pill injection.Injecting or inject the liquid recommended dose at every turn at pill is the 1-5mg/kg body weight, and every 8-12 hour once, until patient's stable disease.

Embodiment 4

The therapeutic scheme of SARS

Pharmaceutical composition 1 (Superdex G25):, can use following scheme at patient's SARS single therapy.

3 months pregnant woman urines of 1L gestation are filtered and concentrate 13 times (according to embodiment 1).This provides about 75ml spissated pregnant 3 months pregnant woman urines (desalination).With spissated pregnant 3 months pregnant woman urines of this 75ml fractionated on Superdex G25, the sample volume that at every turn turns round is 6ml.Set active fraction and lyophilizing.

Pharmaceutical composition 2 (Superdex G25):, can use following scheme at patient's SARS single therapy.

With 150, the commercial hCG goods of 000IU are dissolved in the 2-4ml 50mM ammonium bicarbonate.With hCG goods fractionated on Superdex G25 of this 2-4ml, set active fraction and lyophilizing from least 3 runnings.

Pharmaceutical composition 3 (GPC 300A):, can use following scheme at patient's SARS single therapy.

3 months pregnant woman urines of 1L gestation are filtered and concentrate 13 times (according to embodiment 1).This provides about 75ml spissated pregnant 3 months pregnant woman urines (desalination).With spissated pregnant 3 months pregnant woman urines of this 75ml fractionated on GPC 300A, the sample volume that at every turn turns round is 50-100 μ l.Set active fraction 3 and lyophilizing.This lyophilized products is used for the treatment of or on GPC 60A post further fractionated, and with fraction 3.2 and 3.3 lyophilizing and be used for the treatment of.

Pharmaceutical composition 4 (GPC 300A):, can use following scheme at patient's SARS single therapy.

With 150, the commercial hCG goods of 000IU are dissolved in the 6ml 50mM ammonium bicarbonate.The hCG goods of this 6ml are separated set active fraction 3 and lyophilizing at GPC 300A column fractionation.This lyophilized products is used for the treatment of or on GPC 60A post further fractionated, with fraction 3.2 and 3.3 lyophilizing and be used for the treatment of.

Pharmaceutical composition 5 (GPC 60A):, can use following scheme at patient's SARS single therapy.

3 months pregnant woman urines of 1L gestation are filtered and concentrate 13 times (according to embodiment 1).This provides about 75ml spissated pregnant 3 months pregnant woman urines (desalination).With spissated pregnant 3 months pregnant woman urines of this 75ml fractionated on GPC 60A, the sample volume that at every turn turns round is 50-100 μ l.Set active fraction 3 and lyophilizing.

Pharmaceutical composition 6 (GPC 60A):, can use following scheme at patient's SARS single therapy.

With 150, the commercial hCG goods of 000IU are dissolved in the 6ml 50mM ammonium bicarbonate.The hCG goods of this 6ml are separated at GPC 60A column fractionation, and the sample volume that at every turn turns round is 50-100 μ l, set active fraction 3 and lyophilizing.

Pharmaceutical composition 7 (Superdex 75):, can use following scheme at patient's SARS single therapy.

3 months pregnant woman urines of 1L gestation are filtered and concentrate 13 times (according to embodiment 1).This provides about 75ml spissated pregnant 3 months pregnant woman urines (desalination).With spissated pregnant 3 months pregnant woman urines of this 75ml fractionated on Superdex 75, the sample volume that at every turn turns round is 50-100 μ l.Set active fraction 3-5 and lyophilizing.

Pharmaceutical composition 8 (Superdex 75):, can use following scheme at patient's SARS single therapy.

With 150, the commercial hCG goods of 000IU are dissolved in the 6ml 50mM ammonium bicarbonate.The hCG goods of this 6ml are separated at Superdex 75 column fractionations, and the sample volume that at every turn turns round is 50-100 μ l, set active fraction 3-5 and lyophilizing.

Pharmaceutical composition 10 (peptide):, can use following scheme at patient's SARS single therapy.

Use single peptide (seeing embodiment 3 tables) or peptide mixer to treat.It below is the peptide mixer example that is used for the treatment of: peptide 1 and 2, peptide 1,2 and 4, peptide 1,2 and 3, peptide 3 and 5, peptide 4 and 6, peptide 4,6 and 7, peptide 3,6 and 7, peptide 1,2 and 6, peptide 1,2, with 7, peptide 1,2 and 9, peptide 1,2, with 10, peptide 1,2, with 11, peptide 1,212, peptide 4,5, with 6, peptide 4,5, with 46, peptide 4,5 and 48, peptide 4,6, with 46, peptide 4,6, with 48, peptide 66 and 6, peptide 66 and 7, peptide 66 and 5, peptide 64 and 65, peptide 4 and 68, peptide 66 and 76, peptide 9 and 11, peptide 10 and 12, peptide 46,49 and 59, peptide 48,49 and 59, peptide 46,48,49 and 59, peptide 48 and 60, peptide 46,48,49,59 and 60, peptide 14 and 48, peptide 14 and 46, peptide 14 and 7, peptide 14 and 6, peptide 14 and 3, peptide 14 and 4.Single peptide or peptide mixer also can with 3 months pregnant woman urine fraction of gestation, commercial hCG goods or the associating of reorganization hCG goods or mix and use.In addition, 3 months pregnant woman urine fraction of gestation also can mix with commercial hCG fraction.In a preferred embodiment, in the pill injection, give patient 1: 1: 1 blended peptide AQGV, LQGV and VLPALP attacking treatment stage with the 5mg/kg body weight, after 4 hours, regularly reach subsequently and continue to inject above-mentioned Ringers lactic acid or hypotonic medium, contain each in three kinds of peptides of 100mg/l in the described solution, flow velocity is 20-200ml/ hour.Cytokine scattergram in the preferred monitor therapy patient blood plasma, as TNF-α, IL-10 or IL-6 level, NO, and arachidonic level, and when think that these levels are in normal range, just finish to treat having only.

Embodiment 5

Bioanalysis is to determine the adaptability of pharmaceutical composition in being used for the treatment of SARS in the body

Zoopery

Animal is raised (laboratory animal 28:1-24,1994) to animal health in scheme described in the report (FELASA) of European laboratory animal scientific institution according to research group under special pathogen-free domestic condition.

Embodiment 5

Determine pharmaceutical composition bioanalysis in being used for the adaptive body of SARS treatment

1.LPS induce inflammation: be to determine anti-inflammatory activity, with the female BALB/c mouse (Harlan) in 8-10 age in week through i.p. injection 30mg/kg LPS (E.coli 055:B5; DifcoLab, Detroit, MI, USA) or 9-10mg/kg LPS (E.coli 026:B6; Difco Lab, Detroit, MI, USA).Control group mice (10) was handled with 100 μ lPBS (pH 7.4) behind lps injection in 2 hours, and in other group mice (10) with 100 μ l (1mg/ml) fraction, the commercial hCG of 100-600IU (Pregnyl, Profasi) or peptide (0.05-5mg/kg) handle through i.p..In other experiment, mice was used fraction in 24 hours or 36 hours behind lps injection, commercial hCG goods or peptide are handled.At experimental session, observe the disease situation since 0 time point (before the lps injection).Afterwards, mice is only observed its further rehabilitation situation.In this experiment, LPS causes the PBS group dead in 48-60 hour behind injection LPS.When mortality rate when 100% reduces at least 40%, think fraction, commercial hCG goods or peptide are active.

2.TSST-1 inductive inflammation: be to determine anti-inflammatory activity, female BALB/c mouse (Harlan) peritoneal injection in 8-10 age in week is dissolved in 20mg D-galactosamine in the 100 μ l sterile saline solution (9%).Then percutaneous down injection give them and be dissolved in 4 μ g TSST-1 in the 100 μ l sterile saline solution (9%), about two positions of 0.5cm under every side omoplate, injection site.Matched group is used does not have 4 μ g TSST-1 subcutaneous injections of D-galactosamine, or only handles with D-galactosamine.Balb/c mice group to D-galactosamine sensitization is also used 100 μ l (1mg/ml) fraction after the TSST-1 injection, commercial hCG goods (Pregnyl, Profasi) of 100-600IU or peptide (0.05-5mg/kg) are handled.In other experiment, mice was used fraction in 24 or 36 hours behind injection TSST-1, commercial hCG goods or peptide are handled.At experimental session, observe the disease situation since 0 time point (before the TSST-1 injection).Afterwards, mice is only observed its further rehabilitation situation.In this experiment, TSST-1 causes the PBS group dead in 48-60 hour behind injection TSST-1.When mortality rate when 100% reduces at least 40%, think fraction, commercial hCG goods or peptide are active.

3. monkey experiment: in this preclinical study, only use to be used to the monkey of testing first, to get rid of any interaction with first pre-treatment.Described animal is anaesthetized with ketamine hydrochloride (ketamine).With the animal via oral intubation and it is freely breathed.Use O ₂/ N ₂O/isoflurane keeps the Animal Anesthesia state.Give the animal atropine as O ₂/ N ₂The pretreatment medicine of O/isoflurane anesthesia.Kept the surgical anesthesia level at colibacillary 2 hours during the infusion, and after escherichia coli are attacked 6 hours, take out the respiratory tract interpolation pipe, and make described animal euthanasia.Before importing antibacterial, carry out 1 hour pre-infusion heart rate and monitoring of blood pressure.

4. with two Rhesus Macacus infusions 10 ¹⁰CFU/kg gram negative bacteria escherichia coli are to produce fatal septic shock.A monkey is accepted placebo treatment, and behind the described antibacterial of infusion, put to death in 7 hours, from anesthesia, do not recover, second monkey accepted described test compounds handle, and put to death at same time point.For example, test compounds is the mixture of 3 kinds of peptides in an experiment; Behind the infusion escherichia coli 30 minutes, for animal is used pill injection in a kind of azygos vein, a kind of mixture that contains following peptide: FLQGV (5mg/kg), AQGV (5mg/kg) and VLPALP (5mg/kg).These peptides are dissolved in 0.9% sodium chloride solution with injection (N.P.B.I., Emmer Compascuum, The Netherlands).When mortality rate when 100% reduces at least 40%, think fraction, commercial hCG goods or peptide are active.

Embodiment 6

The external biological analysis is to determine that pharmaceutical composition is at the adaptability that is used for the SARS treatment

1.NF-κ B analyzes: with mouse macrophage (RAW 264.7 mouse macrophages) at 37 ℃, 5%CO ₂, contain 10% or the DMEM of 2%FBS, penicillin, streptomycin and glutamine in cultivate.The cell that with cumulative volume is 1ml was planted 2 hours (3 * 10 in 12 hole flat boards ⁶Cell/ml), use LPS (escherichia coli 026:B6 then; DifcoLaboratories, Detroit, MI, USA) and/or peptide (1 μ g/mL), fraction (1 μ g/mL) or hCG goods (100-300IU/mL) stimulate.Behind the incubation 30 minutes, centrifugal and collecting cell is to examine extraction with flat board.

2. nuclear extracts: prepare nuclear extract and EMSA (Schriber etc., 1989, nucleic acids research 17) according to described methods such as Schreiber.With cell harvesting in test tube and 4 ℃ with 2000rpm (rev/min) centrifugal 5 minutes (Universal 30RF, HettichZentrifuges).With freezing Tris buffer saline (TBS pH 7.4) flushing precipitation, and resuspending (10mM HEPES pH 7.9,10mM KCl, 0.1mM EDTA, 0.1mM EGTA, 1mM DTT, 0.5mM PMSF and protease inhibitor cocktail (Complete in 400 μ l hypotonic buffer liquid A ^TMMini Roche), placed 15 minutes on ice.Add 25 μ l10%NP-40 and with sample centrifugal (2 minutes, 4000rpm, 4 ℃).Collect supernatant (kytoplasm fraction) and be stored in-70 ℃.To contain nucleolate precipitation with 50 μ l buffer A flushings and resuspending in 50 μ l buffer C (20mM HEPES pH 7.9,400mM NaCl, 1mM EDTA, 1mM EGTA, 1mM DTT, 0.5mM PMSF and protease inhibitor cocktail and 10% glycerol).Sample was shaken 60 minutes at 4 ℃ at least.At last, sample is centrifugal and supernatant (nuclear fraction) is stored in-70 ℃.

Use Bradford reagent (Sigma) to determine final protein concentration in the extract.

3.EMSA: at electrophoretic mobility shift assay, synthetic a kind of oligonucleotide performance NF-κ B binding sequence (5 '-AGC TCA GAG GGG GAC TTT CCG AGA G-3 ').

With 100pl have justice and antisense scant polymer annealing and according to manufacturer instruct use T4 polynucleotide kinase γ- ³²The P-dATP labelling (Promega, Madison, WI).With nuclear extract (5-7.5 μ g) with the 75000cpm probe in association reaction mixture (20 μ l) room temperature incubation 30 minutes, described mixture contains poly-dI-dC (Amersham Pharmacia Biotech) of 0.5 μ g and binding buffer liquid BSB (25mM MgCl ₂, 5mM CaCl ₂, 5mM DTT and 20%Ficoll).The DNA-protein complex by electrophoresis (150V, 2-hour) in the 4-6% polyacrylamide gel, is dissociated from free oligonucleotide.Then with this gel drying and be exposed to X-ray.

4.NO measure: will derive from American type culture collection (Manassas, VA, RAW 264.7 mouse macrophages USA) at 37 ℃ at 5%CO ₂In cultivate with DMEM, contain 10% hyclone (FCS) among the described DMEM, the 50U/ml penicillin, 50 μ g/ml streptomycins, the 0.2M Sodium Pyruvate, 2mM glutamine and 50 μ M 2 mercapto ethanols (Bio Whittaker, Europe).Changed a subculture in per 2 days.

Nitrite is measured: measure nitrite and produce in RAW 264.7 macrophage supernatants.With cell (7.5 * 10 ⁵/ ml) in 48 hole flat boards, in 500 μ l culture medium, cultivate.Cell uses LPS (10 μ g/ml) and/or peptide or fraction (1pg/ml, 1ng/ml, 1 μ g/ml) to stimulate 24 hours, collects culture medium then.In 100 μ l media samples, add 100 μ l Griess reagent (Sigma) and measure nitrite.Use the microplate reader to measure OD ₅₄₀, by with the OD that uses the standard sodium nitrite solution in culture medium, to produce ₅₄₀The calculating nitrite concentration of comparing.

PM1T-cell line or RAW 264.7 derive from American type culture collection

(ATCC, Manassas, VA), and at 37 ℃ at 5%CO ₂The middle cultivation.At the genome experiment, with PM1T-cell (2 * 10 ⁶/ ml) in 6 hole flat boards with the phytohemagglutinin (PHA, 10 μ g/mL) of 2ml and IL-2 (200IU/ml) or PHA, IL-2 and peptide LQGV (10 μ g/mL) incubation for example.After cultivating 4 hours, flushing 10 * 10 ⁶Cell and preparation are to carry out the experiment of gene chip probes array.At macrophage, RAW 264.7 cells for example LQGV (10 μ g/mL) incubation of LPS (1 μ g/mL) or LPS and peptide.After cultivating 1 hour, with 10 * 10 ⁶Cell washes and prepares to carry out the experiment of gene chip probes array.Carry out gene chip expression analysis (expression analysis technological guidance, Affymetrix gene chip) according to manufacturer's guidance.Following key step has been summarized the gene chip expression analysis: 1) target preparation, 2) target hybridization, 3) experiment and fluidics equipment, 4) probe array flushing and dyeing, 5) probe array scanning and 6) data analysis.

5.Th1/Th2 polarographic analysis: with mice through i.p. with the chemical compound of PBS or test (fraction: 1-100 μ g, peptide: 1-100 μ g, hCG:100-300IU) handled 3 days.Under aseptic condition, take out splenocyte then and prepare the single cell suspension.Purification CD4+T cell from spleen passes through with heat stable antigen (HAS), CD16/32, II type MHC (BALB/c, M5/114; NOD, M10/216) and the antibody of GR-1 obtain through the complement loss.Use the magnetic active cell to be further purified in cell through the sorting of biotinylated mAbs mixture; described mAbs is anti-CD11b (M1/70), B220 (RA3 6B2), CD8 (YTS-169) and CD40 (FCK-45.5) antibody; microballon (the MiltenyBiotech that puts together with streptavidin subsequently; Bergisch Gladbach, Germany) incubation.The CD4 that is used for testing ⁺Cell is measured through flow cytometry and is generally 90-95% purity.

At polarographic analysis, tentatively stimulate the CD4 of purification by following steps ⁺: in the flat flat board in 96 holes (Nalge Nunc Int., Naperville, IL., USA), cultivate 1 * 10 ⁵Cells/well, and with in conjunction with dull and stereotyped anti-CD3mAb (145-2C11; 25 μ g/ml) there is soluble CD28mAb (37-51; 10 μ g/ml) and under IL-2 (50U/ml) situation stimulate.At the Th1 cell differentiation, will resist IL-4mAb (11B11; 10ng/ml) and IL-12 (10ng/ml) add in the culture.Cause the Th2 cell with IL-4 (35ng/ml) and anti-IFN-γ mAb (XMG 1.2,5microg/ml).Non-polarised culture only contains anti-CD3, anti-CD28 and IL-2.

In preliminary experiment to all dosage optimizations.After cultivating 4 days, with cell flushing 3 times, and move in the 96 hole flat boards of new anti-CD3 bag quilt, and under the situation that has IL-2 (50U/ml) and anti-CD28 (10 μ g/ml), stimulate again.After 48 hours, collect supernatant and pass through elisa assay IL-4, IFN-γ and IL-10 production are as Th1/Th2 polarization readout.

6.TNF-α, IFN-γ expresses with MHC II: separate the splenocyte that derives from BALB/c for example or NOD mice, and with stimulating under the situation that has IL-2 (50U/ml) or IL-2 and fraction or peptide in conjunction with dull and stereotyped anti-CD3mAb (145-2C11,25 μ g/ml).After 48 hours, collect culture supernatant and pass through elisa assay IFN-γ.In other experiment, with splenocyte, isolating macrophage, DC or antigen-presenting cell (APC) stimulated 24 hours with LPS or LPS and fraction, peptide or hCG.Measure TNF-α or measure MHC II expression by ELISA then by fluidic cell quantitative analysis (FACS).

Human bronchial epithelial cell line BEAS 2B

Be characterised in that the most of crowd of sickness influence of respiratory inflammation.These diseases comprise SARS.Reply stimulation, infection and inflammatory mediator and the cytokine and the somatomedin that produce, in the acute and beginning chronic respiratory inflammation, continue and suppress in play an important role.

Some media that inflammatory cell and inhabitation cell discharge in respiratory inflammation and the respiratory tract and the excessive generation of cytokine and active relevant.Know that now epithelial cell is not only an important function target of inflammatory mediator, and in self inflammation process, also be an active participant.Bronchial epithelial cell can replenish inflammatory cell by discharging chemoattractant in respiratory tract, expression guiding inflammatory cell by cell adhesion molecule moves through epithelium, and regulate other cellular inflammation activity by release medium, described medium such as cytokine, chemotactic factor, Semen arachidis hypogaeae 4 olefin(e) acid metabolite and lax and contraction factor.

Bronchial epithelial cell not only forms the passive barrier of one deck, but also plays a role in immunne response.They can produce many media and produce short inflammation or antiinflammatory action.In addition, bronchial epithelial cell can be expressed the adhesion molecule of many dissimilar cells, thereby helps it to raise.

The TNF-α that is produced by inflammatory cell that exists in the respiratory tract can cause other inflammatory cytokine and chemotactic factor such as RANTES and IL-6.It can also bear the generation of regulating anti-inflammatory cytokines, thereby and destroys epithelial barrier function.Transcribing of most cytokines that the glucocorticoid inhibition is relevant with asthma and chemotactic factor comprises IL-6, RANTES, IL-4.Thisly suppress relevant with the therapeutical effect of glucocorticoid to small part.

BEAS 2B cell is existed under the situation of test compounds with TNF-α or TNF-γ stimulation.The result that we illustrate the active fraction that derives from pregnyl at this as an example.As shown in figure 12, our result illustrates dexamethasone and can suppress that the inductive IL-6 of TNF-α and RANTES produce in the BEAS 2B cell line.As what test at this, a kind of pharmaceutical composition of treatment SARS provided by the invention also can suppress the inductive inflammatory cytokine of TNF-α and IL-6.In addition, dexamethasone can recover the negative adjusting of the inductive antiinflammatory TGF-of TNF-α β cytokine, and fraction not only recovers TGF-β generation, but also promotes this anti-inflammatory cytokines.In addition, dexamethasone and compositions all can suppress the inductive RANTES generation of IFN-γ.TNF-α also can inducing cell adhesion labelling such as lip-deep HLA-DR of epithelial cell and ICAM-1, it replenishes inflammatory cell then.In this mode, epithelial cell also can be as antigen-presenting cell (APC).Our result illustrates the pharmaceutical composition of dexamethasone and treatment SARS all can bear adjusting inductive HLA-DR of TNF-α and ICAM-1 expression.

Claims

1. at least a gene regulatory peptides or its functional analogue production for treating suffer from or the pharmaceutical composition of the doubtful object of suffering from severe acute respiratory syndrome (SARS) in purposes, wherein said peptide or analog are selected from LQG, AQG, LQGV, AQGV, LQGA, VLPALPQVVC, VLPALP, ALPALP, VAPALP, ALPALPQ, VLPAAPQ, VLPALAQ, LAGV, VLAALP, VLPALA, VLPALPQ, VLAALPQ, VLPALPA, GVLPALP, GVLPALPQ, LQGVLPALPQVVC, VCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL, RPRCRPINATLAVEK, EGCPVCITVNTTICAGYCPT, SKAPPPSLPSPSRLPGPS, SIRLPGCPRGVNPVVS, LPGCPRGVNPVVS, LPGC, MTRV, MTR, VVC and QVVC.

2. due to the purposes of claim 1, wherein said severe acute respiratory syndrome are infected by SARS virus.

3. claim 1 or 2 purposes, wherein said object is in to be suffered from or doubtful that suffer from late period and may be in the danger of SARS in the stage that causes death, SARS described late period and that may be the stage that causes death is characterised in that and is selected from one or more following clinical sign or chemical examination finding: the lactic dehydrogenase enzyme level raises, the creatine kinase level raises, neutrophilic leukocytosis, thrombocytopenia, prolonged prothrombin, animal migration pneumonia and histanoxia.

4. each purposes among the claim 1-3, wherein said peptide or its analog can derive from gravid woman's urine.

5. the purposes of claim 4, wherein said women is in preceding 3 months of its gestation.

6. each purposes among the claim 1-3, wherein said peptide or its analog can derive from the promoting sexual gland hormone goods.

7. the purposes of claim 6, wherein said promoting sexual gland hormone comprises human chorionic gonadotropin (hCG).

8. each purposes among the claim 1-3, wherein said peptide or its analog can obtain by chemosynthesis.

9. the purposes of claim 8, wherein said compositions comprises the mixture of peptide LQGV, AQGV and VLPALP.

10. each purposes among the claim 1-9 is wherein tested the immunoregulatory activity of described compositions in the bioanalysis in a kind of body.

11. each purposes among the claim 1-10, the wherein immunoregulatory activity of the described compositions of test in a kind of external biological is analyzed.

12. each purposes among the claim 1-11, the existence of wherein testing the chemical compound that has immunoregulatory activity in the described compositions whether.