CN101065146B - Method of preventive treatment of allergy by mucosal administration of an allergy vaccine - Google Patents

Abstract

The present invention relates to a method of preventive treatment of allergy to an allergen in a subject comprising administering an allergy vaccine containing the allergen as active substance to a mucosal surface of the subject, a) wherein the subject to be treated is sensitised so as to exhibit an IgE response specific to the allergen, b) wherein the subject to be treated is free of clinical symptoms of the allergy associated with the allergen, and c) wherein the preventive treatment is aimed at preventing or reducing subsequent clinical symptoms of the allergy associated with the allergen.

Description

The anaphylactoid method of mucosa delivery prophylactic treatment through allergy vaccine

Invention field

The present invention relates to that prophylactic treatment is to the anaphylactoid method of anaphylactogen in subject, the mucomembranous surface that is included in object is used allergy vaccine.

Background of invention

Anaphylaxis is the main health problem that adopts Western lifestyle country.In addition, the popular of anaphylactic disease increases in these countries day by day.Although in general, anaphylaxis is not considered to the disease of dangerous life, and annual asthma can cause a large amount of death.In the teenager about 30% unusual popular, caused quality of life, the remarkable reduction of working day and money, and constituted the main type of health problem in the Western countries.

Anaphylaxis is complicated disease.Caused irritated incident by multiple factor, comprising the susceptibility of the individuality that is determined by influencing each other of fully understanding as yet between the some kinds of genes.Another key factor is the anaphylactogen that contact surpasses certain threshold value.It possibly be important that some environmental factorss are arranged in sensitization process, comprises pollution, childhood infection, parasitic infection, enteric microorganism etc.In case individual by sensitization, and set up allergic immune response, just the existence of minute amounts of allergen just can effectively change symptom into.

The natural process of anaphylactic disease is accompanied by increasing the weight of on two kinds of levels usually.At first be the development and the severity of disease of symptom, and advancing of disease, for example, be transformed into asthma from Hay Fever.Secondly, the propagation of the anaphylactogen of invasion is modal to be to cause the multiple reaction of anaphylaxis.Chronic inflammatory disease causes the overall weakening of mucosal defense mechanisms, has caused nonspecific stimulation, and the said mucosal tissue of final failure.Thereby maybe be main because food, i.e. milk product and allergy causes eczema or gastrointestinal disorder; But, modal is that they above-mentioned symptom spontaneously occurs.Said baby can suck anaphylactoid danger subsequently in their life.

Most important anaphylactogen source is the modal granule of specific size in our inhaled air.Said source obviously is general, and comprises grass pollen and house dust mite faecal particles, they caused altogether all anaphylactoid about 50%.What have global importance also comprises the animal soft flocks, i.e. cat and Pilus Canitis bits, and other pollen, like Folium Artemisiae Argyi pollen, and micro-fungus, like Alternaria.On region base, also have other pollen to play dominating role, like Northern Europe and Central European birch pollen, the artemisiifolia at eastern united states and middle part, and the japanese cedar pollen of Japan.Insecticide, i.e. Apis and wasp venom, and food account for separately all anaphylactoid about 2%.

Anaphylaxis, i.e. I type allergy are to be caused by the unsuitable immunoreation to the non-morbid substance of external source.Anaphylactoid important clinical performance comprises asthma, Hay Fever, eczema, gastro intestinal disorders.Anaphylaxis takes place rapidly when the anaphylactogen of contact invasion, and within 20 minutes, peaks.In addition, anaphylaxis is specific, and promptly specific individuality is responsive to specific anaphylactogen, and said individuality not necessarily produces anaphylaxis to notifying other materials that cause anaphylactic disease.The characteristic of allergic phenomena is the remarkable inflammation of the mucosa of Target organ, and the allergen specificity antibody of IgE type is in blood circulation and in mastocyte and the lip-deep existence of basophilic granulocyte.

Allergic attack is reacted by external source anaphylactogen and allergenic specific IgE antibody and causes, this moment, said antibody combined with mastocyte and the lip-deep IgE specific receptor of basophilic granulocyte with high affinity.Said mastocyte and basophilic granulocyte comprise preformed amboceptor, i.e. histamine, trypsinlike enzyme and other materials, and they are when the IgE of two or more receptors bind is antibody linked, to discharge.IgE antibody be combine through an allergen molecule time crosslinked.Therefore, follow the principle that the allogenic material that has only an antibodies epi-position can not cause allergic reaction.Be combined in receptor crosslinked of the lip-deep IgE of mastocyte, also caused being responsible for the eosinophilic granulocyte, the cytotaxis of allergen specificity T-cell and other types is to the release of the signal transduction molecule of irritated reactive site.Said cell and anaphylactogen, IgE and effector lymphocyte interact, and cause after contact allergy is former occurring once more in 12-24 hour allergic symptom (late phase reaction).

Anaphylactic disease control comprises diagnosis and treatment, comprises prophylactic treatment.Anaphylactoid diagnosis relates to checking allergenic specific IgE and definite anaphylactogen source.Under many circumstances, conscientious anamnesis just is enough to diagnosis of allergy, and confirms the anaphylactogen source material of invasion.But, modal is that diagnosis is through objective determination, supports like skin penetrating test, blood count or provocative test.

The treatment option is divided into three kinds of main types.First kind possibly be to avoid anaphylactogen or reduce contact.And avoid anaphylactogen is conspicuous, for example, is exactly like this for food allergen.But also possibly be difficulty or expensive, as for the house dust mite anaphylactogen, or perhaps it be infeasible, as for pollen allergens.Second kind and the most widely used Therapeutic Method are out the typical symptomatic treatment medicine in place, like anti--histamine and steroid.The symptomatic treatment medicine is safe and effective; But they can not change the natural cause of disease of diseases related, can not control disease propagate.The third therapeutic scheme is a specific allergy vaccination, under most of situation, does like this and can alleviate or alleviate the allergic symptom that is caused by relevant anaphylactogen.

Traditional specific allergy vaccination is the causal treatment of anaphylactic disease.Its interfere fundamental immunity mechanism causes the persistency of patient's immune state to be improved.Therefore, the protective effect of specific allergy vaccination extended to above treatment time, and is opposite with the symptomatic drugs treatment.Some patient who receives treatment has been cured, in addition, most of patient experience disease severity with experience the alleviation of symptom, or suppressed advancing of disease at least.Therefore, specific allergy vaccination has preventive effect, can reduce the risk that hay fever develops into asthma, and has reduced and new anaphylactoid risk occurs.

Still do not understand the details of the amynologic mechanism of supporting successful allergy vaccination.Known, specific immune response is the adaptive immunity reaction like the production of antibodies of anti-special pathogen.This reaction is different from the innate immunity reaction, and the innate immunity reaction is the nonspecific reaction to pathogen.Allergy vaccine is devoted to solve the problem of adaptive immunity reaction, and it comprises cell and the molecule with antigenic specificity, like T-cell and the B-cell that produces antibody.If do not have the help of corresponding specific T-cell, the B-cell can not be ripe for producing the cell of antibody.The T-cell of participating in the allergic immune response attack mainly is the Th2 type.Having proposed equilibrated foundation new between Th1 and the Th2 cell already is favourable with important for the amynologic mechanism of specific allergy vaccination.Whether this is to reduce through the Th2 cell, the skew from Th2 to the Th1 cell, or the rise of Th1 cell caused be suspicious.Recently, having proposed regulatory T-cells already is important to the mechanism of allergy vaccination.According to this model regulatory T-cells, i.e. Th3 or Tr1 cell have been reduced and have been had specific Th1 of corresponding antigen and Th2 cell.Although there are these uncertainties, it is generally acknowledged that active vaccine must possess the allergen specificity of stimulation T-cell, the ability of preferred TH1 cell.

Although possess advantage, special allergy vaccination is not widely used, and this mainly is owing to two reasons.A reason is, the inconvenience relevant with the traditional vaccine vaccination regimen, and this scheme comprises multiple vaccination, promptly injects in the time at some months.Another prior reason is the danger of allergic side reactions to occur.To the common vaccination of infectious agent through once or heavy dose of several times immunity inoculation effectively carry out.But, this method can not be used for allergy vaccination, because the pathologic immunoreation had taken place already.

Therefore, conventional specific allergy vaccination is carried out through repeatedly subcutaneous immunity inoculation in a long time.This process can be divided into two stages, escalated dose stage and maintenance stage.In the escalated dose stage, begin from little dosage, increase dosage of inoculation, continue 16 time-of-weeks usually.When reaching the maintenance dose of recommendation, adopt this dosage in the maintenance stage.Usually per six weeks injection once.After per injection, the patient must accept 30 minutes medical treatment nurse, because there is the danger of anaphylaxis side reaction, although very rare, this in principle side reaction maybe life-threatening.In addition, the equipment of clinic should support emergency treatment.There is no doubt that, can eliminate or reduce having now based on the vaccine of different administration approach, and help using widely, even can carry out self-vaccination at home based on the inherent anaphylaxis side reaction of subcutaneous injection vaccine.

Improve the trial of specific allergy vaccination and carried out more than 30 years already, and comprise various methods.Existing some kinds of methods concentrate on anaphylactogen itself through changing the IgE reactivity.

(Int.Arch.Allergy Immunol such as Fanta; 1999; 120:218-224) relate in grass pollen allergic patients group the research that changes through the inductive immunology of SLIT, said patient is according to following Standard Selection: have clinical symptoms (rhinitis and/or seasonal bronchial asthma) season in the grass pollen loose powder, the grass pollen extract is shown positive skin prick test; With the specific IgE to grass pollen, this confirms through RAST-CAP.SLIT carries out through the drop that sublingual administration is used the anaphylactogen extract.

Holt etc. (" Suppression of IgE responses following inhalationof antigen "; Immunology Today; Vol.8, No.1,1987) mentioned the following fact; As to contact reaction that suck or the anaphylactogen that mouth and nose splash into, induced corresponding to the toleration that passes through the inductive reaction of dietary antigens at gastrointestinal tract at upper respiratory tract.

Holt etc. (" Sublingual allergen administration.I.Selectivesuppression of IgE production in rats by high allergen doses "; Clinical Allergy; 1988; Volume18 pages229-234) relates to continuous seven days the rat that is used to first test is carried out the sublingual administration of anaphylactogen (ovalbumin), the last time after the sublingual administration 5 days; Carry out parenteral with ovalbumin and attack, the result shows the selective inhibitory that IgE is special to ovalbumin.By inference, said treatment mechanism relates to the attack that anaphylactogen-specificity suppresses cell.It is relevant that the document has also disclosed the animal that the treatment that is proposed is used to test first, and should be different from the Sublingual desensitization method.

WO95/17208 has disclosed the method for Polyglucan property disease; Comprise the anaphylactogen of using doses to former unsensitized object; This dosage effectively induced hypersensitivity former-foundation of the stable colony of specificity T-auxiliary-1-appearance memory lymphocyte, said cell can suppress anaphylactogen-specificity T-auxiliary-lymphocytic activity of 2-appearance.Preferred 3 months to 7 years old of the said object that will treat is big.For example, can be dermatophagoides pteronyssinus as anaphylactogen, grass pollen and trees pollen.The administration of said anaphylactogen can oral, intranasal, and mouth and nose, rectum, intradermal, intramuscular or subcutaneous route are carried out.

For example; Homepage www.immunetolerance.org has disclosed not the preventative-therapeutic planned clinical research to the child of inhalant sensitization on (on October 11st, 2004); Wherein, Use the Sublingual drop that contains any allergen (dermatophagoides pteronyssinus, timothy grass and cat) for said child, and wherein, anaphylaxis appearred in said child at 3 years subsequently.The said child that this research is recruited has atopic dermatitis or food anaphylaxis reaction medical history, and they biology mother or father or a compatriot have the specific reaction history of venereal disease.

The purpose of this invention is to provide improving one's methods of prophylactic treatment individuality, particularly child.

Summary of the invention

Realized this purpose through the present invention, the present invention relates in subject prophylactic treatment to the anaphylactoid method of anaphylactogen, the mucomembranous surface that is included in object is used and is comprised the allergy vaccine of said anaphylactogen as active substance,

A) wherein, the said object that will treat is reacted the special IgE of said anaphylactogen so that show by sensitization,

B) wherein, the said object that will treat do not have the relevant anaphylactoid clinical symptoms of said anaphylactogen and

C) wherein, said preventative-therapeutic purpose is prevention or alleviates the relevant anaphylactoid clinical symptoms subsequently of said anaphylactogen.

The present invention is based on following new discovery; Can prevent in individuality, to occur symptoms of allergic to said anaphylactogen; The immune system of said individuality had contacted anaphylactogen already; But wherein, said immunoreation does not also develop into and clinical symptoms, like rhinitis, conjunctivitis, rhinorrhea, nasal obstruction, sinusitis, sneeze, atopic dermatitis, pruritus, shed tears, rhinorrhea, the state relevant of stridulating with skin irritation.Believe always that up to now the individuality that will treat should be by sensitization in order to obtain anaphylactoid effective prophylactic treatment.In addition, believe with the relevant mechanism of Polyglucan reaction it is to induce and up to now through dietary antigens inducing at gastrointestinal tract inductive reaction corresponding oral cavity toleration always.But,, confirm amazingly that through treating sensitization already, but the individuality that does not show any clinical symptoms as yet can obtain preventive effect according to the present invention.The preventive effect that it is believed that acquisition works through certain mechanism, and this mechanism is partially or completely similar with the mechanism of the desensitization that in specific allergy vaccination (immunotherapy), takes place.

Believe that also it is the most effective after sensitization, beginning further before anaphylaxis Th2 cell effect development, to carry out prophylactic treatment as soon as possible at immune system response.In other words, it generally is favourable after child's contact allergy already is former, when they are as far as possible little, treating.In addition, it is believed that because the effect of early prevention property treatment that treatment can be carried out with littler dosage application times and/or shorter treatment time still less, more than relatively be the adult's specific allergy vaccination that has occurred clinical symptoms relatively.Because the soft property of prophylactic method, it is applicable to the general method of vaccination of all children or large-scale particular child colony.

Other advantages of the inventive method are that sensitized individuals is that anaphylactoid individuality appears in most probable, so the individuality of sensitization has constituted and carries out preventative-therapeutic maximally related crowd.

The invention still further relates to anaphylactogen is used to produce and be used for the purposes of in the subject anaphylactoid mucosal vaccine of prophylactic treatment,

A) wherein, the said object that will treat is reacted the special IgE of said anaphylactogen so that show by sensitization,

B) wherein, the said object that will treat do not have the relevant anaphylactoid clinical symptoms of said anaphylactogen and

C) wherein, said preventative-therapeutic purpose is prevention or alleviates the relevant anaphylactoid clinical symptoms subsequently of said anaphylactogen.

Brief description of drawings

Figure 1A representes the sensitized mice clinical data (sneeze number of times) with 5000SQ Ph1p or the attack of buffer intranasal.

Serum IgE level in the sensitized mice body that Figure 1B representes to attack with 5000SQ Ph1p or buffer intranasal.

BALIgE level in the sensitized mice body that Fig. 1 C representes to attack with 5000SQ Ph1p or buffer intranasal.

NALIgE level in the sensitized mice body that Fig. 1 D representes to attack with 5000SQ Ph1p or buffer intranasal.

NAL eosinophil levels in the sensitized mice body that Fig. 1 E representes to attack with 5000SQ Ph1p or buffer intranasal.

Fig. 2 A representes to carry out SLIT or buffer and handles, and with Ph1p carry out that intranasal attacks the clinical data (sneeze number of times) of sensitized mice.

Fig. 2 B representes to carry out SLIT or buffer is handled, and carries out the respiratory tract anaphylaxis reaction of the sensitized mice of intranasal attack to the reaction (weighing through the Penh value) of MCH attack with Ph1p.

Fig. 3 A representes to carry out SLIT or buffer and handles, and carries out serum IgE level in the sensitized mice body that intranasal attacks with Ph1p.

Fig. 3 B representes to carry out SLIT or buffer and handles, and carries out serum IgG in the sensitized mice body that intranasal attacks with Ph1p ₁Level.

Fig. 4 A representes to carry out SLIT or buffer and handles, and carries out BAL IgE level in the sensitized mice body that intranasal attacks with Ph1p.

Fig. 4 B representes to carry out SLIT or buffer and handles, and carries out NAL IgE level in the sensitized mice body that intranasal attacks with Ph1p.

Fig. 4 C representes to carry out SLIT or buffer and handles, and carries out BAL IgA level in the sensitized mice body that intranasal attacks with Ph1p.

Fig. 4 D representes to carry out SLIT or buffer and handles, and carries out NAL IgA level in the sensitized mice body that intranasal attacks with Ph1p.

Fig. 5 A representes to carry out SLIT or buffer is handled, and carries out the BAL eosinophile peroxidase level of the sensitized mice of intranasal attack with Ph1p.

Fig. 5 B representes to carry out SLIT or buffer is handled, and carries out the NAL eosinophile peroxidase level of the sensitized mice of intranasal attack with Ph1p.

Fig. 6 A representes to carry out SLIT or buffer and handles, and carries out the T cell proliferation in the sensitized mice spleen that intranasal attacks with Ph1p.

Fig. 6 B representes to carry out SLIT or buffer is handled, and carries out the intracellular T cell proliferation of sensitized mice lymph node (LN) that intranasal is attacked with Ph1p.

Detailed description of the invention

Anaphylactogen

The anaphylactogen of preparation of the present invention can be that reported already when contact is individual repeatedly can induced hypersensitivity, i.e. anaphylactoid any naturally occurring albumen of IgE mediation.The example of naturally occurring anaphylactogen comprises pollen allergens (trees, medicinal herbs, weeds and grass pollen allergens); Insect hypensensitiveness former (inhalant, saliva and venom allergens; For example, acarid anaphylactogen, Blatta seu periplaneta and midge anaphylactogen, hymenopteran (hymenopthera) venom allergens), animal hair and dandruff allergens are (for example; From Canis familiaris L., cat, horse, rat, mice etc.), and food allergen.From trees, grass type and the pollen allergens of medicinal herbs be: from following purpose plant on the taxonomy: Fagales, Fructus oleae europaeae order (Oleales), pinales and Platanaceae comprise that birch (Betula), alder (Folium Et Cacumen Alni Japonicae), hazelnut (Corylus), Carpinus (CarpInus) and Fructus Canarii albi (Olea), Cedrus deoclar (Roxb.) G. Don (Cryptomeria and Chinese juniper genus), plane tree (oriental plane tree), Poales (Poales) comprise that Lolium, ladder forage spp, annual bluegrass genus, bermuda grass genus, timothy grass, Holcus, phalaris arundinacea, Semen Fagopyri Esculenti and sorghum, chrysanthemum order and Urticales comprise the medicinal herbs of Ambrosia, artemisia and Parietaria.Other important inhalant allergens are from the family dirt demodicid mite with the subordinate: Dermatophagoides and Europe demodicid mite belong to, the storage demodicid mite belongs to, for example, and LepIdoglyphys; Glycyphagus and Tyrophagus are from the anaphylactogen of Blatta seu periplaneta, midge and flea, for example; Blatella, Periplaneta, Chironomous and Ctenocepphalides; And,, comprise as stinging or biting insects from stinging like anaphylactogen, the venom allergens of cat, Canis familiaris L. and horse from mammal; The Hymenoptera of Tathagata on taxonomy comprises the anaphylactogen of Apis (Superfamily Apidae), wasp (Superfamily wasp section) and Formica fusca (Superfamily Formicidae).From the important suction anaphylactogen of fungus, like anaphylactogen from alternaria and cladosporium genus.

In particular of the present invention, said anaphylactogen is Bet v 1, Aln g 1, Cor a 1 and Car b 1, Que a 1, Cry j 1, Cry j 2, Cup a 1, Cup s 1, Jun a 1, Jun a 2, jun a 3, Ole e 1, Lig v 1, Pla 11, Pla a 2, Amb a 1, Amb a 2, Amb t 5, Art v 1, Art v 2 Par j 1, Par j 2, Par j 3, Sal k 1, Ave e 1, Cyn d 1, Cyn d 7, Dac g 1, Fes p 1, Hol 11, Lo1 p 1 and 5, Pha a 1, Pas n 1, Ph1 p 1, Ph1 p 5, Ph1 p 6, Poa p 1, Poa p 5, Sec c 1, Sec c 5, Sor h 1, Der f 1, Der f 2, Der p 1, Der p 2, Der p 7, Der m 1, Eur m 2, Gly d 1, Lep d 2, Blo t 1, Tyr p 2, Bla g 1, Bla g 2, Per a 1, Fel d 1, Can f 1, Can f 2, Bos d 2, Equ c 1, Equ c 2, Equ c 3, Mus m 1, Rat n 1, ApIs m 1, ApI m 2, Ves v 1, Ves v 2, Ves v 5, Dol m1, Dil m 2, Dol m 5, Pol a 1, Pol a 2, Pol a 5, Sol i 1, Soli 2, Sol i 3 and Sol i 4, Alt a 1, CIa h 1, Asp f 1, Bos d 4, Mal d 1, Gly m 1, Gly m 2, Gly m 3, Ara h 1, Ara h 2, Ara h 3, Ara h 4, Ara h 5 or from above-mentioned any one reorganization (shufflant) heterozygote of molecule Fan Zhi.

In a preferred embodiment of the invention; Said anaphylactogen is selected from following one group: trees pollen allergens, grass pollen allergens, medicinal herbs anaphylactogen and zoo-anaphylactogen, preferred said anaphylactogen is selected from grass pollen allergens, dust mite allergen, Ambrosia anaphylactogen, Cedrus deoclar (Roxb.) G. Don pollen, cat anaphylactogen and birch anaphylactogen.

In another embodiment of the invention, said preparation comprises at least two kinds of dissimilar anaphylactogens, and said anaphylactogen is from identical anaphylactogen source or from different anaphylactogen sources; For example, 1 type and 5 types grass type anaphylactogen, or 1 type and 2 type acarid anaphylactogens; From different acarids and grass class, weeds antigen is like short and small and huge Ambrosia anaphylactogen respectively; Different fungus anaphylactogens; Like alternaria and cladosporium genus, trees anaphylactogen, like birch, hazelnut, carpinus turczaninowii, Oak Tree and Folium Et Cacumen Alni Japonicae anaphylactogen, food allergen, former like Semen arachidis hypogaeae, Semen sojae atricolor and milk allergy.

The anaphylactogen that mixes in the said preparation can be a form of extract, the anaphylactogen of purification, the anaphylactogen of modification, the mutant of recombinant allergens or recombinant allergens.The anaphylactogen extract can naturally contain one or more heterogeneous of identical anaphylactogen, and recombinant allergens is only represented a kind of heterogeneous of anaphylactogen usually.In preferred embodiments, said anaphylactogen is a form of extract.In another kind of preferred embodiment, said anaphylactogen is a recombinant allergens.In another kind of preferred embodiment, said anaphylactogen is that naturally occurring low IgE-combines variant or the low IgE-of reorganization to combine variant.

Anaphylactogen can exist with identical molal quantity or the ratio of existing anaphylactogen can preferably fluctuate 1:20.

In another embodiment of the invention, it is like WO99/47680 that said low IgE combines anaphylactogen, the described anaphylactogen of WO02/40676 or WO03/096869A2.

Prophylactic treatment

Since this century already with specific allergy vaccination (SAV), be referred to as specific active immunotherapy or desensitization in the past, be used to treat the anaphylactic disease of 1 type IgE mediation.

The general advantage that obtains through SAV comprises: a) alleviate allergic conditions and Medical Consumption, b) at eyes, improve toleration and c to said anaphylactogen in nose and the lung) alleviate dermoreaction property (in early days and late phase response).

The basic mechanism of the improvement that obtains through SAV is not still understood, and but, can from the multinomial SAV research of in the past decade carrying out, extract multinomial common trait: 1) total IgE quantity does not change during treating; 2) during escalated dose; Quantity short time of allergenic specific IgE increases, and falls back to initial (pretreatment) level then again, 3) epitope specificity and the affinity of IgE remain unchanged; 4) allergen specificity IgG; IgG4 particularly raises 5 fast during SAV) started new Th0/1/Reg reaction and 6 on the surface) as if not change of Th2 reaction.There is not dependency by SAV between the outbreak of inductive effect and specific IgG.

SAV has induced new immunoreation, and it is ripe (raised the Th0/1T-cell, started allergen specificity IgX (X possibly be A1, A2, G1, G2, G3, G4, M or D)) during treating.Affinity (or quantity/affinity) as new antibody response; IgX was ripe already; IgX maybe with the said anaphylactogen of IgE effective competition, suppress the anaphylactic reaction of the normal ＂ Th2 of ＂ type, be characteristic with the crosslinked of IgE of mastocyte and the lip-deep receptors bind of basophilic granulocyte.Therefore, clinical symptoms can alleviate gradually.

It is believed that the prophylactic treatment that carries out among the present invention can work through the mechanism identical with SAV mentioned above at least to a certain extent.

The mucosa of using said allergy vaccine can be any suitable mucosa; And route of administration comprises in the oral cavity (through the digestive system mucosa), nasal cavity, vagina, Sublingual, eyes, rectum, urethra, mammary gland, lung, in ear (promptly passing through ear) and buccal administration, preferably buccal or sublingual administration (mouth mucosa drug administration).Said allergy vaccine can be preparation, vagina holder (vagitories), suppository or pessulum (uteritories) form in spray, aerosol, mixture, suspension, dispersion liquid, emulsion, gel, paste, syrup, emulsifiable paste, ointment, implant (ear, eyes, skin, nose, rectum and vagina), the breast.

By inference, preferably carry out the mucosa delivery of vaccine through mucosa, said mucosa contacts antigen preparation naturally.Therefore, for the anaphylaxis that gas carries the mucosa antigen preparation, preferably pass through the respiratory system administration, preferred mouth mucosa drug administration.Correspondingly, for the anaphylaxis of mucosa antigen preparation, it will contact with the mucosa of digestive system, preferably adopts oral administration.

In one embodiment of the present invention, said object has been accepted the vaccination scheme, comprises using said vaccine every day.In another embodiment of the invention, said vaccination scheme comprises per two days, per three days, or used one time vaccine in per four days.For example, said vaccination scheme comprises that using said vaccine surpassed for 4 weeks, preferably surpasses for 8 weeks, more preferably surpasses for 12 weeks, more preferably surpasses for 16 weeks, more preferably surpasses for 20 weeks, more preferably surpasses for 24 weeks, more preferably surpasses for 30 weeks, most preferably surpasses for 36 weeks.

Administration time can be continuous time.Perhaps, administration time is the discontinuous time of being interrupted by one or more not administration phases.Preferably, (always) time of (always) time ratio administration of not administration is short.

In another embodiment of the invention, used vaccine one time to trying individuality every day.Perhaps, use vaccine twice for every day the said individuality that tried.Said vaccine can be the vaccine of single dose.

Mouth mucosa drug administration

Mouth mucosa drug administration can be used any existing mouth mucosa drug administration preparation, comprises solution, suspension, and the fast-dispersing type, drop and lozenge carry out.

In a preferred embodiment of the invention, adopted Sublingual immunotherapy (SLIT), in this case, fast-dispersing type, drop and lozenge are preferred preparations.

The example of fast-dispersing type can be referring to US-A-5, and 648,093; WO00/51568, WO02/13858, WO99/21579; WO00/44351, US-A-4,371; 516 and EP-278877, and DK PA 2,003 00279 and DK PA 2,003 00318 to be examined, all be with the name application of assignee ALK-Abell ó A/S.Preferred fast-dispersing type is produced through lyophilization.The preferred substrate of producing preparation is isinglass and modified starch.

Typical increment dosage hyposensitization can be used for the present invention, wherein, is increased to the dosage of the anaphylactogen of fast dispersing solid dosage form to greatest extent a certain.The preferred effectiveness of the UD of said dosage form is the 150-1000000SQ-u/ dosage form; Preferred effectiveness is the 500-500000SQ-u/ dosage form; Preferred effectiveness is the 1000-250000SQ-u/ dosage form, more preferably 1500-125000SQ-u/ dosage form, most preferably 1500-75000SQ-u/ dosage form.

In another embodiment of the invention, said dosage form is multiple single dose, preferably in 1500-75000SQ-u/ dosage form scope.

Sensitization

The object of treating is by sensitization, so that show the IgE reaction special to using anaphylactogen.In the present invention, phrase " shows the reaction to the special IgE of said anaphylactogen " being illustrated in can detected anaphylactogen-specific IgE antibody level at least a immunoassay.The detection of said anaphylactogen-specific IgE antibody can be carried out with any routine immunization assay method, for example, and referring to WO94/11734 and WO99/67642.

In specific embodiments of the present invention, further said object is carried out sensitization, so that show the Th2 cell effect special to said anaphylactogen.

In specific embodiments of the present invention, further said object is carried out sensitization, so that in skin prick test (SPT), show positive anaphylactogen-specific reaction.

In other specific embodiments of the present invention, the age of said object preferably less than 30 years old, was more preferably less than 20 years old less than 40 years old, most preferably was 2-10 year.

Clinical symptoms

The object of treating does not have the relevant anaphylactoid clinical symptoms of said anaphylactogen.

The anaphylactoid clinical symptoms that said anaphylactogen is relevant can be any common sympton, comprises rhinitis, conjunctivitis, rhinorrhea, nasal obstruction, sinusitis, sneeze, atopic dermatitis, pruritus, sheds tears, rhinorrhea, stridulates and skin irritation.

It is that anaphylactoid index takes place that multiple factor is arranged, and in life, shows clinical symptoms subsequently.Show one or more these the indication factor following object be known as high-risk object.The indication factor of high-risk object is and the relevant anaphylactoid clinical symptoms of one or more anaphylactogens except the anaphylactogen of vaccine.Other indication factors of high-risk object are in one of father and mother or grand parents or both or one or more anaphylaxiss of existence in one or more Sibling.Prophylactic treatment of the present invention is specially adapted to high-risk object.But, the object that treat can also be the object that does not show the indication factor of high-risk object, for example, does not have the anaphylactoid clinical symptoms to other anaphylactogens.

The preparation of allergy vaccine

The allergy vaccine that is used for the inventive method can be to be fit to any dosage form that mucomembranous surface is used, and comprises spray, aerosol, mixture, tablet (enteric and non-enteric coated tablet), capsule (hard and soft, enteric and non-enteric), suspension, dispersion liquid, granule, powder, solution, emulsion, chewable tablet, drop, gel, paste, syrup, emulsifiable paste, lozenge (powder, granule, tablet), preparation, vagina holder, suppository or pessulum in dispersible tablet, instillation liquid, gas, steam, ointment, bar, implant (ear, eyes, skin, nose, rectum and vagina), the breast fast.

Should be understood that preparation of the present invention also comprises other adjuvants and other excipient that are fit to said preparation type.Said other adjuvants and excipient are conventionally known to one of skill in the art, and comprise, for example, and solvent, emulsifying agent, wetting agent, plasticizer, coloring material, filler, antiseptic, viscosity-controlling agent, buffer agent and mucosa emplastic etc.The example of compound method is well known to those skilled in the art.

Adjuvant

Said mucous membrane irritability reaction vaccine can comprise adjuvant; It can be any conventional adjuvant; Comprise oxygen metal salt; Thermal instability enterotoxin (LT), cholera toxin (CT), b subunit of cholera toxin (CTB); The polymerization liposome; The saltant toxin, for example, LTK63 and LTR72; Microcapsule; Interleukin (for example, IL-1 β, IL-2, IL-7, IL-12, INF γ), GM-CSF, MDF derivant, CpG oligonucleotide, LPS, MPL, phosphophazenes, , glucosan, antigen preparation, liposome, DDE, DHEA, DMPC, DMPG, DOC/ Alumen complex, incomplete Freund,

, LTOral adjuvant, muramyldipeptide, monophosphoryl lipid A, muramyl-tripeptide and PHOSPHATIDYL ETHANOLAMINE.

Said oxygen metal salt can be any oxygen metal salt, as long as the effect that needs can be provided.In preferred embodiments, the metal cation of said oxygen metal salt is selected from following one group: Al, K, Ca, Mg, Zn, Ba, Na, Li, B, Be, Fe, Si, Co, Cu, Ni, Ag, Au and Cr.In preferred embodiments; The anionic metal of said oxygen metal salt is selected from following one group: sulfate, hydroxide, phosphate, nitrate, iodate, bromate, carbonate, hydrate, acetate, citrate, oxalates and tartrate, and their mixed form.Example has aluminium hydroxide, aluminum phosphate, aluminum sulfate, aluminum acetate, aluminium potassium sulfate, calcium phosphate, Maalox (mixture of aluminium hydroxide and magnesium hydroxide), beryllium hydroxide, zinc hydroxide, zinc carbonate, zinc chloride and Barium Sulfate.

Referring to WO04/047794, the allergy vaccine of aqueous solution or quick dispersible tablet form is particularly suitable for buccal and sublingual administration.

Use the method for the effect of mucosa delivery assessment immune modulating treatment method

The invention still further relates to and in the experimental animal body, assess the method for immune modulating treatment method to the anaphylactoid effect of anaphylactogen, this method may further comprise the steps

A) make said animal to said anaphylactogen sensitization,

B) through contact in nasal cavity or the trachea said animal is carried out the anaphylactogen attack first time,

C) use mouth mucosa drug administration that said animal is implemented said Therapeutic Method,

D) level of biomarker is measured and

E) use said measurement result to assess the effect of said Therapeutic Method.

Herz etc. (Methods32 (2004) 271-280) are one piece of review articles, have disclosed several animal models.In a kind of such model,, make mouse sensitization through peritoneal injection rBet v1 with the aerosol challenge of Bet v1 extract referring to 3.1 parts.Then, obtain immunoregulation effect through injecting immune advantage peptide or through mucosal administration rBetv1, this carried out before or after sensitization.Intranasal or oral administration rBet v1 have caused the inhibitory action of the anaphylactogen-specific antibody level of all isotypes, reduced IL-4, IL-5 and IFN-γ, and suppressed the respiratory inflammation and the respiratory tract anaphylaxis reaction of experimental animal and sensitized animal first.Herz etc. have disclosed multiple other models; Referring to table 2; Comprise equally with anaphylactogen and carry out various types of parenterals; Intranasal and oral cavity sensitization, for example, administration of antibodies, cytokine are attacked and treatment, tuerculoderma, histamine release, eosinophilic granulocyte, respiratory inflammation and T cell or the like.

The present invention just is being based on so any, can attempt mouth mucosa drug administration is used in assessment immune modulating treatment method appraisal procedure to the anaphylactoid effect of anaphylactogen in experimental animal.

The principle of animal model test method of the present invention is, makes experimental animal to anaphylactogen sensitization, promptly handles; So that show allergic immune response, attack with said anaphylactogen then, so that the induced hypersensitivity reaction to said anaphylactogen; Can measure and assess then; Wherein, further said animal is implemented Therapeutic Method, can study of the influence of said method then anaphylactic reaction.

Said experimental animal can be any animal that is used as experimental animal usually, comprises rodent, for example, and mice, rat, Cavia porcellus and rabbit, pig, Canis familiaris L., cat and monkey.

In specific embodiments of the present invention, said mouth mucosa drug administration is sublingual administration (Sublingual immunotherapy (SLIT)).The said anaphylactogen and the preparation that are used for said Therapeutic Method can be any anaphylactogen and the preparations that the anaphylactoid prophylactic method of top combination is disclosed.

When carrying out mouth mucosa drug administration, should guarantee that said preparation can keep the scheduled time at the predetermined position in oral cavity.In specific embodiments of the present invention, can prevent that said experimental animal from swallowing.For example, preventing to swallow can be through realizing with the fixing said animal of hands.For example, for rodent, can be fixed on through scrimp between two fingers so that the skin around the fastening head prevents to swallow with skin of neck.In addition, can also prevent to swallow, as sucking anesthetis through using anesthetis; For example, ether, Hal and sevoflurane, or injecting narcotic; For example, the mixture of fentanyl, fluanisone and midazolam, fentanyl, fluanisone and stabile mixture; Restrain his life and the mixture of medetomidine, restrain him and order and the mixture of xylazine atipamezole, urethane and tribromoethanol.

In another embodiment of the invention, said Therapeutic Method carries out before the anaphylactogen attack after sensitization and in the first time.Perhaps, said Therapeutic Method carried out after the anaphylactogen attack in the first time.In a kind of example in back, attack and after said Therapeutic Method, to carry out through the optional anaphylactogen second time that contact in nasal cavity or the trachea is carried out.

In another kind of preferred embodiment of the present invention, said biomarker is selected from following one group: anaphylactogen-specific antibody, clinical symptoms and effector lymphocyte.Said antibody can be any kind, and hypotype or their combination comprise IgA, IgA1, IgA2, IgD, IgE, IgG, IgG1, IgG2, IgG3, IgG4, IgM.The detection of the antibody specificity of said anaphylactogen is to use any routine immunization assay method to carry out, and preferred method of immunity can be referring to WO94/11734 and WO99/67642.Clinical symptoms can be any symptom that is usually used in animal model, comprises the sneeze number of times, wipes nose number of times etc.For example said effector lymphocyte is selected from following one group: the eosinophilic granulocyte, and mastocyte, basophilic granulocyte, the B cell, the T cell, antigen-presenting cell (APC ' s) and by above-mentioned cell-derived cell.In one embodiment of the present invention, effector lymphocyte's level is the level determination through the measuring effect cell marking.Said labelling is preferably selected from following one group: secretion molecule, molecule in surface molecular and the cell.Preferably, said secretion molecule is selected from following one group: amboceptor, cytokine, cytotoxic protein and soluble recepter.

The effect of said Therapeutic Method is assessed according to above-mentioned measurement result, and measurement result is to carry out according to the general scientific knowledge to the successfully performance of the immunoreactive biomarker of treatment.

Definition

In the present invention, used to give a definition:

Term ＂ mouth mucosa drug administration ＂ representes to be placed on below the tongue said dosage form or route of administration Anywhere in the oral cavity, makes active component contact with the mucosa of patient oral cavity or throat, so that obtain the local of said active component or systematicness effect.The example of mouth mucosa drug administration approach is a sublingual administration.

Term ＂ sublingual administration ＂ representes route of administration, wherein, dosage form is placed on below the tongue, so that obtain the part or the systemic effect of active component.

Term ＂ SQ-u ＂ representes SQ-unit: SQ-unit measures according to ALK-Abell ó A/S ' s ＂ SQ biopotency ＂-standardized method, and wherein 100,000SQ unit equals the subcutaneous maintenance dose of standard.Usually, the extract of 1mg contains 100,000-1, and 000,000SQ-unit, this depends on the source and the employed production method of anaphylactogen.Can confirm definite allergen content through immunoassay, promptly total main allergen content and total allergen activity.

Phrase " immune modulating treatment " expression can be regulated the immunoreactive treatment of the object of receiving treatment.

Specific embodiments

The effect of example 1:SLIT on the mouse model of rhinitis

Ultimate principle:

Set up the rhinitis model, so that check Sublingual immunotherapy (SLIT) is in the effect that has on the mouse model of clinical manifestation.

Method:

Animal

To 6-10 week big female BALB/c mouse carry out stable breeding, and can keep with the special diet of the composition of the antiserum cross reaction of timothy grass (Ph1p) with not containing.Each experimental group comprises 8-10 animal.

Zoopery

The Ph1p extract that is adsorbed on the Alumen through three intraperitoneal (i.p.) injection carries out sensitization to mice, carries out Sublingual treatment 6-9 time-of-week with Ph1p-extract or buffer then.With the Ph1p-extract mice is carried out intranasal subsequently and attack two time-of-weeks, and by following methods analyst clinical symptoms.After slaughtering blood-letting, collect bronchovesicular liquid (BAL), nasopharynx liquid (NAL), spleen and lymphonodi cervicales are used for analyzing.

Clinical data

Sneeze and wiping nose: after intranasal administration Ph1, observed mice, and add up the sneeze number of times and wipe the nose number of times with 10 minute time.

Respiratory tract allergy: adopt whole health plethysmogram (pletysmograph), block through the concentration induced draft that strengthens aerosolized MCH.Pulmonary air flow blocks through using after the MCH in 6 fens clock times enhanced pause (penh) to be measured.

IgA analyzes

The Estapore magnetic bead (Estapore IB-MR/0,86) that is combined on the goat anti-mouse IgA is cultivated with BAL or NAL.Wash then and cultivate with biotinylated anaphylactogen.Wash then and cultivate, and after washing, measure luminous with photometer (Magic Lite Analyser EQ) with the LITE preparation of streptavidin labelling.

IgE analyzes

Be combined in Estapore magnetic bead (Estapore IB-MR/0,86 on the goat anti-mouse IgE; A0201) cultivate with mice serum.Wash then and cultivate with biotinylated anaphylactogen.Wash then and cultivate, and after washing, measure luminous with photometer (Magic Lite Analyser EQ) with the LITE preparation of streptavidin labelling.

IgG, IgG1 and IgG2a analyze

Ph1p (10 μ g/ml) extract is added in the dull and stereotyped hole of ELISA, and under 4-8 ℃, said flat board is left standstill next day.At room temperature on vibration table, sealed one hour then with the flat board of buffer washing, and with 2% casein buffer through coating.After removing the casein buffer, the blood serum sample that dilutes is added on the said flat board, and at room temperature on vibration table, cultivated 2 hours.Wash said flat board, and with the biotinylated rabbit with 0.5%BSA buffer dilution anti--mice IgG/IgG1/IgG2a adds in each hole, and at room temperature on vibration table, placed one hour.After washing, the streptavidin-HRP that is diluted in the 0.5%BSA buffer is added in each hole, and at room temperature on vibration table, placed one hour.Developed said dull and stereotyped 20 minutes with TMP substrate (3,3 ', 5,5 '-tetramethyl benzidine), stop with 0.5M H2SO4 then.Resulting reactant mixture carries out spectrophotometer measurement under the terminal point of 450nm.

The T-cell proliferation test

Spleen chosen be broken into single-cell suspension liquid, and with culture medium washing three times.The statistics cell quantity, and adjust to 1.67x10 ⁶Cell/mL.With 3x10 ⁵Cell adds in each hole of the flat culture plate in 96 holes, and attacks said cell with 0,10 and 40 μ g/mL Ph1 p extracts.At 37 ℃ and 5%CO ₂The middle cultivation the 6 day time of said cell added in each hole through the last 18 hours 3H-thymus pyrimidines with 0.5 μ Ci at cultivation stage, and the radioactive label of harvesting and statistics integration is measured propagation then.

The result

Inducing of rhinitis

From Figure 1A-E, can find out, after peritoneal injection sensitization, mice carried out intranasal (IN) attack and show clear and definite rhinitis symptom two weeks with Ph1p extract (5000SQ/ mice/sky).At first, the obvious mice many (Figure 1A) of the number of times of their sneeze than the sensitization of attacking with buffer.Secondly, at serum, has higher IgE level (Figure 1B-D) among BAL and the NAL.The 3rd, the granulocytic flux enhancement of the eosinophilic granulocyte of these mices in NAL (Fig. 1 E).

The effect of SLIT on the rhinitis mouse model

In order to assess the effect of SLIT on the muroid model of above-mentioned rhinitis, with 125.000SQ Ph1p extract or buffer sensitized mice is carried out the Sublingual and handle 9 time-of-weeks, carry out intranasal with Ph1p then and attack.In addition, one group of sensitized mice does not carry out the Sublingual to be handled, and with buffer they is carried out intranasal and attack, and therefore is used as negative control.

Referring to Fig. 2, the SLIT-treatment can alleviate clinical symptoms, because compare with the mice of buffer-processing, the sneeze number of times significantly reduces (Fig. 2 A).Similarly, MCH is attacked the air flue allergy property (through the measurement of Penh value) react alleviate, particularly with the dosage of 5-15mg/mL when hour attacking (Fig. 2 B).

In addition, the antibody horizontal in the serum receives the influence of SLIT-treatment.Referring to Fig. 3, compare with the mice that buffer is handled, in the mice body that SLIT-handles, the serum levels of Ph1p specific IgE (Fig. 3 A) and IgG1 (Fig. 3 B) significantly reduces.

The specific IgE level reduces equally in the BAL of the mice that SLIT-handles and NAL liquid, and specificity IgA level reduces in BAL, still not obvious in NAL liquid (referring to Fig. 4 A-C).

In addition, above result shows, compares with the mice that buffer is handled, and in the mice that SLIT-handles, eosinophilic granulocyte's granulocyte level of BAL and NAL all significantly reduces (referring to Fig. 5 A-B).

At last, checked the stripped reactivity of spleen and lymphonodi cervicales (LN) cell (tongue drain) when attacking again with the Ph1p extract.Although in spleen, there is the trend of Ph1p-specificity T-cell effect downward modulation,, this trend not obvious on statistics (Fig. 6 A).In contrast, compare with the mice of buffer-processing, the T-cell effect (Fig. 6 B) to Ph1p among the cervical region LNs in drain in the mice that SLIT-handles significantly weakens.

Conclusion

Above result shows that it is clinical that the SLIT treatment can palliate a disease, serology and cell characteristic on the muroid model of rhinitis.

Claims (11)

1. anaphylactogen preparation comprises the purposes of said anaphylactogen as the allergy vaccine of active substance, and said allergy vaccine is the form of skin and mucocutaneous administration, said vaccine in subject prophylactic treatment to the anaphylaxis of anaphylactogen,

A) wherein, the said object that will treat is reacted the special IgE of said anaphylactogen so that show by sensitization,

B) wherein, the anaphylactoid clinical symptoms that the said object that will treat is not relevant with said anaphylactogen and

C) wherein, said preventative-therapeutic purpose is prevention or alleviates anaphylactoid subsequently the clinical symptoms relevant with said anaphylactogen;

Wherein, said anaphylactogen is selected from following one group: grass pollen allergens, Ambrosia anaphylactogen, dust mite allergen, birch anaphylactogen and cat anaphylactogen.

2. purposes as claimed in claim 1, wherein, said object is by the object of sensitization, so that show the Th2 cell effect special to said anaphylactogen.

3. like the purposes of claim 1 or 2, wherein, said object does not have rhinitis, conjunctivitis, rhinorrhea, nasal obstruction, sinusitis, sneeze, atopic dermatitis, pruritus, sheds tears, rhinorrhea, stridulate and skin irritant clinical symptoms.

4. like the purposes of claim 1 or 2, wherein, the age of said object was less than 40 years old.

5. purposes as claimed in claim 4, wherein, the age of said object was less than 30 years old.

6. purposes as claimed in claim 4, wherein, the age of said object was less than 20 years old.

7. purposes as claimed in claim 4, wherein, the age of said object is 2-10 year.

8. like the purposes of claim 1 or 2, wherein, said allergy vaccine is that trans-oral is passed through in digestive system mucosa, nasal cavity, vagina, Sublingual, eyes, rectum, urethra, the mammary gland, the dosage form of lung, in ear or buccal administration.

9. purposes as claimed in claim 8, wherein, said allergy vaccine is buccal or sublingual administration dosage form.

10. like the purposes of claim 1 or 2, wherein, the said object that will treat has and the relevant anaphylactoid clinical symptoms of one or more anaphylactogens except the anaphylactogen of said vaccine.

11. anaphylactogen production is used for the purposes in the anaphylactoid mucosal vaccine of prophylactic treatment in subject,

A) wherein, the said object that will treat is reacted the special IgE of said anaphylactogen so that show by sensitization,

B) wherein, the anaphylactoid clinical symptoms that the said object that will treat is not relevant with said anaphylactogen and

C) wherein, said preventative-therapeutic purpose is prevention or alleviates anaphylactoid subsequently the clinical symptoms relevant with said anaphylactogen;

Wherein, said anaphylactogen is selected from following one group: grass pollen allergens, Ambrosia anaphylactogen, dust mite allergen, birch anaphylactogen and cat anaphylactogen.

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