CN110167588A - Reduce the smooth muscle contraction response induced by inflammatory contracting agent - Google Patents

Abstract

This patent is related to a kind of discovery: TMEM16A inhibitor reduces the contraction response of the smooth muscle cell induced by one or more inflammatory contracting agents.Embodiment herein includes to contact target cell and the medicament of a effective amount of inhibition transmembrane protein 16A (" TMEM16A ") activation or signal transduction.

Description

Reduce the smooth muscle contraction response induced by inflammatory contracting agent

Technical field

The invention belongs to medical domains, in particular it relates to reduce the smooth muscle contraction induced by inflammatory contracting agent Response.

Background technique

Mammal air flue is dominated the excitation for causing smooth muscle contractility by cholinergic nerve fibers, therefore adjusts air flue Contraction.In asthma airways, smooth muscle can be infiltrated by inflammatory cell (such as mast cell, lymphocyte), this can be generated Large amount of cell factor, chemotactic factor (CF), growth factor and inflammatory mediator.Those inflammatory factors may influence multiple mistakes of asthma pathology Journey.It is interesting that some media in the medium also directly cause ASMC to shrink and serve as other than mediated immunity response Airway contraction agent.Effect of this effect in asthma pathology and they how to adjust smooth muscle contraction and it is not immediately clear.

Summary of the invention

Embodiment of the disclosure is related to a kind of contraction for reducing the target cell induced by inflammatory contracting agent in vitro or in vivo The method of response, the method includes making target cell and the activation of a effective amount of inhibition transmembrane protein 16A (" TMEM16A ") or signal The medicament of conduction contacts.

Some embodiments in the embodiment are needing reduction to put down as caused by inflammatory contracting agent further to one kind The method that the reduction is carried out in the patient of sliding Muscle cell contract response, the method includes making vascular system and a effective amount of suppression Transmembrane protein 16A (" TMEM16A ") activation processed or the medicament contact of signal transduction.

In some embodiments, the inflammatory contracting agent may include in 5-HT, histamine, prostanoid or CysLT It is at least one.

In some embodiments, the medicament is the TMEM16A binding molecule for inhibiting TMEM16A function.

In some embodiments, the medicament is the antibody for TMEM16A.

In some embodiments, the medicament may include T16A_inh- A01 or NFA or combinations thereof.

In some embodiments, the target cell is smooth muscle cell.

In some embodiments, the smooth muscle is airway smooth muscle cells or vascular smooth muscle cells.

There is provided the content of present invention is to introduce will further describe in the following specific embodiments in simplified form Some concepts.The content of present invention is not intended to the key features or essential features for identifying theme claimed, also not purport In the range for limiting theme claimed.

Detailed description of the invention

Fig. 1 is shown, and TMEM16A mediates the inward electric current of the agonist induction in ASMC.(a-b) left: CTR and TMEM16A The representative trace of the electric current of 5-HT (a) and U46619 (b) induction in the ASMC of KO mouse.It is right: result it is quantitative (5-HT: The n=26 cell from 4 mouse for every kind of genotype；U46619: the n=20 from 4 mouse for CTR Cell, the n=22 cell from 4 mouse for KO).(c-d): the cavy of left DMSO and T16Ainh-A01 pre-incubation Histamine (c) and LTD in ASMC₄(d) the representative trace of the electric current induced.It is right: the quantitative (histamine: for every group of result The n=14 cell from 3 cavys；LTD₄: for the n=21 cell of DMSO and for the T16A from 4 cavys_inh- N=23 cell of A01 group.(e) left: the representative trace of the electric current of the MCh induction in the ASMC of CTR and KO mouse.It is right: knot Quantitative (the n=12 cell from 3 mouse for every kind of genotype) of fruit.Holding current potential in all these records For -60mV.The peak value of blue block instruction outward current, pink colour circle indicate the peak value of inward electric current.Bar chart indicates average value ± standard error of the mean (s.e.m.), by double tail student t examine, P < 0.05 *, P < 0.01 * *, P < 0.001 * * *, * * * * P < 0.0001。

Fig. 2 (Fig. 2 (A) and Fig. 2 (B)) is shown, and TMEM16A is necessary to the contraction response of contracting agent.(a) CTR is come from It is dyed with the representative H and E of the extrapulmonary bronchus of TMEM16A KO mouse.Scale bar: 200 μm.(b) TMEM16A is lacked not Change the thickness of extrapulmonary bronchus.Bar chart indicates average value ± standard error of the mean, is examined by double tail student t, NS P > 0.05.(c-f) TMEM16A KO (flox/flox Cre+), CTR (flox/+Cre+) and (flox/flox Cre-) bronchus Smooth muscle is to high K⁺With dosage-shrinkage curve of different agonists.Bar chart indicates average value ± standard error of the mean, in figure Sample size (n) is marked, is compared KO (flox/floxCre+) and CTR (flox/+Cre+) by using ANOVA Compared with P < 0.0001 * * * *.(g) internal dosage-Rrs (respiratory resistance) curve of TMEM16A KO and CTR mouse and 5-HT.Bar shaped Figure indicates average value ± standard error of the mean, and sample size (n) is marked in figure, passes through ANOVA, P < 0.001 * * *.(h-i) DMSO and T16A_inhThe guinea pig tracheal smooth muscle and histamine (h) and LTD of-A01 pre-incubation₄(i) dosage-shrinkage curve.By institute There is force value to be expressed as the percentage of the reference induced by 60mM KCl contraction.Bar chart indicates average value ± standard error of mean Difference is marked sample size (n) in figure, passes through ANOVA, P < 0.0001 * * * *.(j) DMSO and T16A_inhThe people of-A01 pre-incubation Dosage-shrinkage curve of bronchial smooth muscle and histamine.All force value are expressed as the reference induced by 60mM KCl contraction Percentage.Bar chart indicates average value ± standard error of the mean, and sample size (n) is marked in figure, by ANOVA, * P < 0.05。

Fig. 3 (Fig. 3 (A) and Fig. 3 (B)) shows TMEM16A and passes through VDCC mediate contractile.(a-d): 60mMKCl (a), 3 μM The peak value (left side) and duration (right side) induction CTR and KO bronchus of MCh (b), 300nM 5-HT (c) and 3 μM of 5-HT (d) are smooth Ca in flesh²⁺Signal.Bar chart indicates average value ± standard error of the mean, and sample size (n) is marked in figure, passes through double tails Student t is examined, and P>0.05 NS, P<0.05 * pass through ANOVA, P<0.0001 * * * *.(e-f) nifedipine of pre-incubation is to CTR With the influence of MCh (e) and 5-HT (f) dosage-shrinkage curve of KO bronchial smooth muscle.It is left: representative trace, it is right: quantitative knot Fruit.Bar chart indicates average value ± standard error of the mean, sample size (n) is marked in figure, by ANOVA, by CTR nitre benzene Horizon is compared with CTR, P < 0.0001 * * * *.

Fig. 4 is shown, and TMEM16A is necessary to air flue hyperresponsiveness.(a) TMEM16A missing reduce 5-HT and The humidification that U46619 stimulates MCh.Bar chart indicates average value ± standard error of the mean, and sample size is marked in figure (n), by using double tail student t inspections are matched, the lines of key group are compared,^##P<0.01、^###P < 0.001, by not It matches double tail student t inspections to be compared with CTR, P < 0.05 *.(b) anaphylactogen (OVA) sucking is induced asthma (OVA group) and non- The Rrs of asthma (PBS group) CTR and KO mouse is increased.Bar chart indicates average value ± standard error of the mean, and sample is marked in figure CTR OVA is compared by product size (n) by ANOVA with CTRPBS, P < 0.0001 * * * *, by ANOVA, by KO OVA It is compared with CTR OVA,^####P<0.0001.(c) MCh gradient sucks induced asthma (OVA group) and non-asthma (PBS group) CTR It is increased with the Rrs of KO mouse.Insertion figure: by by Δ Rrs to suppression percentage and the corresponding MCh dosage for knocking out TMEM16A Average delta Rrs value be compared to make value to normalize.Bar chart indicates average value ± standard error of the mean, is marked in figure Sample size (n) is examined by double tail student t and is compared with CTR PBS, and P < 0.05 *, P < 0.01 * * pass through double tail student t Inspection is compared with CTR PBS,^#P<0.05。

Fig. 5 shows the schematic diagram for showing the signaling conduction model based on TMEM16A of hypothesis.Inflammatory contracting agent combines it Corresponding GPCR and successively activate G_q-PLC-IP₃Axis.IP₃Activate IP₃Rs, induction sarcoplasmic reticulum (SR) Ca²⁺Release.It is discharged from SR Ca²⁺With direct activation MLCK or TMEM16A-VDCC axis, mediation Ca can be passed through²⁺It flows into, amplifying cells solute Ca²⁺Signal And pass through this indirect more MLCK of pathway activation.

Fig. 6 (Fig. 6 (A) and Fig. 6 (B)) shows the representative record trace of the electric current of the agonist induction in ASMC.(a) By the electric current of 5-HT (using 3 μM in pipette) induction in mouse ASMC.For CTR, 11/26 cells show goes out interior to electricity Stream, wherein 1 cell shows the inward electric current of oscillation；For KO, only 2/26 cells show goes out inward electric current.(b) mouse By the electric current of U46619 (using 1 μM in pipette) induction in ASMC.For CTR, 15/20 cells show goes out inward electric current, Wherein outward current is also shown in 2 cells and 1 cell shows the inward electric current of oscillation；For KO, whole 22 cells are all Inward electric current is not shown, and 8 cells shows go out outward current.(c) it is lured in cavy ASMC by histamine (using 3 μM in pipette) The electric current led.For the cell of DMSO pre-incubation, all 14 cells show that inward electric current；For 10 μM of T16A_inh-A01 The cell of pre-incubation, 11/14 cell shows reduced inward electric current, and apparent electric current is not shown in 3/14 cell. (d) by LTD in cavy ASMC₄The electric current of (using 100nM in pipette) induction.For DMSO group, 18/21 cell shows interior To electric current；For T16A_inh- A01 group, 18/23 cell also shows that inward electric current, but amplitude is suppressed.(e) mouse By the electric current of MCh (using 3 μM in pipette) induction in ASMC.For CTR, all 12 cells show that inward electric current, In 5 cells shows go out outward current and 1 cell shows the inward electric current of oscillation；For KO, showed all 12 Cell only shows outward current.

Fig. 7 is shown, and the expression of agonist receptor does not change in TMEM16A KO ASM.(a) protein prints Mark shows TMEM16A, mAChR M3,5-HT_2AR, TP R and rouge valve arrangement albumen -1 the branch gas outside TMEM16A KO and CTR lung Expression in pipe smooth muscle, beta-actin are used as molecule reference.(b) mAChR in CTR and KO extrapulmonary bronchus smooth muscle M3 (n=6), 5-HT_2AThe relative quantity of R (n=3), TP R (n=5) and rouge valve arrangement albumen -1 (n=6) quantify.Bar chart Indicate average value ± standard error of the mean.

Fig. 8 shows TMEM16A Cl in ASM_CaThe specific deficiency in channel.(a) Western blotting (left side) shows and props up outside lung More than 90%KO efficiency (quantitative data in right figure) in tracheae.(b) it is bronchial to show intrapulmonary for representative immunofluorescence dyeing High KO efficiency in smooth muscle layer.(c)Cl_CaElectric current is significantly inhibited in KO ASMC.It is left: representative record trace, right: The I-V curve of CTR and KO ASMC.Apply 600nM [Ca by pipette solution²⁺]_i, E_Cl=0mV.Bar chart indicates average value ± standard error of the mean, 10 cells of the CTR:n=from 3 mouse, 11 cells of the KO:n=from 3 mouse lead to Cross ANOVA, P < 0.0001 * * * *.(d) Cl of 10mM caffeine induction is eliminated in KO ASMC_CaElectric current.It is left: representative record Trace, it is right: quantitative result.Bar chart indicates average value ± standard error of the mean, CTR:n=49 from 10 mouse thin Born of the same parents, 52 cells of the KO:n=from 10 mouse are examined, P < 0.0001 * * * * by double tail student t.

Fig. 9 shows the recording trace of ASM contraction and airway obstruction.(a-d) KCl (a), MCh (b), U46619 (c) and 5- The representative record trace that TMEM16A KO and the CTR bronchial smooth muscle of HT (d) gradient induction is shunk.(e) CTR and KO mouse In 5-HT sucking induction airway obstruction representative record trace.

Figure 10 is shown, and TMEM16A missing inhibits the contraction of low-level MCh induction.(a) low (300nM) but not high (3 μM) The bronchial smooth muscle of the MCh induction of concentration is shunk to be suppressed by TMEM16A missing.The power of each time point is expressed as putting down Mean value ± standard error of the mean indicates, sample size (n) is marked in figure.(b) in CTR and TMEM16A KO bronchus The quantitative result of the maximum, force of 300nM and 3 μM of MCh induction.Bar chart indicates average value ± standard error of the mean, and sample is big Small (n) is identical as figure (a), is examined by double tail student t, P < 0.05 *.

Figure 11 is shown, the T16A in ASM_inh- 01 is the specific inhibitor and effective inhibitor of TMEM16A.(a) pre- training The 10 μM of T16A educated_inh- 01 inhibits contraction of the left extrapulmonary bronchus of 3 μM of 5-HT induction in CTR, but does not inhibit TMEM16A KO (CTR:n=6, KO:n=7).(b) 10 μM of T16A of pre-incubation_inh- 01 does not inhibit 3 μM of MCh inductions in both CTR and KO Contraction (CTR:n=5, KO:n=5).Bar chart indicates average value ± standard error of the mean, is examined by double tail student t, NS P>0.05、**P<0.01。

Figure 12 shows T16A_inhEffect of-the A01 in cavy and people ASM.(a) DMSO that is induced by histamine gradient or T16A_inhThe representative record trace that the guinea pig tracheal smooth muscle (TSM) of-A01 pre-incubation is shunk.(b) by LTD₄Gradient induction DMSO or T16A_inhThe representative record trace that the cavy TSM of-A01 pre-incubation is shunk.(c)10μM T16A_inh- A01 induces globefish The relaxation of mouse TSM basal tension.Upper figure: representative record trace, the following figure: quantitative result.DMSO:n=4, T16Ainh-A01:n= 6, it is examined by matching double tail student t, P < 0.01 * *.(d) recording trace of three kinds of human bronchial smooth muscle samples, whole are shown T16A out_inhInhibiting effect of-the A01 in the contraction that histamine induces.

Figure 13 is shown, the Ca in stimulation triggering CTR and TMEM16A KO bronchial smooth muscle²⁺It increases.(a)60mM KCl, (b) 3 μM of MCh, (c) 300nM 5-HT and (d) 3 μM of 5-HT trigger Ca²⁺It increases.Ca²⁺The dynamics of signal is by the glimmering of GCaMP3 Light percentage (%, Δ F/F0) indicates.Bar chart indicates average value ± standard error of the mean, and sample size is marked in figure (n)。

Figure 14 shows the representative record trace of reinforcing effect.100nM 5-HT and 100nM U46619 are clearly enhanced Contraction response of the extrapulmonary bronchus smooth muscle to 100nM MCh, while knocking out TMEM16A and inhibiting enhanced power.With colour Bar chart indicates the addition (100nM MCh: black, 100nM 5-HT: green, 100nM U46619: blue) of agonist.

Figure 15 is shown from asthma and non-asthma CTR and TMEM16A^SMKOThe histopathology of the slice of lung.Arrow refers to To the position with inflammatory cell infiltration, bar chart=100 μm (length) or 50 μm (short).

Specific embodiment

It summarizes

Embodiment of the disclosure is related to a kind of discovery: the reduction of TMEM16A inhibitor is lured by one or more inflammatory contracting agents The contraction response for the smooth muscle cell led.These inflammatory contracting agents are released from inflammatory cell (comprising mast cell, at fiber finer Born of the same parents, smooth muscle cell and other immunocytes) and induce the contraction response of smooth muscle cell.In some embodiments, by inflammation Property contracting agent induction contraction response it is associated with inflammatory shrinkage disease.For example, the example of inflammatory shrinkage disease included Quick property asthma, apoplexy, burn, cytokines release syndrome etc..Embodiment of the disclosure can make ASM relaxation to treat allergy Property asthma, alleviate the cerebral angiospasm as caused by secondary inflammatory contracting agent with treat apoplexy and alleviate by cytokine storm The spasm of the thin vessels of caused damaged skin is to treat burn.

Definition

Unless otherwise defined, otherwise all technical terms and scientific terms used herein have and neck belonging to the present invention The identical meaning of the meaning that the those of ordinary skill in domain is generally understood.Although it is similar or be equivalent to it is those of described herein side Method and any method and material of material may be used to practice or test the present invention, but depict preferred method and material Material.For purposes of the present invention, following term is defined below.

Article " one and one kind (a/an) " used herein refers to one or more of the grammatical object of the article In one (i.e. at least one).For example, " element " means an element or more than one element.

" about " mean relative to reference quantity, level, value, number, frequency, percentage, size, size, amount, weight or length The number of degree variation up to 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% Amount, level, value, number, frequency, percentage, size, size, amount, weight or length.

Through this specification, unless the context otherwise requires, otherwise word " comprising " and "comprising" will be understood as implying The step of including stated or element or step group or element group, but be not excluded for any other step or element or step group or want Plain group.

" by ... form " mean include and be limited to phrase " by ... form " after any content.Therefore, phrase " by ... form " show that listed element is required or enforceable, and other elements are not present.

" substantially by ... form " is intended to be included in any element listed after the phrase, and is limited to not interfere Or promote other elements for the activity of listed element assignment or effect in the disclosure.Therefore, phrase " substantially by ... Composition " shows that listed elements are required or enforceable, but these other elements are optional and may exist or can To be not present, this depends on its activity for whether influencing listed elements or effect.

" pharmaceutically acceptable carrier, diluent or excipient " is including but not limited to by U.S.'s food and drug pipe Reason office be approved as can be used for any adjuvant of the mankind or domestic animal, carrier, excipient, glidant, sweetener, diluent, preservative, Dyestuff/colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent or emulsification Agent.In some embodiments, pharmaceutically acceptable carrier may include one or more inactive pharmaceutical ingredients.Pharmaceutically may be used Inactive pharmaceutical ingredient in the carrier system of receiving may include stabilizer, preservative, additive, adjuvant, aerosol, compression Air or other suitable gases are prepared other suitable non-live together with therapeutic compounds (that is, active constituent (API)) Property drug ingedient.

Pharmaceutically acceptable carrier (for example, sucking carrier) may include known in the art for various inhalant dosage forms Pharmaceutically suitable non-active ingredient, such as aerosol propellants (for example, HFA Hydrofluoroalkane propellant), surfactant, addition Agent, suspending agent, solvent, stabilizer etc..Alcohol can be considered to be active or inactive ingredient according to its product configuration product Good example.

As used herein, inhalant dosage form including but not limited to as under stress pack and contain API and carrier system Drug products aerosol, valve system appropriate activation when discharge the API and carrier system, it is intended to topical application In olfactory epithelium.Inhalant dosage form can also be delivered to oral cavity (tongue and sublingual aerosol) or lung (Inhaled Aerosol)；Foam gas is molten Glue is the dosage form containing one or more API, surfactant, aqueous or non-aqueous liquid and propellant, as a result, if promoted Agent is in interior (discontinuous) phase (that is, oil-in-water type), then stable foam is discharged；And if propellant is in outer (continuous) Spraying or rapid disruption foam is then discharged in phase (that is, water-in-oil type)；Dosing aerosols are and allow to pass in each activation The pressurized dosage form for sending the spraying metered dose valve of even amount to be used together；Powder aerosol is to pack and contain under stress In the product for the API of powder type discharged when activating valve system appropriate；And aerosol spray is to utilize compressed gas Body is provided as propellant using product as power and the liquid medicine suitable for aqueous solvent needed for wet spray discharge Aerosol product.

" pharmaceutically acceptable salt " includes acid-addition salts and base addition salts.

" pharmaceutically acceptable acid-addition salts " refer to that salt below, the salt retain the biological effectiveness and property of free alkali Matter, is not biologically or other aspects are undesirable, can be formed by inorganic acid, such as, but not limited to, hydrochloric acid, hydrobromic acid, sulphur Acid, nitric acid, phosphoric acid etc.；And organic acid, such as, but not limited to, acetic acid, 2,2- dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, Aspartic acid, benzene sulfonic acid, benzoic acid, 4- acetaminobenzoic acid, camphoric acid, camphor -10- sulfonic acid, capric acid, caproic acid, octanoic acid, carbon Acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulphate, ethane -1,2- disulfonic acid, ethane sulfonic acid, 2- hydroxyl second sulphur Acid, formic acid, fumaric acid, galactosaccharic acid, gentianic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2- oxo-glutaric acid, phosphoglycerol, glycolic, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, Malonic acid, mandelic acid, methanesulfonic acid, glactaric acid, naphthalene -1,5- disulfonic acid, naphthalene-2-sulfonic acid, 1- hydroxy-2-naphthoic acid, niacin, oleic acid, Orotic acid, oxalic acid, palmitinic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-ASA, decanedioic acid, Stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-methyl benzenesulfonic acid, trifluoroacetic acid, undecenoic acid etc..

" pharmaceutically acceptable base addition salts " refer to salt below, the salt keep free acid biological effectiveness and Property, is not biologically or other aspects are undesirable.It can be made by the way that inorganic base or organic base are added into free acid These standby salt.Salt derived from inorganic base including but not limited to sodium salt, sylvite, lithium salts, ammonium salt, calcium salt, magnesium salts, molysite, zinc salt, Mantoquita, manganese salt, aluminium salt etc..Preferred inorganic salts are ammonium salt, sodium salt, sylvite, calcium salt and magnesium salts.Salt derived from organic base includes But salt not limited to the following: primary amine, secondary amine and tertiary amine, substituted amine (including naturally occurring substitution amine), cyclammonium and alkali Property ion exchange resin, as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropyl amine (TPA), diethanol amine, ethanol amine, decyl alcohol, Dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexyl amine, lysine, arginine, histidine, coffee Alkali, procaine, hydrazine amine (hydrabamine), choline, glycine betaine, benzene second Bian amine, benzyl star, ethylenediamine, aminoglucose, methyl Portugal Osamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidines, N-ethylpiperidine, polyamino resin etc..It is particularly preferred Organic base is isopropylamine, diethylamine, ethanol amine, trimethylamine, dicyclohexyl amine, choline and caffeine.

Crystallization would generally generate the solvate of the compound of the disclosure.As used herein, term " solvate " refers to The aggregation of one or more compound molecules and one or more solvent molecules including the disclosure.Solvent can be water, In this case, solvate can be hydrate.Alternatively, solvent can be organic solvent.Therefore, the chemical combination of the disclosure Object can exist in the form of hydrates, include monohydrate, dihydrate, semihydrate, times semihydrate, trihydrate, four Hydrate etc. can also exist with corresponding solvation form.The compound of the disclosure can be real solvate, and In other situations, the compound of the disclosure can only retain the mixture that allogenic water either water adds some external source solvents.

" pharmaceutical composition " refers to the preparation of the compound of the disclosure and what is usually received in the art is used to give birth to Object reactive compound is delivered to the medium of mammal (for example, people).This medium includes its all pharmaceutically acceptable load Agent, diluent or excipient.

" substantially (substaintially) " mean almost all or completely, such as certain given quantity 95%, 96%, 97%, 98%, 99% or higher.

As used herein, term " treatment " is for being directed toward subject's application or application for the treatment of agent or applying with subject With or application program or mode with obtain disease or with health related symptom treatment benefit, and include: (1) preventing lactation The disease or symptom occurred in animal specifically is diagnosed as when this mammal is susceptible to suffer from the symptom but not yet suffering from When the symptom；(2) inhibit disease or symptom, that is, prevent its development；(3) mitigate disease or symptom, that is, disease or symptom is caused to disappear It moves back；Or (4) mitigate the symptom as caused by disease or symptom, i.e., alleviate pain in the case where not solving potential disease or symptom Bitterly.

As used herein, any modification of term " prevention ", " inhibition ", " reduction " or these terms includes any can measure Slow down or completely inhibit to realize desired result.For example, compared with normal condition, it is understood that there may be the drop of activity or symptom Low amounts be reduce 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more or in which can derived from any range.

As used herein, term " disease " and " symptom " may be used interchangeably or can be different, the difference is that specific Disadvantage or symptom may without known pathogen (so that the cause of disease is not yet determining), and be not therefore also considered as disease and only It is only undesirable symptom or syndrome, wherein clinician has identified multiple or less specific symptom group.

As used herein, term administering " and " delivering " are for describing to subject, target (for example, cell, tissue, device Official, a part of system etc.) application or delivering or with target directly side by side place the disclosure composition process.Some In embodiment, " application " and " delivering " may include subject sucking so as to subject, target (for example, cell, tissue, Organ, a part of system etc.) delivering or with target directly side by side place the disclosure composition process.In some implementations In example, the composition of the disclosure can be taken orally, intravenously, in peritonaeum, subcutaneous, intramuscular, it is intrathecal, intradermal, intranasal, apply by rule of thumb With, vaginal suppository, suppository.For example, target can be a part of airway smooth muscle fiber or the air flue of subject.Term " is applied With " and " delivering " be used interchangeably.

As used herein, when being applied to target (for example, cell, tissue, organ etc.), term " contact " and " exposure " are used In the process for the compound for describing directly to place the disclosure side by side to target application or delivering or with target.Term administering " and " delivering " can be used interchangeably with " contact " and " exposure ".

As used herein, term " patient ", " subject " and " individual " is used interchangeably herein, and means wait control Mammalian subject that is treating and/or therefrom obtaining biological sample.Mammal include people and domestic animal, as cat, dog, pig, ox, Sheep, goat, horse, rabbit etc..

As used herein, term " effective " means to be enough to realize desired, expected or plan result.For example, " having Effect amount " can be the amount for being enough to generate the compound for the treatment of benefit.

As used herein, term " treatment is effective " or " treating beneficial " refer to promotes in terms of the therapeutic treatment of symptom Or the anything of the health of enhancing subject.This including but not limited to reduce disease S or S breaking-out, frequency, hold Continuous time or severity.

As used herein, term " the effective amount for the treatment of " means effectively to generate the as described herein of desired therapeutic response Composition amount.The amount for constituting the compound of the disclosure of " therapeutically effective amount " will be according to compound, symptom, its severity With age of mammal to be treated and change, but routinely can by those of ordinary skill in the art in view of its from It is determined in the case where body knowledge and the disclosure.

As used herein, " side known to persons of ordinary skill in the art can be identified by various reference books and database Method ".Synthesis, which is described in detail, can be used for preparing the reactant of the compound of the present invention or provides the suitable of reference to the article of description preparation Reference book and paper including, for example, " synthetic organic chemistry (Synthetic Organic Chemistry) ", John Wiley Father and son publishing company (John Wiley&Sons, Inc.), New York；S.R.Sandler et al., " organo-functional group preparation (Organic Functional Group Preparations) ", second edition, Academic Press (academic press), knob About, 1983；H.O.House, " modern synthesis (Modern Synthetic Reactions), second edition, W.A.Benjamin, Inc., California door Lip river Parker (Menlo Park), 1972；T.L.Gilchrist, " jeterocyclic chemistry Learn (Heterocyclic Chemistry) ", second edition, John Wiley father and son publishing company, New York, 1992；1.March, " Advanced Organic Chemistry: reaction, mechanism and structure (Advanced Organic Chemistry:Reactions, Mechanisms, and Structure), the 4th edition, Willie international scientific publishing company (Wiley-Interscience), knob About, 1992.It can also be by by (China can be contacted in most of libraries and college library, university library, academic library and by online database The special zone Sheng Dun American Chemical Society, www.acs.org obtain more details) obtain ' chemical abstracts of American Chemical Society takes Be engaged in (the Chemical Abstract Service of the American Chemical Society) ' preparation it is known The index of chemicals identifies specific and similar reactant.It can be prepared by customizing chemical Synesis Company known in catalogue But not commercially available chemicals, it is fixed that many standard chemical supply companies (for example, those of outlined above) provide Composite service processed.

As used herein, term " diagnosis " means to identify the presence or property of pathological condition.

As used herein, term " safe dose and effective quantity " refers to when as described herein in use, being enough to generate the phase The therapeutic response of prestige and without the excessive adverse side effect to match with reasonable benefit/Hazard ratio, (such as toxicity, stimulation or allergy are answered Answer) component amount.Specific safe dose and effective quantity or therapeutically effective amount will change with factor, such as the specific disease treated Shape, the physical condition of patient, the type of treated mammal or animal, the duration for the treatment of, concurrent therapy property (such as If fruit has) and used specific preparation and compound or derivatives thereof structure.

Method and composition

Embodiment of the disclosure is related to a kind of contraction for reducing the target cell induced by inflammatory contracting agent in vitro or in vivo The method of response, the method includes making target cell and the activation of a effective amount of inhibition transmembrane protein 16A (" TMEM16A ") or signal The medicament of conduction contacts.For example, target cell is smooth muscle cell.

Some embodiments in the embodiment are needing to reduce caused by inflammatory contracting agent smoothly further to one kind The method that the reduction is carried out in the patient of Muscle cell contract response, the method includes making vascular system and a effective amount of inhibition Transmembrane protein 16A (" TMEM16A ") activation or the medicament contact of signal transduction.For example, smooth muscle be airway smooth muscle cells or Vascular smooth muscle cells.

In some embodiments, the contraction response of the target cell induced by inflammatory contracting agent is related to inflammatory shrinkage disease Connection, and therefore embodiment of the disclosure can be related to the method for treating inflammatory shrinkage disease.

Inflammatory shrinkage disease herein, which refers to, to be caused by inflammatory contracting agent and is expected to cause impaired and/or stimulated The disease or symptom of the abnormal contraction response of the smooth muscle (for example, ASM and blood vessel) of tissue.For example, inflammatory shrinkage disease Example includes allergic asthma, apoplexy, burn, cytokines release syndrome etc..Embodiment of the disclosure can make ASM relaxation To treat allergic asthma, alleviate the cerebral angiospasm as caused by secondary inflammatory contracting agent to treat apoplexy and alleviate by thin The small blood vessel spasm of damaged skin caused by intracellular cytokine storm is to treat burn.

Inflammatory mediators refer to the mediation inflammation relevant to inflammatory shrinkage disease discharged by various cells (for example, smooth muscle) The signal transduction molecule of property response.Inflammatory mediators do not include the cholinergic excitement for the neural response for mediating the cell such as smooth muscle Agent.The receptor of inflammatory mediators is usually located at immunocyte.For example, many inflammatory mediators are from by infiltrating in asthma airways Cell in release, some of mediators may directly cause smooth muscle spasm.

Inflammatory contracting agent is the inflammatory mediators for referring to cause smooth muscle contraction response.For example, smooth muscle is receiving inflammation Contraction (for example, hyperresponsiveness contraction) is shown after the stimulation of property contracting agent, this causes hollow organ (for example, ASM and blood Pipe) lumen narrow.The example of inflammatory contracting agent includes 5-HT, TXA2, LTD4 and histamine.As used herein, inflammatory contracting agent Different from cholinergic agonist (for example, MCh), the cholinergic agonist is released from the cynapse of cholinergic nerve and may draw It rises and shrinks response.The response as caused by cholinergic agonist reflects the physiology nervous activity carried out on smooth muscle, because such as Fruit does not excite, then will not shrink response.

TMEM16A is the Cl expressed in smooth muscle, skeletal muscle, heart, epithelium and liver etc._CaThe member in channel, and lead to Cross generation ICl_Ca(ref) various physiological actions are played.It is interesting that TMEM16A is expressed with tissue-derived in smooth muscle cell And change, such as its in air flue great expression, in the blood vessel appropriateness expression, hardly express in enteron aisle.TMEM16A activation It is very sensitive to cytosol calcium.In air flue and musculus sphincter ani internus smooth muscle, even being released from the inherent Ca2+ sparks of SR all TMEM16A can be activated to a certain extent, or even causes to shrink (IAS ref).In this report, it has been found that some Asthma inflammatory contracting agent can be used as the potent activator of TMEM16A, and the hyperresponsiveness of airway smooth muscle is thus mediated to receive Contracting.This shrinkage character may cause asthma hyperresponsiveness.Mechanism study shows that this effect passes through GPCR/IP₃/Ca²⁺/ TMEM16A/VDCC signal transduction cascade is realized.Our result emphasizes inflammatory contracting agent in the weight formed in asthma hyperresponsiveness The property wanted, wherein TMEM16A is the critical therapeutic target of asthma.

In some embodiments, the medicament is the TMEM16A binding molecule for inhibiting TMEM16A function.For example, medicament is For the antibody of TMEM16A, such as T16Ainh-A01 or NFA or combinations thereof.Additional letter relevant to the antibody of TMEM16A is directed to Breath can be found in U.S. Patent number US 9,045,546, and be incorporated to it by introducing.

Shrink response and inflammatory contracting agent

In some embodiments, the inflammatory contracting agent may include in 5-HT, histamine, prostanoid or CysLT It is at least one.Such as histamine, 5-HT, prostanoid and cysteinyl leukotriene (CysLT) inflammatory contracting agent directly cause ASMC shrinks and as airway contraction agent.Effect of the current this effect in asthma pathology and they how to adjust smoothly Flesh shrinks unclear.In some embodiments, the g protein coupled receptor that these contracting agents can be corresponding is (for example, be directed to The 5-HT of 5-HT₂A receptor, the H for histamine₁Receptor, the Cys-LT for CysLT₁Receptor and be directed to TxA₂With other forefront The TP receptor of parathyrine class) it combines and activates G_q/G_iHeterotrimeric G protein and other signals conduct module, wherein the G activated α_qPLC/IP may be passed through with G β γ₃Calcium is caused to discharge from ER.Then, such as PKC, Erk downstream module is activated respectively, this is right It is required for various immune responses.In smooth muscle cell, activating this signal transduction cascade may cause calcium from activation flesh Release in sarcoplasmic reticulum (SR) required for immunoglobulin light chains kinases (MLCK) and initiation are shunk.It is interesting that with cholinergic excitement Agent is compared, even if minimal amount of contracting agent still is able to induce apparent contraction.The expected production of signal transduction activation during this process Raw IP3 molecule wants much less, therefore calcium release is less, is not enough to direct activation MLCK.

In general, smooth muscle contraction can be caused by acetylcholine and depolarising.The depolarising of cell membrane has activated electricity Press dependence Ca²⁺Channel (VDCC) and then cause from extracellular Ca²⁺It flows into.Raised cytosol calcium is then living Change MLCK and be catalyzed myosin light chain phosphorylation (RLCp), the intersection bridge (cross-bridge) for then causing thickness silk moves It is dynamic.Acetylcholine activates G by GPCR_qAnd generate inositol 1,4,5 triphosphate (IP₃) and be released from the Ca of sarcoplasmic reticulum²⁺.It rises High calcium is by activation MLCK come mediate smooth muscle contraction.Meanwhile the G of activation_12-13ROCK, CPI-17 and MYPT1 module are raised, And the contraction of mediate calcium sensitization.Due to structure and biochemistry difference, different smooth muscles can pass through each signal transduction Cascade is to play its Shrinkage behavior.In air flue, in addition to contractile protein such as MYPT1, CPI-17 of differentiation expression, smooth muscle Cell great expression TMEM16A, is Ca²⁺The Cl of activation^-(Cl_Ca) channel key molecule.

Some inflammatory contracting agents may directly cause airway smooth muscle (ASM) to shrink and serve as airway contraction agent, still Adjustment mechanism be potential and Correlation with Pathology still have it is to be determined.Applicant illustrates herein, such as 5-HT, histamine, U46619 It is induced in apparent in airway smooth muscle cells (ASMC) to Cl- electric current with contracting agents such as LTD4, and inhibition can be passed through TMEM16A albumen (i.e. the chloride channel of calcium activation) effectively inhibits this effect.In vitro and internal inhibition can also be passed through TMEM16A inhibits the contraction as caused by these contracting agents.Electrophysiology discloses, these contracting agents mainly pass through GPCR- TMEM16A-VDCC signaling axis come mediate airway smooth muscle (ASM) shrink.Being lacked by TMEM16A blocks this axis significantly to press down The synergistic effect of cholinergic stimulation and contracting agent has been made, has been shunk to reduce internal asthma airways.

In asthma airways, many inflammatory mediators are released from the cell by infiltration, and some of mediators may Directly cause smooth muscle spasm.Potential adjustment mechanism and its Correlation with Pathology are illustrated for understanding Pathogenesis of Asthma and treatment Development is vital.Applicant has found herein, such as 5-HT, T_XA₂, the inflammatories contracting agent such as LTD4 and histamine can pass through activation TMEM16A chloride channel is interior to Cl in ASMC to induce_CaElectric current.Gained Cl_CaElectric current makes ASMC membrane depolarization, this causes Calcium is flowed into via VDCC.Raised cytosol calcium causes smooth muscle contraction.A small amount of inflammatory contracting agent not only independently induces gas Road is shunk, but also enhances (or sensitization) cholinergic activity.This effect can be explained well why when the allergy for suffering from asthma Air flue can become more sensitive to neural stimulation when property inflammation.Therefore, it is received by the ASM that GPCR/TMEM16A/VDCC axis mediates Contracting may be the basis of asthma hyperresponsiveness.

The receptor of inflammatory mediators can generally be located at immunocyte and be answered by multiple signal cascades come mediated immunity It answers.In these cascades, mediator can successively activate G_q, PLC and IP₃/IP₃R, and discharge calcium from SR.Then, it increases Calcium can activate necessary to various responses signaling module (such as PKC, calmodulin, troponin C, calcium tune phosphoric acid downstream Enzyme B ...).TMEM16A is less likely to participate in calcium release event in the process, because it is not expressed in these cells.Gas Road smooth muscle cell expresses inflammatory mediators receptor and TMEM16A albumen.This characteristic expression means two kinds of letters of calcium release Number approach: enter (TMCI) from the calcium current of intracellular release (IRFE) and TMEM16 mediation in ER.Both approach assign air flue Smooth muscle shows contraction and non-constricted response (such as cell Proliferation, differentiation) simultaneously.Due to inhibiting TMEM16A usually will not be complete Inhibit the power of contracting agent to generate, and inhibit to change with inhibitor concentration, therefore IRFE approach and TMCI approach can be It is coexisted or co-activating in the smooth muscle cell stimulated with contracting agent.Both approach coexist can aid in and more efficiently adjust It shrinks.When air flue receives the stimulation of a small amount of contracting agent, the calcium discharged by IRFE approach can activate TMEM16A and pass through TMCI approach expands Ca2+ oscillations, at the same many contracting agents may be come by enough Ca2+ oscillations for being generated by IRFE approach it is direct Cause and shrinks.However, it should be noted that TMCI changes in the functional contribution shunk in response with contracting agent, such as 5-HT tight TMCI is depended on again.The working model of the inflammatory contracting agent for adjusting airway smooth muscle contraction is shown in Fig. 5.In this model In, contracting agent combines its corresponding receptor and successively activates G_qAlbumen and PLC cause to generate IP from ER₃It is discharged with calcium.It rises High cytosol calcium causes TMEM16A activate, is interior to Cl in turn^-Electric current generates, necessary to VDCC activation and activation MLCK Calcium current enters.It in the process, is critical event by local Ca effective activation TMEM16A.In fact, several from what is reported before this A evidence shows the space compartment (for example, TMEM16A) for promoting ER and TMEM16A interaction and the ER in neuronal cell IP₃Receptor (IP₃R it) common location and directly interacts, and gained local Ca can be activated with TMEM16A and is coupled；It is flat The IP of sliding myocyte₃R can be distributed at the peripheral SR of plasma membrane, to be IP₃The calcium of induction provides mutual with TMEM16A The space opportunity of effect.In addition, G_qSmooth muscle is knocked out with TMEM16A, and I is also supported to the similar contraction phenotype of the response of shrinker The model that is proposed.It should be further understood that there are three features for this GPCR-TMEM16A-VDCC signal transduction model tool.Firstly, Due to the abundant expression of TMEM16A, ASM mainly uses the model.Secondly, under physiology or pathological condition, this model can be with The mechanism of contracting agent is served as, because of the amount of the contracting agent discharged from bronchus or lung under tranquillization or induction state not Ying Yushen The maximum concentration used in vitro test of asking someone is equally high.Third, this mechanism can with the mechanism coexistence of direct activation MLCK, Although 5-HT may be exception.Latter mechanism can promote to make response to the contracting agent of high concentration under extreme conditions.

The unique property of asthma is that ASM becomes more sensitive to the cholinergic stimulation on the basis as hyperresponsiveness.It crosses Quick property inflammatory mediators and the synergistic effect or enhancement effect of cholinergic transmitter are considered as a possible cause of this effect.? In this report, applicant observes two kinds of contracting agents and the apparent enhancement effect of MCh really.Therefore, TMEM16A activity is blocked Airway smooth muscle relaxation can not only directly be made, but also can be reduced hyperresponsiveness.

Each embodiment as described above can be combined to provide other embodiment.If it is necessary, then can be right The various aspects of embodiment, which are modified, provides other embodiment with concept using each patent, application and publication.

In view of description as detailed above, embodiment can be carried out herein with other changes.In general, in following following claims In, used term is not necessarily to be construed as claim being limited to the specific implementation disclosed in description and claims Example, but should be interpreted whole models comprising all possible embodiment together with equivalent required by this claim It encloses.Therefore, claim is not limited by the disclosure.

Example

Example 1

Inflammatory contracting agent induces the Cl of ASMC by TMEM16A_CaElectric current

By generating Cl_CaElectric current, ASMC film can be depolarized by GPCR agonist.In order to whether test this electric current It is generated by TMEM16A, applicant with the ASMC cell of various contracting agents processing fresh separated and passes through repairing cell membrane first To record Cl_CaElectric current.After with 3 μM of 5-HT (the main shrinkage agent discharged from mouse hypertrophy cell) processing, applicant is measured The obvious inward electric current (Fig. 1 a and Fig. 6 a) of 11 smooth muscle cells in 26 smooth muscle cells.It can also be used using this method The similar Cl after being handled with 1 μM of U46619_CaThe measurement of electric current, the U46619 are that another effective inflammatory of asthma is shunk Agent, the inflammatory contracting agent are the thromboxane As from intra platelet free calcium₂(TXA2) stable derivatives (Fig. 1 b and Fig. 6 b).Due to Mouse ASMC is almost without histamine H₁Receptor and Cys-LT₁Receptor, and applicant fails be used as effective airway contraction agent Histamine and LTD₄Cl is measured after the two processing_CaElectric current, application is rich in histamine H to applicant instead₁Receptor and Cys-LT₁The globefish of receptor Mouse ASMC measures histamine and LTD₄The Cl of induction_CaElectric current.As desired by applicant, two kinds of contracting agents all with 5-HT and The similar fashion of U46619 significantly induces interior to Cl^-Electric current, although Cl^-The amplitude of electric current change (Fig. 1 c, Fig. 1 d and Fig. 6 c, Fig. 6 d).It is interesting that also can effectively induce Cl down to 0.3 μM of methacholine_CaElectric current, it means that cholinergic agonist and Inflammatory contracting agent Cl_CaElectric current, which generates, has common mechanism (Fig. 1 e and Fig. 6 e).In order to determine induce by contracting agent it is this interior Whether mediated to electric current by TMEM16A, applicant apply ASM (seeing below) from TMEM16A knock-out mice and TMEM16A、T16A_inhThe specific inhibitor of-A01., it is surprising that TMEM16A missing is almost completely eliminated, and 10μM T16A_inh- A01 largely inhibits the inward electric current (Fig. 1 a-e and Fig. 6 a-e) induced by contracting agent.This effect It should can not be lacked secondary to TMEM16A, because corresponding receptor is in mutant ASM without changing (Fig. 7).This observation result Show the Cl of the ASMC induced by inflammatory contracting agent strongly_CaElectric current is mediated by TMEM16A.

In order to obtain TMEM16A deficiency smooth muscle, applicant makes Tmem16a^flox/floxMouse and SMA-Cre transgenosis Mouse hybrid.Gained mouse is Tmem16a^flox/flox；SMA-Cre is used as knock-out mice (Tmem16a^SMKOOr KO), and Tmem16a^flox/+；SMA-Cre littermate is used as control mice (CTR).Tmem16a^SMKOThe outer ASM of lung in only about 10% TMEM16A remains (Fig. 8 a).The immunofluorescence dyeing of lung sections is shown, and is not influencing table of the TMEM16A in epithelium In the case where reaching, most of TMEM16A lacks (Fig. 8 b) in intrapulmonary bronchial smooth muscle.In order to confirm the Cl in ASM_CaChannel Whether due to TMEM16A, applicant is using CTR the and KO ASMC newly separated, by being applied with pipette solution 600nM Free Ca²⁺Concentration carries out impulse stimulation or records full cell Cl by focal be perfused to cell addition caffeine^- Electric current.The result shows that the typical Cl of outward rectification_CaElectric current and caffeine induction it is interior to both Cl- electric currents in KO ASMC all Receive very big inhibition (Fig. 8 c, Fig. 8 d).

Example 2

TMEM16A is necessary to the contraction response of inflammatory mediators

In order to test effect of the TMEM16A in the contraction that contracting agent mediates, applicant measures TMEM16A deficiency ASM and T16A_inhThe contraction of the ASM of-A01 processing.Before measuring, applicant has checked the bronchial thickness of lung, and it was found that It is not substantially change with CTR (P > 0.05, double tail student t are examined) comparison.After with 60mM KCl or 10 μM of MCh stimulations, mutation Muscle generates and compares the comparable power (flox/+ of muscle；Cre⁺Or flox/flox；Cre^-) (Fig. 2 c and Fig. 9 a, Fig. 9 b), this table Bright this muscle has normal contraction organ.It is interesting that being induced when being mutated muscle with the MCh processing down to 300nM Power significantly less than control (examined by double tail student t, KO:2.58 ± 0.68mN is to CTR:5.66 ± 0.95mN, P < 0.05, Figure 10) this shows that TMEM16A is required, this and ICl for the contraction response of weak cholinergic stimulation_CaThe result of induction is consistent. Inflammatory contracting agent U46619 induced in a manner of dose response ASM shrink (Fig. 2 e and Fig. 9 c), and the maximum, force induced with MCh is quite (for MCh, about 12mN；For U46619, about 10mN).After TMEM16A missing, the power of about 34%-76% by It significantly inhibits (by ANOVA, P < 0.0001, Fig. 2 e), and inhibition level changes with dosage.At with 300nM U46619 When reason mutation muscle, the power more than 76% is suppressed, and simultaneously for 10 μM of U46619, about 34% power is suppressed (figure 2e).This result shows that, by U46619 generate an average of at least 56% power by TMEM16A mediation.After being handled with 5-HT, Control smooth muscle induces obvious contraction in a manner of dose response, but maximum tension is about 5mN to 6mN, about for by U46619 Or 50% (Fig. 2 f) of MCh induction., it is surprising that when with the 5-HT of each dosage stimulation mutation muscle or even dosage Increase to 100 μM, power is all nearly eliminated (by ANOVA, P < 0.0001) (Fig. 2 f and Fig. 9 d).In the 5-HT of all dosage Under, average inhibition is 73.37 ± 1.70%.This observation indicate that, by 5-HT induction power mainly pass through TMEM16A be situated between It leads.This effect can further be confirmed in vivo because with 5-HT excitation mouse raised respiratory resistance (Rrs) by TMEM16A missing significantly inhibits.(passing through ANOVA, p < 0.001, Fig. 2 g and Fig. 9 e).Because cavy ASM is good rich in reaction H₁Receptor and Cys-LT₁Receptor, so applicant is measured using this muscle to histamine and LTD₄Contraction response.Two kinds of contractions Agent all generates power (Fig. 2 h, Fig. 2 i) in a manner of dose response.Note that 0.01nM LTD₄Or 30nM histamine is adequate to bring about power It generates.Applicant then uses T16A_inh- A01 inhibitor assesses TMEM16A in histamine and LTD₄Work in the contraction of induction With.Before our subsequent experiments, by using Tmem16a^SMKOMouse confirms specificity (figure of this inhibitor in ASM 11).Histamine and LTD₄Valence available significantly inhibit (Fig. 2 and Figure 12).T16A_inhThe addition of-A01 almost disappears In addition to power (Fig. 2 h, Fig. 2 i and Figure 12 a, figure of the histamine (10 to 300nM) by low dosage and LTD4 (0.01 to 0.3nM) induction 12b).Enable us surprisingly, the basal tension of cavy ASM is also significantly inhibited (Figure 12 c).With contraction agent dose Increase, T16A_inhThe inhibiting effect of-A01 declines rapidly.When histamine and LTD₄Dosage respectively reach 3 μM and when 10nM, T16A_inh- A01 lies in less than obvious inhibiting effect.This result shows that, TMEM16A is by histamine and LTD₄It is weak stimulate into Necessary to capable sensitization response.If applicants assume that the physiological level of contracting agent remains essentially in basal tension, TMEM16A inhibitor also supports this conclusion to the inhibition of basal tension.Applicant is also tested for T16A_inh- A01 is to expression H₁By Body and Cys-LT₁The influence of people's tracheal smooth muscle of receptor.Fresh tracheae is isolated from the lung bioplsy of operation of lung cancer patient Then sample measures strip of muscle through stress.When being handled with histamine, strip of muscle is with the dose response similar with cavy smooth muscle Mode generates apparent power (Fig. 2 h, Fig. 2 j).Add 10 μM of T T16A_inh- A01 also causes power tension to be significantly inhibited (Fig. 2 j With Figure 12 d).In short, our observation is the results show that the missing of TMEM16A or inhibition lead to the power caused by inflammatory contracting agent Substantially reduce, this show contracting agent mediate contraction in need TMEM16A.

Example 3

Inflammatory contracting agent passes through GPCR/TMEM16A/VDCC signaling axis mediate contractile

Applicant has speculated the working model that the power of contracting agent generates herein, shows TMEM16A activation by GPCR receptor simultaneously And with it is resulting it is interior to Cl electric current depolarized cells film trigger, then cause calcium current to enter by VDCC.In order to verify this it is assumed that Shen Ask someone to have checked the signal transduction module in GPCR/TMEM16A/VDCC axis.Enter to measure the calcium current of KO smooth muscle cell, Shen It asks someone to hybridize TMEM16A knock-out mice with Ai38D mouse (a kind of strain of the expression with GCaMP3 calcium instruction).Application People tests the functional status of this calcium instruction first.After being handled with 60mMKCl, GCaMP3 muscle is shown in the initial stage Strong calcium flash of light, followed by the sustained period (Figure 13 a) of relatively weak signal.Applicant is also tested for be pierced in response to MCh This sharp instruction is sub and obtains similar result (Figure 13 b).In TMEM16A deficiency smooth muscle, such as in CTR muscle It observes, 60mM KCl depolarising increases (Fig. 3 a and Figure 11 a) induction of steady and lasting calcium.3 μM of MCh induces calcium liter Height, wherein with (p=0.167) indifference is compareed, average delta F/F0 value is 39.67 ± 3.44% (Fig. 3 b and Figure 13 b).This calcium Lift mode seems consistent (Figure 10) with corresponding contraction response.Since 5-HT is the main and typical contracting agent of mouse, Shen It asks someone the calcium release action for therefore substantially measuring 5-HT.After handling mutation smooth muscle with 300nM 5-HT, the peak Δ F/F0 Value drops to 6.56 ± 1.02%, while the Δ F/F0 peak value of CTR is that 13.20 ± 2.15% (Fig. 3 c) (are examined by double tail student t It tests, p < 0.05).When with (3 μM) the processing muscle of 5-HT of relatively high dose, Δ F/F0 peak value is 23.00 ± 3.29%, this Peak value, which is also significantly lower than, compares (34.35 ± 4.31%) (examining by double tail student t, p < 0.05).By the 5-HT of matched doses The Δ F/F0 value of the sustained period of induction is significantly reduced in KO (by ANOVA, P < 0.001, Fig. 3 c, Fig. 3 d, Figure 13 c, figure 13d).Other contracting agents that applicant also passes through in TMEM16A deficiency smooth muscle or the smooth muscle of inhibitor processing measure Calcium discharges and obtains similar trend (data are not shown).This result discloses, and TMEM16A is in response to the calcium in contracting agent Necessary to raising.In order to whether determine this calcium release by VDCC mediation, applicant applies a kind of nifedipine (L-type Ca²⁺The specific inhibitor in channel (VDCC)).After adding 1 μM of nifedipine, control smooth muscle is shown by MCh initiation The slight change of power, and observe substantially changeing by the 5-HT power caused.However, adding 1 μM in KO bronchial smooth muscle Nifedipine and the dose response curve (Fig. 3 e, Fig. 3 f) for having not been changed both MCh and 5-HT.Total result shows that two kinds are shunk The MCh of agent and low dosage may adjust ASM contraction by GPCR/TMEM16A/VDCC axis.

Example 4

TMEM16A enhances the contraction response of cholinergic agonist, is necessary to asthma hyperresponsiveness

In asthma, ASM becomes more sensitive to cholinergic stimulation, this is considered as the main original of asthma hyperresponsiveness Cause.Applicant's conjecture, this process may relate to activate TMEM16A by inflammatory contracting agent.Applicant is therefore in vitro and internal Measure contracting agent and the enhancement effect of MCh.In isolated experiment, the contracting agent (5-HT:100nM of applicant's low dosage； U46619:100nM) pretreatment of mice ASM does not cause obvious contraction, then at the MCh (100nM) of low dosage Reason, it is also independent to cause obvious contraction (Figure 14).It has been found that the combination addition of contracting agent and MCh cause power to generate significantly Increase (Fig. 4 a and Figure 12).Add 100nM MCh to mouse ASM addition 100nM 5-HT, induction is significantly higher than independent 5-HT/MCh Double addition power (100nM 5HT+100nMMCh:6.19 ± 0.79mN to 100nM 5-HT+100nM 5-HT:2.22 ± 0.57mN examines, all P < 0.05 100nM MCh+100nM MCh:4.12 ± 0.80mN by matching double tail student t)；Add Significant higher power tension (100nM U46619+100nM MCh:5.88 can be induced by adding 100nM U46619 to add 100nM MCh also ± 0.91mN to 100nM U46619+100nM U46619:2.08 ± 0.65mN to 100nM MCh+100nM MCh:4.11 ± 0.80mN is examined, all p < 0.05 by matching double tail student t).TMEM16A missing significantly suppresses all these enhancing effects Answer (Fig. 4 a is examined, P < 0.05 by double tail student t).The inhibiting rate of 5-HT and U46619 is respectively 42.17 ± 10.51% Hes 42.29 ± 10.16%.This result shows that, the contraction response of cholinergic stimulation can be enhanced in contracting agent, and TMEM16A joins accordingly With.

In order to determine possibility contribution of this enhancement effect in asthma contraction, applicant is established by chicken egg white (OVA) acute asthma model induced.Although both CTR mouse asthma and KO mouse asthma show similar inflammatory infiltration and Epithelium swelling (Figure 15), but the liter as desired by applicant is shown with the CTR asthma animal that anaphylactogen (5%OVA) is excited High respiratory resistance (Rrs), and KO asthma animal shows significant lower Rrs (by ANOVA, P < 0.001, Fig. 4 b). For example, after excitation 5-45 minutes when, the Rrs (Δ Rrs) of KO mouse asthma is only about the 50% of CTRRrs.This result shows that, Individual anaphylactogen seems to be enough induced asthma contraction, and TMEM16A missing is able to suppress this process.In addition, working as with each When the MCh of dosage excites asthma animal, knock-out animal also shows that the inhibiting effect (Fig. 4 c) to airway contraction.Applicant pays attention to It arrives, inhibition level changes with excitation MCh dosage used.When MCh dosage is less than 30 μ every mouse of g, it is mutated mouse asthma It shows that about 40% Δ Rrs inhibits, while when dosage increases to 100-300 μ every mouse of g, not showing inhibition (Fig. 4 c Insertion figure).Total result shows that the TMEM16A activation mediated as contracting agent is necessary to asthma hyperresponsiveness.

Example 5

Genetic mouse model

Zoopery in this research is according to Nanjing University animal pattern research center animal welfare and to use the committee What the guilding principle of (Nanjing of China) carried out.The flox mouse of Tmem16a is established via those skilled in the art, including TMEM16A CTR(Tmem16a^flox/+；SMA-Cre⁺)、KO/Tmem16a^SMKO(Tmem16a^flox/flox；SMA-Cre⁺) and Tmem16a^flox/flox.To CTR, KO and the Tmem16a in 8 to 20 weeks of two kinds of genders^flox/floxMouse carries out a series of analyses, In comprising Western blotting, immunofluorescence, patch-clamp, isometric tension and respiratory resistance measurement.By 8-20 weeks with two kinds of genders Ai38D mouse hybrid TMEM16A CTR mouse and KO mouse be used for Ca²⁺Measurement.All mouse are 129:B6 congeric strains Background.

6. western blot analysis of example

As described in Alison J.Davis et al., keep the strip of muscle separated from tracheae and CTR mouse and KO small The extrapulmonary bronchus of mouse is homogenized (20mM Tris alkali, 137mM NaCl, 2mM EDTA, 1%NP- in 100 μ L lysis buffers 40,10% glycerol, protease inhibitor cocktail (Roche Holding Ag (Roche)), pH=8).Then homogenate is cultivated 15 on ice Minute, and be centrifuged at 3000g to remove cell fragment.80 μ L supernatants of each sample are transferred in new pipe.With two Quinolinecarboxylic acid (BCA) kit (BCA Protein Assay Kit, Pierce) measures protein concentration.By protein and 5 × sample Savor buffer (10%SDS, 20% glycerol, 0.05% bromophenol blue, 10mM beta -mercaptoethanol, 200mM Tris-HCl, 8M urea) Mixing, is then denaturalized 5 minutes at 95 DEG C.After being parsed by SDS-PAGE electrophoresis, by Protein transfer to pvdf membrane and With the 5% skimmed milk closing in TBST, the second level for then successively cultivating Primary antibodies and horseradish peroxidase (HRP) conjugation is anti- Body.Primary antibodies used in Western blotting are summarized in table 1.Keep trace visual using ECL (Sudogen, Co.Ltd.) Change.

Table 1

Example 7

Immunofluorescence

The paraffin section (with a thickness of 10 μm) of mouse lung lobus sinister is used for immunofluorescence dyeing.By containing 1%BSA and The PBST buffer of 5% nonimmune lowlenthal serum cultivates slice to block the non-specific binding of Primary antibodies.By rabbit-anti TMEM16A polyclonal antibody (being diluted with 1:200, ab53212, Ai Bokang company) and the anti-SMA monoclonal antibody of mouse (with 1: 500 dilutions, clone 1A4, Thermo Fisher Scientific Inc. (Thermo)) it is used as Primary antibodies.It will be diluted with 1:250 Alexa Fluor 488- conjugation goat anti-rabbit antibodies (hero company (Invitrogen)) and with the diluted Alexa of 1:250 The goat anti-mouse antibody (hero company) of Fluor 555- conjugation is used as secondary antibody.Nucleus is dyed with DAPI. Image is captured by Laser Scanning Confocal Microscope (FV-1000, Olympus (Olympus)).

Example 8

The separation of airway smooth muscle cells

By TMEM16A CTR mouse and KO mouse (8 to 20 weeks, two kinds of genders) or hartley (Hartley) cavy (250 To 300g, male) tracheae and extrapulmonary bronchus prepare smooth muscle, and as described previously use papain and glue Protoenzyme is handled.Gained group is woven in dissociation culture medium and is dissected (DM；136mM NaCl,5.36mM KCl,0.44mM KH₂PO₄、4.16mM NaHCO₃、0.34mM Na₂HPO₄, 20mM Hepes, 5.56mM glucose, 4.9mM MgCl₂、pH 7.1).With containing 30U/mL papain, 0.2mM dithiothreitol (DTT), 0.02mM EDTA without MgCl₂DM carry out first Secondary digestion, and with no MgCl₂DM in contain 3U/mL clostridiopetidase A 1A, 1mg/mL bovine serum albumin(BSA), 0.1mg/ml deoxidation The solution of ribalgilase I carries out second and digests.The pipe of second of digestion is aggressively shaken until most several piece disappears (about 20 points Clock).After being centrifuged 2 minutes at 300g, by being centrifuged 2 minutes at 300g, with no MgCl₂DM wash collected cell one It is secondary.Separated cell is resuspended in DM, is then transferred into culture dish, stored at 4 DEG C.Before the use, dipping bath is used Liquid replaces DM to carry out Patch-clamp techniques.

Example 9

Patch-clamp techniques

Using MultiClamp 700B amplifier, Digidata 1440A and pClamp 10 (molecular device) to record There is electric current.Filter current and the electric current is sampled with 10kHz at 2kHz, and is analyzed using Clampfit 10 Data.In order in 600nM [Ca²⁺]_iUnder the conditions of record Cl^-Electric current, as described previously uses whole-cell patch-clamp.Dipping bath liquid Contain 150mM NaCl, 1mM CaCl₂、1mM MgCl₂, 10mM D-Glucose, 10mM mannitol and 10mM HEPES；It uses NaOH adjusts pH value to 7.4.Pipette solution contains 130mM CsCl, 10mM EGTA, 1mM MgCl₂、8mM CaCl₂、 10mM HEPES,1mgATP；PH value is adjusted to 7.3 using NaOH.Under these conditions, E_Cl=0mV.With 10mV increment record In response to 1 second voltage pulse of -80mV to+100mV and to 700 millisecond pulses of -60mV in the smooth muscle cell newly separated Full cell currents.

In order to record by the electric current of contracting agent and caffeine induction, whole-cell patch-clamp as described previously is used.Leaching Bath foam contains 130mM NaCl, 5mM KCl, 1mM CaCl₂、1mM MgCl₂, 20mM HEPES, 10mM D-Glucose；It uses NaOH adjusts pH value to 7.4.Pipette solution contains 140mM KCl, 1mM MgCl₂、20mM HEPES、0.025mM EGTA；PH value is adjusted to 7.2 using KOH.Under these conditions, E_Cl=0mV.3 μM of 5-HT, 1 μM are given by pipette U46619,3 μM of histamine, 100nM LTD₄, 3 μM of MCh and 10mM caffeines apply focal enhancing perfusion with induced current (ALA Scientific Instruments Corporation (ALA Scientific Instruments)).After destroying diaphragm under pipette, record electric current No more than the 5th minute.

Example 10

Histopathology

Scheme is had modified according to the method.Will tracheae, extrapulmonary bronchus and lung lobus sinister immerse 4%PFA in overnight and It is dehydrated in the ethanol solution of hierarchical sequence.By organization embedding in paraffin.The cutting thickness of slice is 10 μm and is pacified On glass slide.H the and E decoration method of execution standard.It is measured from innermost edge to ragged edge by using Image J software The thickness of the bronchial smooth muscle layer of edge.Three scheduled bronchiole sites (near both ends of two sites close to cartilage, One site is among smooth muscle layer) at assess extrapulmonary bronchus smooth muscle layer thickness.3 slices of every mouse are carried out Measurement.

Example 11

Isometric contraction measurement

Power is measured, ice-cold Cray uncle this (Krebs) solution (118.07mM NaCl, 4.69mM KCl, 2.52mM CaCl₂、1.16mM MgSO₄、1.01mM NaH₂PO₄、25mM NaHCO₃, 11.1mM glucose) in, from the group of surrounding Middle left extrapulmonary bronchus of the removing from TMEM16A CTR mouse and KO mouse is knitted, the ring of about 1.5mm long is cut and is installed In linear myograph (610-M；Danish Myo Technology) on and pass through Powerlab recording device (Ai De Instrument International Trading Company Ltd (AD Instruments)) record isometric tension.Bronchial rings are maintained at by O₂-CO₂Mixing Object (95%O₂- 5%CO₂) aoxidized at 37 DEG C Cray uncle this solution in.In 60mM K⁺Stimulation is primary and balances after forty minutes, Suitable preload is given to obtain the rest tension of about 5mN.With 60mM K⁺After stimulating again, measure to different stimulated The contraction response of object.

Guinea pig trachea is shunk and is measured, the tracheae of male hartley cavy (250 to 300g) is quickly removed and is set (mKrebs, 118mM NaCl, 4.6mM KCl, 0.5mM MgCl in ice-cold primary this solution of the Cray by modification₂、 1.8mM CaCl₂、1mM KH₂PO₄、24.9mM NaHCO₃, 11.1mM glucose) and will be cleared out of in its tissue from surrounding It goes.Excision includes the ring of two cartilagines tracheales from tracheae.In order to obtain item, the cartilage opposite with muscle layer is cut.By tracheae One cartilage end of item is fixed to the bottom of muscle dipping bath room；The other end is connected to force snesor (MLT0201, angstrom moral instrument International Trading Company Ltd).Isometric tension is measured by PowerLab (Ai De instrument International Trading Company Ltd) recording device. Tracheal strip is maintained at by O₂-CO₂Mixture (95%O₂- 5%CO₂) in the mKrebs that is aoxidized at 37 DEG C.By rest tension tune Save 1g.Before the experiments, the item is stimulated at least twice with 60mM KCl.By 10 μM of T16A_inh- A01 or mediator (DMSO) Pre-incubation 10 minutes, agonist gradient is then added.

Human bronchial smooth muscle contraction is measured, in ice-cold oxygen saturation HEPES-Tyrode buffer (137mM NaCl、2.7mM KCl、1.8mM CaCl₂、1mM MgCl₂, 5.6mM glucose and 10mM HEPES, pH 7.4) in quickly breeding The sample separated from the operation excision of patients with lung cancer.After being transported to laboratory, the tissue of surrounding is removed, bronchus is put down Sliding muscular tissue is cut into the item of 3mm wide.One end of item is fixed to full of by O₂-CO₂Mixture (95%O₂- 5%CO₂) at 37 DEG C The bottom in the flesh bathroom of this solution of the Cray uncle of lower oxidation, the other end is connected to force snesor, and (MLT0201, angstrom moral instrument are international Trade Co., Ltd).Since the diameter and thickness of the smooth muscle layer in different samples is different, so being surveyed by 60mM KCl stimulation Try suitable rest tension.By 10 μM of T16A_inh- A01 or DMSO mediator pre-incubation 10 minutes, are then added reagent.

Example 12

Ca is carried out using Ai38D report mouse²⁺Signal measurement

By Tmem16a^SMKOMouse and CTR mouse hybrid are at Cre dependence GCaMP3 report mouse (Ai38D).? HEPES-Tyrode (H-T) buffer (137.0mM NaCl, 2.7mM KCl, 1.0mM MgCl₂、1.8mM CaCl₂、10mM HEPES and 5.6mM glucose, pH=7.4) in, the left extrapulmonary bronchus of CTR and KO is mounted on to total focus linear flesh is dynamic to be traced Device (120cw；Danish Myo Technology) on.Agonist is added to self-control gravity perfusion system with 10mL/min flow velocity System.GCaMP3 fluorescence is excited by 488nm argon laser, and every by Laser Scanning Confocal Microscope (FV-1000, Olympus) 2.5 seconds record images.The mean fluorecence for calculating ROI (bronchial smooth muscle range) with SV10-ASW software (Olympus) is strong 1-2 minutes before stimulation average fluorescent strengths are defined as initial fluorescence (F0), are indicated with fluorescence percentage (% Δ F/F0) by degree Ca²⁺The dynamics of signal.

Example 13

With OVA sensitization

Use the mouse of two kinds of genders of 8-10 week old.The 0th day and the 14th day, 4mg is dissolved in by intraperitoneal injection Aluminium hydroxide (alum, Pierce) in the ovalbumin (OVA, Sigma-Aldrich) of 100 μ g make mouse sensitization. Then, it is excited mouse 60 minutes the 24th day, 25 days and 26 days with the 1%OVA being atomized in PBS.It will receive most in 24 hours The mouse excited afterwards is used as acute asthma model.It will receive the diluted 4mg aluminium hydroxide of PBS via intraperitoneal injection and use mist The mouse of the PBS excitation of change is used as non-asthma comparison model.

Example 14

Respiratory resistance measurement and statistical analysis

Applicant measures respiratory resistance using flexure aerating system (SCIREQ).24 after last time OVA or PBS excitation Hour receives mouse and makes its anesthesia by intraperitoneal injection 250-300mg/kg avertin；Then mouse is placed on table simultaneously And it is intubated with No. 18 metal needles of blunt mouth.The needle is connected to the rodent ventilator of flexure aerating system；It will breathing Machine is set as generating the tidal tolerance of 10ml/kg with the frequency of 150 breaths/mins.It is surveyed by the disturbance of entitled SnapShot The resistance (Rrs) of respiratory system is measured, and newton resistance (Rn) is measured by the disturbance of entitled Quick prime-3.In order to measure The variation of respiratory resistance after being induced with 5%OVA is kept for 5 minutes after sucking is atomized OVA, records a Rrs value per minute, directly By the 45th minute.For the respiratory resistance variation of 5-HT excitation, the successive doses (37.5 μ g, 75 μ g, 150 μ g) of 5-HT are atomized To excite air flue, and calculate excitation after in 3 minutes each dosage maximum Rrs to draw dosage-response curve.For MCh The respiratory resistance of excitation changes, and the MCh of successive doses (10 μ g, 30 μ g, 100 μ g, 300 μ g) is atomized to excite air flue, and Calculate the maximum Rrs and Rn of each dosage in 3 minutes after exciting.

Data are expressed as average value ± standard error of the mean.It is examined by unpaired double tail student t or unpaired double Group difference is determined to variance analysis (ANOVA).It matches double tail t and examines the difference (label for being also used for testing in some experiments In the text).Significance setting it is as follows: P>0.05 NS, P<0.05 *, P<0.01 * *, P<0.001 * * *, * * * * P< 0.0001。

Claims (8)

1. the method that one kind reduces the contraction response of the target cell induced by inflammatory contracting agent in vitro or in vivo, the method packet Including contacts target cell and the medicament of a effective amount of inhibition transmembrane protein 16A (" TMEM16A ") activation or signal transduction.

2. a kind of carry out the reduction in needing to reduce the patient of smooth muscle cell contraction response caused by inflammatory contracting agent Method, the method includes making vascular system and the activation of a effective amount of inhibition transmembrane protein 16A (" TMEM16A ") or signal transduction Medicament contact.

3. method according to any one of claims 1 and 2, wherein the inflammatory contracting agent includes 5-HT, histamine, forefront At least one of parathyrine class or CysLT.

4. method according to any one of claims 1 and 2, wherein the medicament is to inhibit TMEM16A function TMEM16A binding molecule.

5. method according to any one of claims 1 and 2, wherein the medicament is the antibody for TMEM16A.

6. method according to any one of claims 1 and 2, wherein the medicament include T16Ainh-A01 or NFA or its Combination.

7. according to the method described in claim 1, wherein the target cell is smooth muscle cell.

8. according to the method described in claim 2, wherein the smooth muscle is airway smooth muscle cells or vascular smooth muscle cells.