CN1367700A - Small peptides and method for downregulation of lgE - Google Patents

Abstract

A method for downregulating IgE levels is described. The method involves administering to a patient an IgE downregulating effective amount of a peptide having the formula f-Met-Leu-X, wherein X is selected from the group consisting of Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr.

Description

Be used to reduce little peptide and the method for IgE

Invention field

The present invention relates to have the method for reducing IgE active little peptide, particularly N-formyl-methionyl peptide and being used for the treatment of the indication of replying generation that mediates by IgE.In particular, these peptides are used in any application of using corticosteroid and replace corticosteroid.

Background of invention

IgE (IgE) is one of five antibody-likes that exist in the human body, just knows that before 30 years it is to be responsible for allergic immunoglobulin.After allergen is invaded human body, generate justacrine IgE by the B cell.Yet IgE only accounts for the sub-fraction (for 50-300ng/ml, by contrast, IgG is 10mg/ml) of the total antibody of human serum, so its quantity not sufficient is with in directly and antigen.On the other hand, its effect has obtained amplification by receptor in target cell, and cause at antigenic extensive cell response, in inflammation, itch, cough, shed tears, bronchoconstriction, mucous secretion, vomiting and diarrhoea, promptly in all symptoms usually relevant, culminate with allergic disorder.

Immediate hypersensitivity is by the high-affinity IgE receptor Fc that finds on mastocyte and basophilic granulocyte _εRI triggers.In conjunction with Fc _εThe allergen of RI conjunction type IgE causes the crosslinked of acceptor molecule on the cell membrane, triggers the threshing of cell then, discharges histamine subsequently and sends out other relevant medium of stage with allergic response speed.The cause inflammation activation of cell of these products of mastocyte threshing, an one-step inducing low-affinity IgE receptor Fc goes forward side by side _εRII (being also referred to as CD23).Activated B cell, multiple inflammatory cell (macrophage, eosinophile granulocyte, platelet, natural killer cell), T cell, follicular dendritic cell (follicular dendritic cell, FDC), can find Fc on the epithelial cell of Langerhans cell and bone marrow and thymus _εRII (people such as Delespesse, immunology progress (Adv.Immun.) 49:149-190,1991; People such as Delespesse, immunology comment (Immunol.Rev.) 125:78-97,1992).At B cell surface, Fc _εRII works in the antigen presentation of T cell relying on IgE, also works in B cell crosslinked.On FDC, Fc _εThe RII great expression relates to thus and raises the B cell and enter secondary follicle germinal center in lymph node and the spleen.Work as Fc _εWhen RII expresses, think that it is responsible for relying on the murder by poisoning cytosis of IgE, such as the phagocytosis of mononuclear cell to immune complex on inflammatory cell.Solubility Fc _εRII (sFc _εRII) growth and the differentiation of precursor that also can be by triggering plasma cell, T cell and basophilic granulocyte are initiated by body fluid and cell-mediated immune responses.

Become in the plasmacytic process of secretion IgE at the B cell differentiation, relate to the sophisticated signal cascade of cytokine and surface molecular, it is believed that this cascade mainly betides the secondary follicle germinal center in lymph node and the spleen.It is essential that surface molecular interacts for the physics that B cell and T cell and mastocyte are provided, and this effect is to trigger IgE to generate desired.These surface moleculars are CD40 aglucon (CD40L) and Fc _εRII.As 2 type t helper cell (Th ₂) when being activated being exposed to antigen-presenting cell (APC) back, their transient expression CD40L.CD40 on CD40L and the B cell interacts, and causes the activation of B cell.Th after the activation ₂The emiocytosis various kinds of cell factor, such as IL-4 and IL-13, the B cell that they act on after the activation makes it to turn to IgE to generate.IL-4 also raises the Fc on B cell and the inflammatory cell _εRII expresses, for thigmic stimulus and soluble growth factor provide more multi-source.

Mastocyte and basophilic granulocyte are also secreted IL-4 and are expressed CD40L, and thus can be when having IL-4 with Th ₂The similar mode of cell is synthetic by the interact IgE that induces the B cell of the physics with the B cell.Consider the tissue distribution of the various kinds of cell type that relates to the IgE generation, it is synthetic that IgE also may take place in skin, lung and the intestinal.

IgE is synthetic go up to be in harmonious proportion save the B of germinal center cell avoid apoptosis can be by B cell membrane conjunction type IgE and complement receptor 2 (CR2 is also referred to as CD21) through sFc _εThe crosslinked mediation of RII.CR2 is the memebrane protein of the high glycosylation found on B cell, FDC and some T cell and basophilic granulocyte.SFc _εRII can participate in the synthetic positive feedback control of IgE by the CR2 that triggers on the B cell, thereby it is synthetic to strengthen IgE, also promotes the survival of IgE typing B cell simultaneously.

The activation that IgE generates can cause two kinds of different situations.Expose the acute inflammation that causes by allergen and comprise that the early reaction of quick active mastocyte, respiratory tract macrophage and bronchial epithelial cell begins certainly, these cells discharge pro-inflammatory mediator, comprise histamine, eicosanoid, platelet activating factor, oxygen-free radical, neuropeptide and cytokine.These media can be induced airway smooth muscle contraction, mucous secretion and vasodilation.Respiratory inflammation causes that microvascular leakage increases, and causes blood plasma to penetrate in the respiratory tract, and then causes the airway walls thickening, and airway lumen narrows down.

In the reaction, peripheral blood cells is raised enters respiratory tract late, thereby forms chronic inflammatory disease.These cells comprise eosinophile granulocyte, lymphocyte and mononuclear cell, and raise the dependent cells factor, such as IL-5 and granulocyte-macrophage colony stimutaing factor (GMC-SF).As if chemotactic factor such as RANTES and eotaxin, also strengthen raising of eosinophile granulocyte.In the inflammation site, these cells are activated, and their survival is by obtaining to increase by reducing such as factor mediated Apoptosis such as GMC-SF.

To treatment of asthma traditionally based on the order of severity and persistent period of this disease.For acute intermittent symptom, treatment generally includes bronchodilators.Bronchodilators comprises beta-adrenergic agonist, methylxanthine and anticholinergic.These reagent can improve the respiratory tract obstruction in the asthmatic patient, but they as if reduce aspect respiratory inflammation or the bronchus overreaction invalid.In recent years, leukotriene inhibitors has been used for the treatment of slight to medium asthma.Leukotriene is produced through 5-lipoxidase metabolic pathway by arachidonic acid, and knows to have strong bronchoconstriction characteristic for a long time.These so-called slow-reacting substance of anaphylaxis (SRS-A) also induce multiple leukocyte to blood vessel migration, adhesion and gathering, and the increase capillary permeability, finally cause lung interstitial edema, leucocyte chemotaxis, mucus generation, mucociliary malfunction and bronchospasm.Leukotriene D particularly ₄(LTD ₄) the as if main this effect of being responsible in the respiratory tract, and the special receptor on asm cell is had an effect.Leukotriene (comprising the cysteinyl leukotriene) is to discharge in the mastocyte threshing course by the IgE mediation.

Leukotriene inhibitors comprises two types: a kind of activity by inhibition 5-lipoxidase (5-LO) is blocked the synthetic of leukotriene, and this activity is the synthetic needed of leukotriene; LTD on the another kind of competitive blocking-up smooth muscle cell ₄Receptor.Zileutin is first kind can supply the 5-fat oxidation enzyme inhibitor of utilization.Zafirlukast is first kind and treats granted LTD4 receptor antagonist, and other reagent such as monelukast and pranlukast, is carrying out clinical trial at present.These leukotriene inhibitors have been used for the treatment of slight lasting asthma so far, but do not prove the more severe form of asthma also effective as yet.

Adopt antiinflammatory to treat more serious and persistent form in the asthma at present.The reagent that classification belongs to antiinflammatory comprises theophylline, corticosteroid, sodium cromoglicate and sodium nedocromil.As if particularly corticosteroid is more effective aspect reduction bronchus overreaction and severe exacerbation.They contain that by the generation that suppresses cytokine and chemotactic factor eosinophile granulocyte raises, and by inducing the eosinophile granulocyte apoptosis to have an effect.They also act on eliminates respiratory tract edema and bronchorrhea, so the induction type corticosteroid is the most common treatment that is used for the chronic asthma patient.The induction type corticosteroid comprises that beclometasone (beclomethasone), 9-remove fluorine fluocinonide (flunisolide), omcilon (triamcinolone), fluticasone (fluticasone) and budesonide (budesonide).For chronic asthma, β ₂-agonist is invalid, although they can temporarily improve bronchial obstruction.Therefore, optimal treatment may be the β of associating induction type corticosteroid and long term ₂-agonist.Yet the potential side effect of corticosteroid comprises oral candidiasis, dysphonia, adrenal suppression, children growth sluggishness, thinning of skin, osteoporosis, glaucoma and cataract.In addition, not clear now, the relation between " effective dose " of these corticosteroid and " the poisonous dosage ".

Except the downstream events with the IgE signal pathway is a target, developing direct intervention IgE and more synthetic treatment New Policies thereof.The middle cardiac status prompting of IgE in causing allergic complex network is that the therapy of target will effectively be prevented all allergic responses to eliminate IgE or blocking-up IgE bind receptor.Though still be in commitment, obtained some successes at the use of the monoclonal antibody of IgE.(U.S. breathes and CC medical journal (Am.J.Respir.Crit.Care Med.) 155:1828-1834 according to people such as Fahy; 1997) report; humanization mouse monoclonal antibody at IgE has reduced free IgE, and successful blocking-up the early stage and late phase responses that allergen is stimulated.Among the targeting IgE in conjunction with Fc _εRI must the zone anti-IgE antibodies not only block combining of IgE and its receptor, also can prevent by being incorporated into Fc on the basophilic granulocyte _εCrosslinked inductive mastocyte threshing of the IgE of RI and mastocyte and anaphylaxis.These two kinds of anti-IgE antibodies are tested (people such as MacGlashan, Journal of Immunology (J.Immunol.) 158:1438-1445,1997 at present; People such as Corne, clinical investigation magazine (J.Clin.Invest.) 99:879-887,1997).As if up to now, they can reduce the IgE concentration in the serum, also can reduce the Fc on the basophilic granulocyte _εThe RI level illustrates that the cyclical level by regulation and control IgE can change replying of dependence IgE.

Treatment up to now focuses on usually by IgE and activates the downstream events that produces.Therefore, need exploitation to treat the Therapeutic Method of replying that mediates by IgE by regulation and control IgE level.Existing report, Chemotactic Peptide such as N-formyl-methionyl-leucyl-phenylalanine and pepstatin, can suppress mastocyte threshing (inflammation (Inflammation) 5 (1): 13-16,1981).Peptide of the present invention is reduced the IgE level, thereby can be used for regulating and control multiple the replying by the IgE mediation.

Summary of the invention

The invention provides use contains the pharmacopedics compositions with downward modulation IgE active N-formyl-methionyl-leucyl (f-Met-Leu) peptide and treats method by the multiple indication of replying generation of IgE mediation in suitable pharmacopedics carrier.Useful especially peptide is those peptides with general formula f-Met-Leu-X, and wherein X is selected from: Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr.Peptide of the present invention is used in any application of using corticosteroid and replaces corticosteroid.

According to the present invention, be used for the treatment of in the mammal the method for replying by the IgE mediation and comprise that with the amount of effective downward modulation IgE administration is had the peptide of general formula f-Met-Leu-X, wherein X is selected from: Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr.

The present invention also provides the method that is used to reduce film conjunction type and solubility IgE receptor, comprises with the amount of effective downward modulation IgE receptor the patient is used the peptide with general formula f-Met-Leu-X, and wherein X is selected from: Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr.

The present invention also provides the method that is used to suppress plasma cell secretion IgE, comprises with the excretory amount of effective inhibition IgE the patient is used the peptide with general formula f-Met-Leu-X, and wherein X is selected from: Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr.

According to another embodiment, the invention provides the method that is used to reduce the CD40L expression, comprise with the amount of effective downward modulation CD40L the patient used the peptide with general formula f-Met-Leu-X that wherein X is selected from: Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr.

In some preferred embodiment of the present invention, the patient can benefit from the co-administered of peptide of the present invention and second kind of active ingredient.For this co-administered useful especially other active ingredient of the present invention is for example anti-leukotriene, β ₂Agonist, corticosteroid, or the like.

The summary of figure

Fig. 1 is the log10 dose response curve, illustrates the influence of the HK-X of multiple dosage to the special serum IgE level of OVA in the acute asthma mice.

Fig. 2 has shown the lung sections of acute asthma mice after using 50 μ g HK-X.(A) and exist limited cell to infiltrate (B), there is limited mucus accumulation in (C).

Fig. 3 has shown the lung sections of acute asthma mice after using 10 μ g HK-X.Have cell (A) and (B) seldom in the respiratory tract, mucus is confined to the surface (C) of airway epithelial cell layer.

Fig. 4 has shown the lung sections of acute asthma mice after using 1 μ g HK-X.Therapeutic effect reduces, and cell infiltrate to increase (A), and being secreted into mucus in the respiratory tract increases (B) and (C).

Fig. 5 has shown with the OVA mice immunized with (B) post-stimulatory lung sections of saline (A) or carrier (0.05%DMSO).In respiratory tract, do not detect mucous secretion (C).

Fig. 6 is that diagram is used to set up the immunity of chronic asthma mouse model and the sketch map of therapeutic scheme.

Fig. 7 is the block diagram of granuloma number in the diagram chronic asthma mouse lung.

Fig. 8 has shown from weekly and reached for 6 weeks and with the histology pictures of the chronic asthma lung tissue of the mice of HK-X or brine treatment with the OVA immunity.(A) shown the lung tissue photo of control mice, (B) shown the histology pictures with the mice of HK-X treatment, (C) having shown with OVA stimulates but the histology pictures of the mice do not treated.

Fig. 9 has shown the light micrograph of chronic asthma mouse lung tissue accumulation collagenous fibrils.(A) shown the lung sections of using brinish control mice, (B) shown lung sections, (C) shown lung sections immune with OVA but the mice do not treated with the mice of HK-X treatment.

Figure 10 has shown the long-term lung sections of also using the mice of brine treatment with the OVA immunity.

Figure 11 has shown the long-term lung sections of also using the mice of carrier (0.5%DMSO) treatment with the OVA immunity.

Figure 12 is the block diagram of the tissue morphology metric method of diagram chronic asthma.

Figure 13 is the block diagram that is illustrated in the frequency of mucous cell in the chronic asthma mouse breathing road, multiple treatment back.

Figure 14 is the block diagram of the multiple treatment of diagram to the influence of eosinophile granulocyte and neutrophil cell infiltration in the chronic asthma mouse lung.

Figure 15 is illustrated in to use HK-X and the immunity of dexamethasone and the sketch map of therapeutic scheme in the acute asthma mouse model.

Figure 16 is comparison intranasal administration dexamethasone and the HK-X block diagram to the influence of OVA specific IgE level.

Figure 17 is illustrated in to use HK-X and the immunity of control peptide and the sketch map of therapeutic scheme in the acute asthma mouse model.

Detailed Description Of The Invention

According to the present invention, find to have general formula f-Met-Leu-X (wherein X is selected from: Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr) some little peptide have surprising activity, namely Downward modulation IgE level. As a result of, these peptides can be used for treating the generation of replying by the IgE mediation Multiple indication. Peptide of the present invention is used in any application of using corticosteroid and replaces skin The matter steroids.

According to the present invention, preferred peptide can reduce blood IgE level, and the blocking-up lymphocyte (such as Macrophage, monocyte, eosinophile granulocyte, neutrophil cell, TNF, like that) IgE activate.

In preferred embodiments, use peptide of the present invention to treat, also can reduce or complete Full cut-off ends the continuous threshing of mast cell and discharges leukotriene, histamine and other cell factor. According to the preferred embodiments of the invention, peptide of the present invention also can reduce eosinophile granulocyte, have a liking for Alkalescence granulocyte and neutrophil cell infiltrate the inflammation tissue. Lymphocyte, eosinophil granules are thin Born of the same parents and neutrophil cell are not showed chemotaxis when replying preferred peptide of the present invention. Therefore, Lymphocyte, eosinophile granulocyte and neutrophil cell to the chemotactic in inflammation site adhere to, Migration and gathering significantly reduce, and the vasopermeability in inflammation site reduces equally. In addition, originally The preferred compound of invention is not showed toxicity to vitals (all after one's own heart, liver and lung).

Can prepare peptide of the present invention by traditional little chemistry of peptides synthetic technology. When using, Under aseptic condition, prepare these peptides with pharmaceutics acceptable carrier or diluent.

The pharmaceutics composition can exist with unit dosage forms easily, and preparation is used for by the IgE mediation Reply the indication to be treated of the each type of generation. Can be well-known by the pharmaceutics field Any method prepares composition. These methods generally include mixes active ingredient of the present invention and comprises The step of the carrier of one or more attached compositions.

For example, the dosage of pharmaceutics composition will be with object indication type and institute's apparatus to be treated Body is used the path and is changed. When treating acute the replying that is mediated by IgE, the dosage model of active peptide Enclose and to be 0.1-100 every day, 000 μ g/kg, more preferably 1-10,000 μ g/kg. Most preferred Dosage range is about 1-100 μ g/kg body weight, more preferably about 1-20 μ g/kg, most preferably 10-20 μ g/kg. When treating chronic the replying that is mediated by IgE, the dosage range of active peptide can Think 0.1-100 every day, 000 μ g/kg, more preferably 1-10,000 μ g/kg. Most preferred dosage Scope is about 1-1000 μ g/kg body weight, more preferably about 1-100 μ g/kg, most preferably 50-70 μ g/kg. According to the order of severity of situation, frequency of administration is normally little to every 4-6 by every day 1 time The time 1 time. For acute situation, preferred every 4-6 hour 1 administration for peptides. For keeping, may be excellent Select and use 1 time or 2 times every day. Preferably, according to the order of severity of using path and situation, Use the about 16mg peptide of about 0.18-every day. Only need to adopt normal experiment method, those of ordinary skill Just can be identified for delivering interval expeced time of the many doses of particular composition.

Use the path comprise oral, parenteral, rectum, intravaginal, part, nose, eye, direct injection, etc.In preferred embodiments, with the amount of effective downward modulation IgE the patient is used peptide of the present invention.Exemplary pharmacopedics compositions is effectively to regulate and control the peptide of the present invention of the amount of IgE, and this peptide can provide the effect of downward modulation IgE, and is contained in usually in the pharmacopedics acceptable carrier.

When being used for reaching hereinafter more complete description herein, term " pharmacopedics acceptable carrier " comprises one or more compatible solids or liquid filler diluent or the encapsulation material that is fit to be applied to people or other animal.In the present invention, term " carrier " thereby refer to be used for molecule associating of the present invention so that the natural or synthetic organic or inorganic composition of using.Term " is effectively regulated and control the amount of IgE " and is referred to that the amount of this pharmacopedics compositions produces the effect of downward modulation IgE to particular condition to be treated.In the preparation of the compositions of mixing identical composition, can use multiple concentration, with the variation in persistent period of the order of severity that adapts to patient's age to be treated, situation, treatment and the pattern used.

Carrier also must be compatible.When being used for herein, term " compatible " refers to that the composition of pharmacopedics compositions can mix with the little peptide of the present invention, and hybrid mode does not each other weaken required pharmacopedics effect basically.

Little peptide of the present invention (pure) is used with itself usually.Yet the form that they can pharmacopedics acceptable salt class is used.The acceptable salt of these pharmacopedicss includes but not limited to the salt that those are equipped with by following processed with acid: hydrochloric acid, hydrobromic acid, sulphuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-methyl benzenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene 2-sulfonic acid and benzenesulfonic acid.Equally, the acceptable salt of pharmacopedics can be prepared into alkalinous metal or alkaline earth salt, such as sodium, potassium or the calcium salt of hydroxy-acid group.Thus, the invention provides and comprise peptide of the present invention and one or more pharmacopedicss can be accepted carrier (with optional any other pharmacopedics composition) to be used for the pharmacopedics compositions of medical usage.

Compositions comprises the compositions that those are applicable to oral, rectum, intravaginal, part, nose, eye or parenteral administration, and all these are used the path and all can be used for material of the present invention.The pharmacopedics compositions that contains peptide of the present invention also can comprise one or more pharmacopedics acceptable carriers, comprise excipient, such as stabilizing agent (to promote long term storage), emulsifying agent, binding agent, thickening agent, salt, antiseptic, solvent, disperse medium, coating, antibacterial agent and antifungal, etc. blend absorption delay agent, like that.The use in the pharmacopedics active substance of these media and reagent is well known in the art.Unless incompatible with peptide of the present invention, any traditional sucrose or the use of reagent in the pharmacopedics preparation contained in this paper.The auxiliary activity composition also can mix compositions of the present invention.

Be applicable to that Orally administered compositions is mixed with induction type aerosol, spray, syrup or tablet usually.Be applicable to that topical application of compositions is mixed with emulsifiable paste, ointment or solution usually.For acute reply of treatment by the IgE mediation, the concentration of peptide active ingredient is usually less than 1000 μ g/ml in these compositionss, more preferably less than 500 μ g/ml, and most preferably about 200-400 μ g/ml.For chronic reply of treatment by the IgE mediation, the concentration of peptide active ingredient is usually less than 3mg/ml in these compositionss, more preferably less than 2mg/ml, and most preferably about 1-1.5mg/ml.

Be applicable to that sucking the present composition of using can be mixed with for example aerosol or inhalant liquid.Be used for the treatment of example by the acute typical aerosol combination of replying of IgE mediation and comprise that being suspended in isceon and two chloro-difluoromethane adds about 0.1-100 μ g crystallite peptide (every dose) in the oleic mixture.The amount of crystallite peptide more preferably 1-50 μ g, most preferably 10-20 μ g (every dose of aerosol combination) in the compositions.Be used for the treatment of example by the chronic typical aerosol combination of replying of IgE mediation and comprise that being suspended in three chloro-fluomethanes and two chloro-difluoromethane adds about 0.1-1000 μ g crystallite peptide (every dose) in the oleic mixture.The amount of crystallite peptide more preferably 1-100 μ g, most preferably 50-70 μ g (every dose of aerosol combination) in the compositions.The example of typical solution comprises dissolving or is suspended in the peptide of the desired amount of Sterile Saline (for the dissolubility needs, optional about 5%v/v dimethyl sulfoxine (DMSO)), benzalkonium chloride and sulphuric acid (being used to transfer pH).

Be applicable to that the Orally administered present composition also can be mixed with discrete unit, such as capsule, flat capsule, tablet or lozenge, each contains with good grounds peptide of the present invention of replying scheduled volume to be treated by the IgE mediation, perhaps can be contained in the liposome, perhaps as the suspension in aqueous solution or the non-aqueous solution, such as syrup, elixir or Emulsion.The example of tablet formulation substrate comprises corn starch, lactose and magnesium stearate, as the non-activity composition.The example of syrup formula substrate comprises citric acid, dyestuff, flavoring agent, hydroxypropyl emthylcellulose, glucide, sodium benzoate, sodium citrate and pure water.

The compositions that is applicable to parenteral administration comprises the sterilized water preparation of molecule of the present invention easily, preferably oozes with blood of receiver etc.Can use those suitable dispersions or wetting agent and suspending agent to prepare this water formulation with reference to known method.Sterile injectable preparation can also be that avirulent parenteral can be accepted sterile injectable solution or the suspension in diluent or the solvent, for example solution in the 1,3 butylene glycol.In acceptable carrier and solvent, adoptable is water, RingerShi liquid and isotonic sodium chlorrde solution.In aqueous solution, can be used for keeping the dissolubility of some peptide up to approximately 10%v/v DMSO or Trappsol.Equally, can tradition adopt aseptic fixed oil as solvent or suspension media.For this purpose, many expressed ois be can adopt, synthetic monoglyceride or diglyceride comprised.In addition, fatty acid (such as oleic acid or neutral fat acid) can be used for preparation

Set up the scheme of using ovalbumin (OVA) thereby in normal Balb/C mice, inducing acute allergen specific pneumonopathy as the model allergen.This scheme comprises the 1st and 14 day (IP) immunity in the mouse peritoneum is dissolved in 100 μ g OVA of adsorbed onto alum adjuvant, the 14th, 25,26 and 27 day single agent intranasal (IN) use the 50-100 μ g OVA that is dissolved in normal saline solution.Control mice is only accepted Alumen by the IP injection, uses by IN and only accepts normal saline solution.The 28th day, with the OVA mice immunized show to by the surprising similar disease of the inductive people's asthma of allergen.This animal model has been used for estimating the effect of medicine in the allergia acute lung diseases.Late period chronic allergen specific pneumonopathy mouse model

Use ovalbumin (OVA) thus in normal Balb/C mice, induce the scheme of chronic allergen specific pneumonopathy in late period as the model allergen, comprise the 1st and 14 day and (IP) immunity in the mouse peritoneum is dissolved in 100 μ g OVA of adsorbed onto alum adjuvant, 14th, 25,26 and 27 days single agent intranasal (IN) use the 50-100 μ g OVA that is dissolved in normal saline solution, reach 6 months once in a week thereafter.Control mice is only accepted Alumen by the IP injection, uses by IN and only accepts normal saline solution.The 28th day, with the OVA mice immunized show to by the surprising similar disease of the inductive people's asthma of allergen.This animal model also can be used for estimating the effect of medicine in chronic allergia pneumonopathy.Material and method

Special reagent: (Rockford IL) obtains the OVA crystal, and (St.Louis MO) obtains aluminium potassium sulfate (Alumen) by the Sigma chemical company by the Pierce chemical company.OVA (500 μ g/m1) is mixed in distilled water with isopyknic 10% (wt/vol) Alumen.With 10NNaOH mixture is transferred to pH6.5, and in room temperature insulation 60 minutes.With mixture centrifugal 5 minutes, precipitation is resuspended in the distilled water of initial volume, and in 1 hour, uses with 750g.

Immunization protocol: immunization protocol comprises that the 1st day intraperitoneal use the 100 μ gOVA that are dissolved in Alumen, and intraperitoneal was used the 100 μ g OVA that are dissolved in Alumen in the 14th day subsequently, and the associating intranasal administration is dissolved in brinish 100 μ g OVA.25th, 26 and 27 days, stimulation was dissolved in brinish 100 μ g OVA to the mice intranasal.For acute asthma research, the 28th day peaceful and comfortable execution animal also downcut lung.For chronic asthma research, weekly thereafter immune mouse reaches 6 months.Analyze

The ELISA scheme of SERUM IgE: spent the night by Immulon2 microtitration plate (DynexTechnologies) in 4 ℃ of bags with the OVA solution that is dissolved in 50mM carbonate-bicarbonate buffer (pH9.6), and sealed 2 hours in room temperature (RT) with 0.1% casein.Before being incubated by plate, all test serums are diluted with containing 0.1% caseic Tris-NaCl buffer (pH8.0) at 1: 100 with the OVA bag.Comprise in measuring each time and knownly contain at the positive serum sample of the IgE antibody of OVA and in contrast from the normal serum sample of immune mouse not.Blood serum sample is incubated 2 hours in room temperature on flat board, and cleans 6 times with PBS.(the sheep anti mice IgE of biotin-conjugated (Binding Site catalog number (Cat.No.) PB 284, lot number 026917) in room temperature insulation 2 hours, and cleans dull and stereotyped 6 times with PBS to add two anti-after the suitably dilution.Add OPD, carbamide and peroxide solutions, in room temperature insulation 30 minutes.Add 2.5M sulphuric acid cessation reaction.Read the OD of 490/630nm.All samples all carries out double.Change sub-average 10% in the sample room of positive control and the sample.

Lung tissue: downcut lung and trachea, and fixing in the formalin of 10% neutral buffered.Investing tissue in paraffin, and be cut into 7 μ m section.After dewaxing and the hydration, will cut into slices, and redye with methylene blue with the dyeing of eosinophilic staining liquid.The alizarin indigo plant of love, toluidine blue and PAS stain have been identified the mucus in the respiratory tract.By optical microscopy inspection tissue.

Bronchoalveolar lavage (BAL): tie left lung at main stem bronchus.With 0.4ml normal saline solution lavation right lung 3 times, and merge fluid.Use hematimeter to measure the total cellular score order.By the centrifugation residual cells, and cell is resuspended in 10%BSA solution.Cell is dropped on the microscope slide, and with eosinophilic staining liquid dyeing (eosin and methylene blue are redyed).

The tissue morphology metric method of lung is analyzed: the following parameters of measuring allergia pneumonopathy herein in Bao Gao the experiment: 1. as former report (people such as Henderson, The Journal of Experimental Medicine (J.Exp.Med.) 184:1483-1494,1996) described, carry out the airway plug scoring.Use by+extremely ++ ++ marking system, the reflection mucous secretion the order of severity.2. estimate total myxocyte by mucous epithelial counting at random in per 100 epithelial cells in the medium-sized extremely large-scale respiratory tract (600-1,000 μ m diameter).To 10 in different lung pages or leaves zone countings.3. by using extremely by o ++ ++ marking system estimate to be arranged in the cell density (neutrophil cell, eosinophile granulocyte, mononuclear cell and lymphocyte) of the infiltration cell of compartment around the blood vessel or respiratory tract contiguous zone.+ refer to the inflammatory cell layer of the individual cell of 3-5 (not containing); ++ refer to the inflammation density of 5-10 cell; +++refer to 10-20 cell inflammation density; ++ ++ refer to the inflammation density of 20-40 cell.4. by each high power field (10x by 40x) counting being quantized the number of various cell types.5. calculate the edema degree by the marking system that uses assessment blood vessel surrounding fluid accumulation degree.

The statistical analysis of tissue morphology metric method data: use SigmaStat 2.0 editions to carry out statistical analysis.Use the suitable posthoc of separate average value to check the difference of analyzing significance (p＜0.05) by ANOVA.Adopt SigmaPlot 4.0 editions or GraphPad Prism to carry out the structure that datagraphic is showed.

EXAMPLE Example 1: the acute asthma mice is replied the therapeutic dosage of HK-X

It is desirable to, the experiment of demonstration therapeutic effect and drug dose dependency will show the behavior district of 3 uniquenesses: 1) in low dose, with readily good therapeutic effect; 2) heavy dose of, therapeutic efficiency is with dependent dose; 3) at the 3rd dosage range of maximum, the therapeutic efficiency of demonstration is no more than the observed result of maximum intermediate range dosage.

Dose response curve is the important information source about the dosage safety of mankind's application.Sometimes, when the drug dose of using surpasses optimal therapeutic dosage, can observe toxic response.Drug administration is especially true during such as intranasal in position.

In order to determine to render a service, select the HK-X of intranasal administration 0.1,1.0,10 and 50 μ g dosage in the treatment of carrying out the f-Met-Leu-Phe-Phe (HK-X) of (the 25th, 26 and 27 day) range of doses of inductive bronchial asthma acute effect stage with OVA repetition immunity.Used HK-X in preceding 30 minutes stimulating with OVA.Matched group comprises with OVA immunity and the mice that stimulates with OVA, and with being dissolved in brinish Alumen immunity and only with the animal of saline stimulation.Stimulated with OVA for the last time, and put to death all animals next day in the 28th day.Measure serum IgE level, and gather serum and lung tissue is used for further analysis.

In order at first to determine effectively to reduce the optimal dose of serum IgE level in the acute asthma model, preceding 15-30 minute of antigenic stimulus in the 25th, 26 and 27 day is with HK-X (being dissolved in 40 μ l saline) the injection lung of 0.1,1.0,10 and 50 μ g dosage.Fig. 1 has described the dose response curve of serum IgE level.

The HK-X that Fig. 2 A-4C has described various dose replys the influence that the acute change answers sexual stimulus to lung tissue.To acute asthma mice intranasal administration 50 μ g HK-X, provide protection to a certain degree (Fig. 2 A-2C) at the effect of acute asthma.Fig. 2 A and 2B have shown around the blood vessel and have had limited inflammatory cell accumulation around the bronchus.Fig. 2 C has shown and has had limited mucus accumulation.

10 μ g HK-X are the most effective dosage (Fig. 3 A-3C) seemingly.Fig. 3 A and 3B have shown the inflammation infiltration of minimum on every side of blood vessel and respiratory tract.Fig. 3 C has shown the mucous secretion degree in the respiratory tract, and mucus is confined to the surface of airway epithelial cell.

When the dosage of HK-X reduces by 10 times during to 1 μ g, therapeutic effect disappears.Around the blood vessel and the seepage discharge of respiratory tract increase (Fig. 4 A).The corresponding increase of the mucous secretion of airway epithelial cell (Fig. 4 B and 4C).

For the purpose that contrasts and contrast, Fig. 5 A-5C has shown through immune mouse using optimum the replying of saline or HK-X carrier (0.05%DMS0).Shown in Fig. 5 A and 5B, around the lung medium vessels or the respiratory tract peripheral region exist and seldom measure detectable inflammation and infiltrate.Accordingly, shown in Fig. 5 C, there is not mucus accumulation at airway lumen or airway epithelial cell surface.

Learn in the measurement in the critical tissue of the acute asthma order of severity, mucous plug, eosinophile granulocyte number and muciparous respiratory tract cell proportion are in the improvement that shows dependent dose with 0.1 μ g-10 μ g HK-X treatment back.10 μ g HK-X make mucous plug scoring reduction the by 70% (p＜0.05).Interesting is that the mucous plug that 50 μ g HK-X cause reduces much lower (p＜0.05).Observing replying of model identical aspect eosinophile granulocyte number and the muciparous respiratory tract cell proportion.Matter eosinophile granulocyte number had descended 57% between the HK-X of 10 μ g dosage showed, significantly than the effect height (p＜0.05) of 0.1 μ g dosage.The eosinophile granulocyte suppression ratio 10 μ g HK-X that cause by 50 μ g HK-X cause half also to lack (p＜0.05).

Muciparous respiratory tract cell proportion also is suppressed (reducing by 37%, p＜0.05) in the mode of dependent dose by 0.1 μ g to 10 μ g.50 μ g dosage cause a small amount of reduction (11%), significantly are not lower than the HK-X of 0.1 μ g dosage.HK-X shows that at 10 μ g and 50 μ g dosage appropriateness reduces to blood vessel surrounding fluid effect of accumulation.Yet, do not have which kind of dosage different with the HK-X of 0.1 μ g dosage.Showing aspect inflammatory cell scoring or the inflammatory cell accumulation that the maximum HK-X dosage that reduces is 10 μ g.

Parameter show dose response effect below these data proofs: serum IgE level and histopathology feature (the eosinophile granulocyte sum in cell infiltration, mucous plug formation and the matter).Compare with one heavy dose of (i.e. 50 μ g) with smaller dose, intranasal administration 10 μ g HK-X are the most effective dosage.Compared with the control, the animal display serum IgE level for the treatment of with 10 μ g HK-X reduces by 60%, and the cell of lung infiltrates and reduces by 50%, and mucous plug forms reduction by 70% and the eosinophile granulocyte number reduces by 67%.Embodiment 2: carry out or do not carry out the chronic asthma that HK-X intervenes

Animal model also can be used for estimating the effect of medicine in chronic allergia pneumonopathy.In this research, 6 months duration of immunity has been induced lasting inflammation, and keeps by weekly the stimulation with the OVA intranasal.In 20 days time, use brine treatment mice 8 times, to estimate the variation that takes place in the lung.As shown in Figure 6, with HK-X treatment chronic asthma mice.IN uses 50 μ g HK-X (be dissolved in 50 μ 1 and contain the saline that is lower than 2.5%DMSO), has delivered 8 doses in 16 days time altogether.Take for the last time behind saline or the HK-X and to put to death animal in 4 days.Comparative experiments result between the mice of using and not treating with HK-X.

Table 1 has shown in the mouse blood that uses or do not stimulate with OVA the antibody IgE level at OVA.Be important to note that initial 6 months with all animals of OVA immunity, yet, to being called the group of " saline ", intranasal administration saline in the end 20 days but stimulate without OVA.Therefore, these IgE levels are from duration of immunity, and value is used to proofread and correct all relatively as a setting.For example, the IgE level has increased by 36% in the animal for the treatment of with saline or DMSO and stimulating with OVA, and by contrast, the IgE level has increased by 14% in the animal for the treatment of with HK-X and stimulating with OVA.According to calculating, the amount of HK-X containment IgE level is about 60%.

Table 1: the IgE value in the chronic asthma mice serum

Treat average OD SE P value

DMSO/OVA 1.115 0.017 @0.111

Saline/OVA 1.113 0.093 @0.111

HK-X/OVA 0.929 0.033 0.049@0.111

Saline 0.814 0.079 @0.111

Note: these numerical value are with from the OD value representation IgE of ELISA test level relatively.

Chronic asthma key character is the appearance of granuloma structure in lung in mouse model.(saline or DMSO) compares with the animal that makes its spontaneous reduction collagen deposition, and effective downward modulation IgE dosage 50 μ g of HK-X are the number and the size (Fig. 7) of (p＜0.05) reduction these structures in treatment animal lung significantly.

In addition, with the OVA immune mouse with in the process that the HK-X of 8 usefulness 50 μ g dosage treats altogether in 20 days time subsequently, do not observe untoward reaction or disease indication.The experimental session mice is active.Disclosed the variation of science of serious pneumonopathy from lung tissue inspection only with the animal groups of OVA immunity, consistent with observed feature in people's chronic asthma.Peripheral boundary at respiratory tract basal layer (a matter district) and blood vessel exists significant inflammatory cell to infiltrate (Fig. 8 C).During with 8 doses of each 50 μ g HK-X treatment animals, respiratory tract and circumvascular inflammatory cell number obviously reduce (Fig. 8 B) in 20 days time.Suck saline with false immunity of saline or contrast mice immunized, cause obvious respiratory tract and blood vessel to have normal appearance (Fig. 8 A).In with the OVA mice immunized, collagen (blueness) accumulation around blood vessel and the respiratory tract continues to increase (Fig. 9 C).Yet, show that with the lung of HK-X treatment the collagen deposition level reduces (Fig. 9 B).In control mice (use and be dissolved in brinish HK-X), there are not inflammatory cell and deposition of fibrotic collagen (Fig. 9 A) in the lung tissue.On the contrary, in an OVA sensitization and a mice for the treatment of with saline (Figure 10 A-10C) or 0.5%DMSO (Figure 11 A-11C), mucus and muciparous cell are arranged in the respiratory tract, use and like that madder blue (pH2.3) shows mucus.Inflammatory cell infiltration capacity to respiratory tract around the blood vessel is similar.In these chronic asthma mice, surpass 60% respiratory tract by mucus thromboembolism (Figure 10 A and 10B, Figure 11 A and 11B) at 6 monthly age

Also estimated the lung tissue in the chronic asthma and changed, to quantize the degree that lasting inflammation, deposition of fibrotic collagen and respiratory tract narrow down with structural change by the method for morphometry.In Figure 12-14, left figure has shown with the OVA immunity and has reached 6 months and with the replying of the animal of plurality of reagents treatment, and right figure has shown the animal with false immunity of saline and treatment.With the animal of HK-X treatment and only give in 20 days to have significant difference (p＜0.05) (Figure 12) between the mucous plug formation degree of brinish animal.With carrier (0.5%DMSO) but not the animal of HK-X immunity compare with HK-X and also do not showing any improvement aspect the mucous plug scoring.The result is identical with brine treatment.For the kinds of experiments treatment, among the respiratory tract and the answer-mode of inflammatory cell on every side accumulation be parallel to mucous plug data (Figure 12).After intranasal in 16 days gives 8 doses of each 50 μ g HK-X (being dissolved in 40 μ l carriers), significantly reduce mucus accumulation in the respiratory tract and the generation (Figure 13) of myxocyte.Only use brinish treatment to show readily good therapeutic effect once more, but HK-X significantly reduce the number (p＜0.05) of mucous cell in the respiratory tract really.This observed result further confirms not have spontaneous reparation after having set up chronic asthma inducing with OVA.In the analysis of the relevant accessibility inflammatory cell number of respiratory tract, eosinophile granulocyte and neutrophil cell on the data unit of display area have also reduced (Figure 14).Intranasal injects brinish animal and keeps high-caliber eosinophile granulocyte, with the animal of DMSO treatment (p＞0.05) too; Yet, to compare with two matched groups, the HK-X treatment significantly reduces the number (p＜0.05) of eosinophile granulocyte.

These studies show that, in this model with the inductive chronic asthma of allergen, exist very faint in respiratory inflammation or myxocyte secretion even do not have spontaneous reduction, unless use HK-X.In this model, make mice to OVA sensitization, and be exposed to OVA once in a week by the intranasal path and reach 5 months, 8 intranasal administration HK-X treat in 20 days time then.This allergen immunity and stimulation protocol cause the chronic infiltration respiratory tract of inflammatory cell of eosinophile granulocyte and other type, and mucus accumulates in respiratory tract, and muciparous cell hypertrophy.Use HK-X with the dosage (i.e. 50 μ g) of effective downward modulation IgE and can reduce hypertrophy of respiratory tract excessive secretion, myxocyte and raising of eosinophile granulocyte and neutrophil cell.These presentation of results also can be reduced by the replying of IgE mediation by use HK-X with the amount of effective downward modulation IgE level, such as at this respiratory tract excessive secretion mucus and the collagen deposition that is taken place in the inductive asthmatic model by allergen.Embodiment 3:HK-X and the dexamethasone effect in acute Mus asthma

Glucocorticoid is by the strong inhibitor of the inflammatory mediator of various kinds of cell type generation, comprises T cell, mastocyte, mononuclear cell, dendritic cell and eosinophile granulocyte.Can effectively treat people's asthma by suction or systemic application glucocorticoid.They can contain that inflammatory cell infiltrates, and verified mucous secretion and the pulmonary edema of reducing.These reply the direct effect of glucocorticoid to bronchial epithelial cell that relate to.It is also important that steroid can reduce bronchus and excessively reply.Because their effect has obtained confirmation, glucocorticoid has been represented the main therapeutant in the treating asthma, and can uncensoredly use, but because the cumulative toxicity of its existing record has limited their value in time.Because they are as the value of the current standard of asthma effect, the noval chemical compound that is used for treating asthma should compare evaluation with glucocorticoid.

This experiment comparison HK-X and dexamethasone (a kind of widely used glucocorticoid) are regulated and control the effect of mucus release, eosinophile granulocyte number, edema and allergen specific IgE level in this mouse model.The compared dosage 10 μ g and the 50 μ g of HK-X and dexamethasone in this research, have been used.Two kinds of medicines are all carried out intranasal administration at all dosage.From the result of chronic asthma model, selected 50 μ g HK-X according to before as heavy dose.Figure 15 has shown the outline of immunity and therapeutic scheme.

Experiment parameter shows that aspect the reduction serum IgE level, intranasal administration 10 μ g HK-X are than 10 μ g or 50 μ g dexamethasone more effective (Figure 16).10 μ g HK-X have reduced by 28% with serum IgE level.Two kinds of histopathology characteristic aspect below improving, HK-X is also more effective than dexamethasone: the eosinophile granulocyte sum in a cell infiltration and the matter.Compare with the OVA contrast, the HK-X of 10 μ g and 50 μ g dosage infiltrates inflammation and significantly reduces by 54% (p＜0.05), than 10 μ g (13%) and 50 μ g (13%) dexamethasone remarkable effectively (p＜0.05).The HK-X of 10 μ g and 50 μ g dosage is than 10 μ g dexamethasone more effective (p＜0.05).10 μ g HK-X have reduced by 57% with the eosinophile granulocyte counting, and the dexamethasone of same dose has reduced by 13% with the eosinophile granulocyte counting.

Histopathology characteristic aspect below reducing, the HK-X of intranasal administration 50 μ g heavy doses is the same effective with two kinds of doses of dexamethasone: bronchoalveolar lavage (BronchoalveolarLavage, BAL) the eosinophile granulocyte number in, mucous plug formation, the shared percentage ratio of muciparous respiratory tract cell, matter eosinophile granulocyte number and edema.These results have determined the comparative effect of HK-X and a kind of glucocorticoid (being dexamethasone) in the Mus asthmatic model.Embodiment 4:HK-X and the effect of relevant control peptide in acute Mus asthma

The effectiveness that compares the relevant member f-Met-Met of HK-X (being called control peptide) in this experiment measurement below: the mucus release in the acute asthma mouse model, cell infiltration, eosinophile granulocyte number, edema, allergen specific IgE level with peptide family.The compared dosage 50 μ g of HK-X and control peptide in this research, have been used.Two kinds of chemical compounds are all carried out intranasal administration.Figure 17 has shown the outline of immunity and therapeutic scheme.

Though it is closely related to belong to the identical family and the molecular size of chemical compound, control peptide is not showed any treatment characteristic of HK-X.The most significant is that 50 μ g HK-X cause that the serum IgE level at allergen OVA reduces by 7% (p＞0.05) in the serum.50 μ g control peptides do not influence serum IgE level (p＞0.05).In addition, in carrier, deliver and be applied to short scorching the increasing of control peptide obvious promotion aspect following parameters of control animal: mucous plug formation, the number of muciparous respiratory tract cell and the degree of a matter inflammatory cell.In the experiment formerly, HK-X is not showing any short scorching variation aspect the Histological parameter of measuring.Therefore, as if the uniqueness of HK-X is formed and to be responsible for it in downward modulation IgE level with by the effect in the replying of IgE mediation.Embodiment 5: the lung tissue of taking high treatment level HK-X is for a long time replied

Whether the high end value of therapeutic dose scope that is exposed to HK-X for definite long-term intranasal exists potential poisonous influence, the weekly intranasal of mice is exposed to 20 μ g HK-X reaches 3 months.In two weeks in the end, the intranasal dose of HK-X increases to 50 μ g.Used for the last time behind the HK-X 24 hours, and gathered lung tissue and be used for histologic analysis.

Weekly intranasal administration 20 μ g HK-X reach 3 months, use 50 μ g subsequently and reach for 2 weeks, do not cause the pathological change of lung tissue.Aspect mucous plug formation and inflammation infiltration, using saline and HK-X does not have difference (p＞0.05).The mucous secretion of using respiratory tract cell behind the HK-X increases, but does not think biologically it is significant.Observing similar phenomena aspect the matter eosinophile granulocyte number.Animal with the HK-X treatment is per 2200 μ ²About 2 eosinophile granulocytes, and be lower than 1 cell of per unit area (p＜0.05) with the animal display of brine treatment.Liver, spleen and kidney are checked that pathology change.Except focus once in a while, do not observe pathology and change from inflammatory cell in the liver of contrast and treatment treated animal.These data have been determined the toleration of super therapeutic dose in mice of HK-X.The immunogenicity of embodiment 6:HK-X and antigenicity

The purpose of this research is to determine whether can produce immunne response (estimating by the antibody generation) at HK-X when several different paths are applied to mice.Thus, immunogenicity relevant and antigenicity in this research, have been estimated simultaneously with HK-X.

HK-X is small-sized tetrapeptide.In most applications, these micromolecular immunogenicities are very low; Yet in vivo, micromolecule can be puted together or adsorb (becoming hapten) bigger protein or hemocyte (carrier).Penicillin, chinidine and alpha-methyldopa allergic response are exactly the example that this hapten is replied.Owing to destroy erythrocyte (carrier), can cause anemia and immune plyability disease at haptenic antibody.

Many hapten are such as the dinitrophenol,DNP (DNP) or the trinitrophenol (TNP) of experiment purposes, covalently bound carrier molecule.The antigenicity of carrier molecule is high more, then causes at the probability of haptenic immunne response big more.Keyhole  hemocyanin (KLH) is widely used carrier, supports the strong antibody response at hapten (as DNP or TNP) usually.

The use of adjuvant has greatly increased the probability that potential immunogen causes immunne response.Complete Freund's adjuvant (CFA) or bacterial peptide polysaccharide have been widely used in stimulation at the very low haptenic immunne response of immunogenicity.

Therefore, after at first determining (not having anti-HK-X reactivity), use, checked the potential immunogenicity of HK-X by coupling KLH and in the antibacterial adjuvant from the utilizability of the antibody in normal drug exposure path.These extreme conditions can determine whether HK-X has immunogenicity.Material and method

The immunogenic conjugate of HK-X: the spacerarm of the carbon of the 12-20 through making an addition to carboxyl terminal makes HK-X put together KLH.Lysine residue on KLH is finished connection.(Seattle WA) prepares this conjugate by UnitedBiochemical.

Immunogenic preparation: will with 0.1mg/ml be suspended in HK-X-KLH conjugate among the PBS in the complete Freund's adjuvant that contains the 1.0mg/ml mycobacterium tuberculosis var bovis (CFA) with emulsifying in 1: 1.

The adjuvant immunity scheme: to Balb/C female mice intradermal immunization 0.1ml Emulsion, booster immunization after 4 weeks, the back blood sampling of 6 weeks.

Solution immunization protocol: the conjugate (volume 0.1-0.2ml) that Balb/C female mice peritoneal injection 100 μ g is not contained adjuvant.Blood sampling after 21 days.

Normal drug exposure path: in the research of therapeutic asthma, gather serum by the animal of using HK-X through the intranasal path.

The mensuration of antibody: by elisa assay at puting together and not puting together the antibody of HK-X.The following HK-X or the HK-X conjugate that are dissolved in PBS in order to 10 μ g/ml are spent the night by Immulon2 microtitration plate (Dynex Technologies, catalog number (Cat.No.) 3455) in 4 ℃ of bags:

Only use the HK-X peptide

HK-X-KLH, the conjugate of peptide and KLH

HK-X-BSA, the conjugate of peptide and BSA

The HK-X-spacerarm contains the peptide of the linear interval arm of 12 carbon.Inferior daily PBS clean-out opening uses diluted sample buffer (0.1M Tris/0.15M NaCl buffer, pH8.0 add 0.1% casein (ICN, catalog number (Cat.No.) 902896, lot number 99333)) in room temperature sealing 30 minutes then.The mice serum sample with same buffer 1: 100 or dilution in 1: 200, is added in the hole, and in room temperature insulation 2 hours.Use the PBS clean-out opening then, and be incubated 2 hours in room temperature with the goat anti-mouse IgG two anti-(Cappel, catalog number (Cat.No.) 55554, lot number 39714) of puting together peroxidase.After cleaning with PBS, with hole and OPD chromagen (Sigma, catalog number (Cat.No.) P-9187, lot number 18H82111) in room temperature reaction 30 minutes.Add 50 μ l 2.5M sulphuric acid cessation reactions.Use BIO-TEK EL800 reader to measure the OD of 490/630nm then.The result

Measure from the HK-X in normal drug exposure path: to from the anti-HK-X reactivity of the serologic test of following several groups of mices: induce asthma and with the HK-X treatment, induce asthma and treat with the contrast of DMSO (carrier) treatment with the saline immunity and with DMSO (carrier) with OVA with OVA.Every other day mice intranasal administration 50 μ g HK-X or carrier are reached 16 days.

Do not observe IgG reactivity at the HK-X that puts together 12C spacerarm (HK-X-spacerarm), KLH (HK-X-KLH) or BSA (HK-X-BSA).Observe IgG reactivity in the useful OVA mice immunized and in 1 control mice at OVA with the saline immunity, and as the contrast of ELISA.In the serum of the animal that comes the personal HK-X-KLH immunity that is dissolved in adjuvant, observe IgG reactivity at HK-X-KLH and HK-X-BSA, with it as these antigen coated contrasts to elisa plate.

The HK-X of solution immunity coupling carrier: with the HK-X-KLH immune mouse of solution form, and blood sampling after 21 days.ELISA result shows that 4/5 blood serum sample and KLH and HK-X-KLH react, do not react but observe, the antibody that does not have to generate carry out immunity with the soluble carrier of coupling HK-X after at the HK-X of coupling KLH is described with HK-X-BSA and HK-X-spacerarm.

The HK-X of adjuvant immunity coupling carrier: in order to impel generation at the antibody of HK-X, with the KLH or the HK-X-KLH immune mouse that are dissolved in complete Freund's adjuvant, booster immunization 1 time, and in the back blood sampling of 6 weeks.ELISA result shows the antibody that has generated at KLH.Also generated antibody at HK-X.This is subjected to the support of following evidence: 1) HK-X-KLH serum is higher 2 times than only using the serum of KLH immunity with the antibody response of HK-X-KLH; And 2) the use by oneself serum and the HK-X-BSA of HK-X-KLH mice immunized reacts, but do not react with BSA.Yet, do not observe antibody response at the HK-X of coupling 12C spacerarm.

According to the result of these researchs, can draw immunogenicity and antigenic several conclusion about the HK-X peptide.At first, after this peptide of therapeutic intranasal administration reached 16 days, mice did not generate the antibody at HK-X.Secondly, when carrying out immunity with the soluble peptide form of puting together immunogenic carrier KLH, mice does not generate antibody.The 3rd, under extreme conditions, during promptly at coupling KLH and with the Freund's complete adjuvant immunity, can impel the antibody of mice generation at HK-X.Yet, even in kind of situation, the antibody response of generation may be the new epi-position that produces at by puting together of HK-X and KLH, because detect less than antibody response at the HK-X that puts together the 12C spacerarm.This conclusion is subjected to the support of following observed result: before being incubated with determined antigen HK-X-KLH, after the free HK-X of adding reaches at least 30 minutes in antiserum, do not reduce the antibody response at HK-X-KLH.

Therefore, as if impossible clinical associated antibodies or other immunne response that causes at HK-X in clinical setting.There are 5 ideas that the observed result support is such.These observed results are: 1) size of HK-X has only 4 aminoacid (less than 600Dal), and this makes it unlikely have immunogenicity; 2) all aminoacid among the HK-X all are hydrophobic, and their characteristic and immunogenicity are irrelevant; 3) in order to have immunogenicity, HK-X immunogenic carrier covalency or the static with bigger in vivo is relevant; 4) antibody that generates at HK-X might point to the epi-position (neoantigen) that forms by joint vector and HK-X; 5) even if pointing to the antibody and the free HK-X of neoantigen can react, also be slight (low-affinity).The primates toxicologic study of embodiment 7:HK-X

This research is to carry out at a tame zooscopy BIOSUPPORT of mechanism (Redmond, Washington) according to the GLP standard.6 bulls and the female Rhesus Macacus that obtain by Charles River have been studied.A group, group comprises 2 animals in contrast, IV gave carrier (buffer saline that contains 3%DMSO) and reached 5 days every day.0-4 and taking a blood sample in 7 days is used for CDC and chemical method analysis.B group comprises 3 animals, and IV took the 20 μ g/kg HK-X that are dissolved in carrier (buffer saline that contains 3%DMSO) and reached 5 days every day.0-4 and taking a blood sample in 7 days is used for CDC and chemical method analysis.The C group comprises other 3 animals, takes 150 μ g/kg with same approach IV every day.D group comprises all 6 animals of B group and C group, finish their scheme at the C treated animal after, use same approach IV every day takes 1000 μ g/kg.During whole research, observe all animals every day, record body weight and general health situation and behavior.When D prescription case finishes, all animals of peaceful and comfortable execution, necropsy, and be used for histologic analysis by the representative tissue sample of following organ collection: liver,kidney,spleen, lung, the heart, lymph node and brain.By obtaining committology and independently carrying out the histopathology evaluation with the associated veterinary pathologist of BIOSUPPORT with the associated histopathologist of Histatek.

These dosage of HK-X are to select according to the dose therapeutically effective 10 and the 50 μ g/kg of HK-X in mouse asthmatic model.

All do not record the significantly unusual of leukocyte, hematocrit/hemoglobin or platelet count at any given day at any dosage level.Similar, all do not record the significantly unusual of chemical score at 3 kinds of dosage levels.Not record the histology subsequently in the spleen that obtains of the animal of 1000 μ g/kg HK-X or vehicle Control group and the representative tissue sample of lymph node unusual be exposed to 20 or 150 μ g/kg by every day.In liver, kidney, the heart and the lung tissue sample of treatment animal and control animal of hanging oneself, all record minimal many kitchen ranges lymphocyte and infiltrate, be judged as thus and treat irrelevant.Slight glomerular injury (being common in old and feeble Rhesus Macacus) is also disobeyed and is treated and demonstration difference, also thinks irrelevant with treatment thus.Other small histology's variation is considered to not remarkable.

Being significantly higher than in the cytometry of 6 Rhesus Macacus of dosage that treatment is considered or the chemical analysis at the dosage level that is exposed to HK-X does not have recognizable toxicity.The small histopathology that records in liver,kidney,spleen, lymph node, the heart and lung changes and disobeys and treat and demonstration difference, is considered to pathological performance of background or the artificial variation relevant with euthanasia.

The therapeutic dosage of this primates research explanation HK-X can be used for treating the people, and does not have overt toxicity or side effect.

This paper has described the present invention in detail by the preferred embodiments of the invention.Yet after considering this description and accompanying drawing, those skilled in the art will understand, and can make amendment and improve in the spirit and scope of the invention by the claim definition.

Claims (6)

1. be used for the treatment of in the mammal method by the indication of replying generation of IgE mediation, comprise that with the amount of effective downward modulation IgE administration is had the peptide of general formula f-Met-Leu-X, wherein X is selected from: Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr.

2. the process of claim 1 wherein and use another kind of active ingredient with described peptide, described active ingredient is selected from: anti-leukotriene, β ₂Agonist and corticosteroid.

3. be used to reduce the method for IgE receptor, comprise with the amount of effective downward modulation IgE receptor and use the peptide with general formula f-Met-Leu-X, wherein X is selected from: Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr.

4. the method for claim 3, wherein the IgE receptor is selected from: Fc _εRI, Fc _εRII and solubility Fc _εRII.

5. be used to reduce the CD40 part and prevent it further to relate to the method that IgE generates thus, this method comprises with the amount of effective downward modulation CD40 part uses the peptide with general formula f-Met-Leu-X, and wherein X is selected from: Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr.

6. be used to suppress the method for plasma cell secretion IgE, comprise described plasma cell is contacted with the peptide with general formula f-Met-Leu-X that can effectively suppress the excretory amount of IgE, wherein X is selected from: Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr.

Images