CN110368385A - Purposes of the indole -3-formaldehyde in preparation treatment atopic dermatitis drug - Google Patents

Abstract

The invention belongs to pharmaceutical technology fields, it is related to the medicinal and skin care item purposes that tryptophan metabolite includes indole -3-formaldehyde (IAId) etc., and in particular to tryptophan metabolite includes purposes of the indole -3-formaldehyde in the drug and skin care item that preparation has immunoregulation effect；Our experiments show that AD sample inflammation and psoriasiform inflammation that the tryptophan metabolites such as described IAId can inhibit MC903 to induce, inhibit CD4 in corium⁺,Gr1⁺The infiltration of positive cell；Inhibit the expression of inflammatory factor；Reduce the degree that skin is lost through the severe edema due to hypofunction of the spleen；Mouse is inhibited to scratch behavior；Experimental result confirms that the tryptophan metabolites such as the IAId have the function of clearly anti-AD sample inflammation, it can be used for preparing the drug and skin care item for preventing and treating atopic dermatitis (eczema), it is especially formed into external preparation, for preventing and treating Acute and chronic inflammation caused by abnormal immune reaction.The prevention and treatment that the present invention is atopic dermatitis in the clinical application of clinical experimental stage sum provide new medicament selection, have extensive commercial applications prospect.

Description

Purposes of the indole -3-formaldehyde in preparation treatment atopic dermatitis drug

Technical field

The invention belongs to pharmaceutical technology fields, are related to the new biological action of tryptophan metabolite, and in particular to tryptophan Metabolite includes the medicinal and skin care item purposes of indole -3-formaldehyde (IAId) etc. more particularly to tryptophan metabolite includes Purposes of the indole -3-formaldehyde in the drug and skin care item that preparation has immunoregulation effect；The tryptophan metabolite Have the function of inhibiting immune response including indole -3-formaldehyde (IAId), is used especially for preparing external curing atopic dermatitis The drug and skin care item of the inflammatory dermatoses such as (eczema), psoriasis.

Background technique

Data disclose atopic dermatitis (atopic dermatitis, AD) be the whole world most often include skin disease it One, disease incidence increases year by year, and the disease incidence in children is up to 20% at present, according to statistics, about the children of a quarter at Nian Houhui development is AD in adults, and about 1/5th AD infant makes further progress as the possibility of asthma.Since at present there is no thorough The means that bottom cures, based on symptomatic treatment, medical practice show clinic, the illness state of an illness usually recurrent exerbation, acutely The difficulty of social activity caused by itch, sleep disturbance and extensive skin lesion causes greatly to perplex to patient's physical and mental health, separately there is research table Bright, the illness rate of depression, anxiety disorder and self-closing disease increases year by year in AD patient, and the social concern thus caused is more and more tighter High, for AD patient while body and mind bears immense pressure, family and society need to put into a large amount of medical resource and health care expense With causing high financial burden.It data show, be up to 5,000,000,000 beauty in the expense that the U.S. is dropped in every year on average on treatment AD Member, the expense for treating AD account for the 10% of family income, cause huge financial burden to patient home and society.It grinds Study carefully and disclose under the overall background that industrialization degree is constantly deepened and living condition greatly improves, the appearance of problem of environmental pollution with And excessive cleaning habit is formed such that and also increases year by year in the disease incidence of China AD, causes greatly to patient, family, society Burden.In the past few decades, although those skilled in the art have done wide research and discussion to the mechanism of AD, so far Until still lack effective treatment means and precautionary measures.

Separately some researches show that skin flora imbalance is the important link of AD morbidity, wherein staphylococcus aureus abundance liter The severity of high degree and AD is positively correlated, and all the time, inquires into skin flora and participates in the mechanism that AD is adjusted, pass through tune The method of section flora treats and prevents the hot spot that AD is this field research, however previous research is directed generally to flora proper constituent Effect study in AD, and related scholar thinks, information provided by opsonigenous substance has more functional meaning, and metabolism produces Object can more reflect the information in terms of the vigor of flora, the functions such as pathogenic.In recent years, the flora and place that opsonigenous substance mediates Main immune system interaction is receive more and more attention, will become the main target for the treatment of AD by the intervention of bacterial metabolism Point, however, research focuses primarily upon therapeutic effect of the intestinal tract flora metabolite to bowl inflammatory diseases at present, so far, still Have no the report of therapeutic effect of the skin flora metabolite to AD.

Tryptophan is the important sources of nitrogen source needed for flora is survived, and indoles and its derivative are a kind of important tryptophan generations Thank product, the horizontal adjusting by related flora level；Indoles substance plays important in adjusting intraor extracellular signal path Effect, it is considered to be link up the key substance between environment, flora and human body three.Indole -3-formaldehyde is a kind of important Yin Diindyl class metabolite, some researches show that indole -3-formaldehydes, and the IL-22 of aryl hydrocarbon receptor (AhR) access can be relied on by promotion Transcription, inhibit the field planting of Candida albicans in enteron aisle, maintain the balance of intestinal flora, inhibit intestinal inflammation, but not yet See the research report of the adjustment effect in relation to indole -3-formaldehyde in AD inflammation.

Status based on the prior art, present inventor is quasi- to provide indole -3-formaldehyde (IAId) new medicinal and shield The purposes of skin product purposes, especially indole -3-formaldehyde in the drug and skin care item that preparation has immunoregulation effect.

Summary of the invention

The purpose of the present invention is the statuses based on the prior art, provide tryptophan metabolite new biological action, relate to And tryptophan metabolite includes the new medicinal and skin care item purposes such as indole -3-formaldehyde (IAId), and in particular to tryptophan generation Thank purposes of the product indole -3-formaldehyde in the drug and skin care item that preparation has immunoregulation effect, especially tryptophan generation Thank to purposes of the product indole -3-formaldehyde in the drug and skin care item that preparation inhibits inflammatory reaction, the tryptophan metabolism produces Object indole -3-formaldehyde can be used for the inflammatory dermatoses such as external application prevention and treatment atopic dermatitis (eczema), psoriasis.

Tryptophan metabolite indole -3-formaldehyde (IAId) of the present invention is a kind of with structure shown in Fig. 7 Indole derivatives, research shows that a variety of tryptophan metabolites have similar biological action, such as IAA, TrA tryptophan Metabolite.

In the present invention, the AD model (MC903 model) induced by the mouse Calcipotriol (MC903) of foundation is carried out The tryptophan metabolites such as indole -3-formaldehyde inhibit the experiment of AD sample inflammation, the results show that

(1) inhibiting effect of the IAId to AD sample dermatitis

1) external preparation for skin IAId mitigates AD sample inflammation

It is continuous to smear 14 days in MC903 model mice ear external application IAId, the result shows that mouse AD sample is scorching after external application IAId Disease is substantially reduced, substantially shows as the red and swollen mitigation of IAId group, the scales of skin that peel off reduces (as shown in Figure 1A)；HE dyeing display intradermal leaching The inflammatory cell of profit reduces (as shown in Figure 2 B)；IAId group ear thickness is substantially reduced (as shown in Figure 1 C), in addition to this, passes through view Frequency record discovery external application IAId can obviously inhibit the scratching behavior (as shown in figure iD, * * * P < 0.001) of mouse；

2) external application IAId can inhibit the expression of inflammatory factor, reduce the total IgE level of peripheral blood,

RT-PCR the results show that IAId obviously inhibit IL-4, IL-5, IL-6, IL-13, TSLP expression and IAId inhibits the total IgE of peripheral blood horizontal (as shown in Fig. 2A~2F) to ELISA as the result is shown；

3) external application IAId can inhibit the infiltration of inflammatory cell,

After external application IAId, the GR1 that is infiltrated in experiment mice otic tissues⁺As shown in Figure 3, (* * * P < 0.001), CD4⁺ The quantity of (as shown in Figure 3D, * * * P < 0.001) cell is significantly reduced compared with MC903 group, and CD8⁺The infiltration quantity of cell has no There is statistical difference (as shown in Figure 3 C).

(2) inhibiting effect of the tryptophan metabolites such as IAA, TrA to AD sample dermatitis and psoriasis

The present invention has also carried out other tryptophan metabolites such as IAA, TrA and has tested to the effect of mouse AD sample scytitis Research, the results show that the scytitis of mouse is substantially reduced (such as Fig. 4 institute after described tryptophan metabolite IAA, TrA effect Show), the inflammatory cell of HE dyeing display intradermal infiltration reduces (as shown in Fig. 3 B)；

The present invention acts on the mouse psoriasiform inflammation mould of the imiquimod induction of preparation using described IAA, TrA Type, as the result is shown., IAA, TrA effect after, psoriasiform dermatitis substantially change it is substantially reduced, histocytology display infiltration Inflammatory cell significantly reduces (as shown in Figure 5), and it is aobvious to show that described tryptophan metabolite IAA, TrA have psoriasis model The effect of the inhibition inflammation of work.

(3) tryptophan metabolites such as IAId, IAA, TrA promote the maintenance of barrier function

Experiment display, IAId, IAA, TrA remarkably promote the table of mice skin tissue barrier relevant molecule such as Filaggrin It reaches, reduces and lose horizontal (as shown in Figure 6) through the severe edema due to hypofunction of the spleen, show that tryptophan metabolite takes part in the adjusting of skin barrier function.

The tryptophan metabolites such as IAId of the invention inhibit AD inflammation and psoriasiform inflammation the experimental results showed that, The AD sample inflammation that the tryptophan metabolites such as IAId can inhibit MC903 to induce inhibits CD4 in corium⁺,Gr1⁺Positive cell Infiltration；Inhibit the expression of inflammatory factor (IL-4, IL-5, IL-6, IL-13, TSLP)；Reduce the degree that skin is lost through the severe edema due to hypofunction of the spleen； Mouse is inhibited to scratch behavior；The wherein psoriasiform skin that tryptophan metabolite IAA, TrA can effectively inhibit imiquimod to induce It is scorching；Experimental result confirms that the tryptophan metabolites such as described IAId have the work of clearly anti-AD sample inflammation and psoriasiform inflammation With can be used for preparing the drug and skin care item for preventing and treating the inflammatory dermatoses such as AD, be further used for preventing and control Treat Acute and chronic inflammation caused by abnormal immune reaction.

In the present invention, the drug and skin care item of the inflammatory dermatoses such as prevention and treatment atopic dermatitis of the IAId preparation can External preparation, including but not limited to liniment or gel is made.

The invention has the following advantages that

The tryptophan metabolites side effect such as IAId of the present invention is minimum, and safety coefficient is very high, can obviously inhibit special The inflammatory reaction of answering property dermatitis, psoriasis；IAId is suitable for being formed with explicitly inhibiting the pharmaceutical preparation of Th2 type inflammatory reaction, Compound medicinal formulation is formed including external preparation is made, or with other medicines, or prevention and day of the skin care item for disease is made Often nursing.

Test result research of the present invention confirms that the tryptophan metabolites such as IAId are inhibiting the effect in Th2 type inflammation, Drug development for tryptophan metabolites such as IAId as treatment AD provides more detailed theoretical foundation and new Research Thinking； The prevention for being AD in clinical experimental stage and further clinical application and treatment provide new medicament selection, have extensive Commercial applications prospect.

Detailed description of the invention

Fig. 1 is that local skin external application IAId of the present invention inhibits AD sample inflammation photo, wherein

A: mouse ear substantially photo；B: mouse ear tissue HE slice dyeing；C: the variation line chart of mouse ear thickness；D: Scratching number of the mouse within half an hour；* 0.001 vs.MC903 group of P < 0.05, * * P < 0.01, * * * P <.

Fig. 2 is that IAId inhibits the expression of inflammatory factor and reduces whole blood IgE level, wherein A-E is followed successively by IL-4, IL- 5, the mRNA relative expression quantity of IL-6, IL-13, TSLP；F: outside mouse

Total IgE is horizontal in all blood；* P < 0.05, * * P < 0.01, * * * P < 0.001vs.MC903 group.

Fig. 3 is that IAId inhibits cell infiltration result figure, wherein

A: the streaming figure of the inflammatory cell infiltrated in mouse ear tissue；B: the number of GR1+ cellular infiltration in mouse ear tissue Amount；C: the quantity of CD8+ cellular infiltration in mouse ear tissue；D: the quantity of CD4+ cellular infiltration in mouse ear tissue；* P < 0.05, * * P < 0.01, * * * P < 0.001vs.MC903 group.

Fig. 4 is IAA, and TrA inhibits AD sample inflammatory consequences,

A: mouse ear photo；B: mouse ear tissue HE dyeing.

Fig. 5 is IAA, and TrA inhibits psoriasiform inflammatory consequences,

Mice Auricle Topical Imiquimod prepares psoriasis model, and auricle skin lesion prepares HE slice.

Fig. 6 is the maintenance situation that IAId, IAA, TrA participate in skin barrier function,

A:RT-PCR detects the expression of filaggrin in mouse tissue, and B: different disposal is to mouse skin through the severe edema due to hypofunction of the spleen Lose the influence of (TEWL), N=5, the * * * vs.MC903 group of P < 0.001.

Fig. 7 is the structure chart of tryptophan metabolite indole -3-formaldehyde (IAId) of the present invention.

Specific embodiment

Embodiment 1

1) it prepares MC903 mouse model and IAId intervenes:

Testing mouse used is ICR female mice, and 4-8 weeks size is raised in SPF rank environment, and mouse feed is SPF Rank；In Mice Auricle external application MC903, once a day, continuous 14 days；While the preparation of MC903 model, outside mouse ear It is continuous to smear 14 days with IAId；

2) record scratching number:

(1) selection is in the state of peace and quiet, with the behavior of cameras record mouse；

(2) 30min video is randomly selected, the total scratching number of every group of mouse is calculated；

3) it the inflammatory cell of flow cytometer detection mouse ear tissue infiltration: prepares mouse ear tissue monocytes suspension: wherein wrapping It includes: being shredded mouse ear tissue using the scissors of disinfection, tweezers；The otic tissues shredded, after 4 DEG C of centrifugations 2000rpm, 5min Remove supernatant；It is poured into 6 orifice plates after the tissue of precipitating is shredded again, the pancreatin of 2ml 0.5% is added in 37 DEG C of insulating boxs Digest 5min；The tissue of digestion is added serum and terminates, and is transferred in streaming pipe after the filtering of 200 mesh filter screens；To streaming Guan Zhongjia Enter the resuspension of 1ml culture medium；10 μ L cell suspensions are drawn from every pipe, are added in cell counting board, carry out cell with microscope It counts；4 DEG C, 1200rpm is centrifuged 5min, adjusts volume according to cell count, and adjusting every solencyte concentration is 10⁶/mL；Streaming Cell analysis: centrifugation 1200rpm, 5min；Supernatant is abandoned, antibody diluent is added, is resuspended, 1200rpm is centrifuged 5 min；The step It need to repeat 2 times, to completely remove culture medium；Supernatant is abandoned, antibody diluent is added to 100 μ L, anti-cell membrane molecule stream is added 5 μ L of formula antibody, rear to be resuspended, 4 DEG C are protected from light incubation, 30min；Streaming antibody cleaning solution is added again, is centrifuged 1200rpm after mixing, 5min, the step need to repeat 2 times, to completely remove streaming antibody；About 5 μ L of streaming antibody is added, 4 DEG C of incubations, keep away after resuspension Light 30min；Streaming antibody cleaning solution is added, 1200rpm, 5min are centrifuged after mixing；The step need to repeat 2 times, to remove completely Remove streaming antibody；Supernatant is abandoned, about 300 μ L fixers are added, is resuspended, 4 DEG C of avoid light places, flow cytomery is carried out；

4) detection of mouse peripheral blood IgE: it is conventional to take eyeball of mouse blood, it is put into EP pipe；Whole blood sample is quiet under room temperature 1h or so is set, is precipitated to serum；Centrifugation: 12000rpm, 6min are sucked out serum and are put into another EP pipe；Coated elisa plate, overnight； Add standard items: being marked with quasi- sample wells on enzyme mark IgE coating plate, sequentially adding the standard items 50ul of various concentration, (each concentration does 2 A parallel hole)；Sample-adding: set respectively blank well (sample and enzyme marking reagent is not added in blank control wells, remaining each step operation is identical), to Test sample sample wells is first loaded 40 μ l of product in sample to be tested hole on enzyme mark coating plate, is then loaded 10 μ l of product Avidin again, and sample-adding will Sample is added on ELISA Plate hole bottom, does not touch hole wall, shakes and mixes；It incubates: incubating 30 points with 37 DEG C of sealing plate film sealing plate postposition Clock；With liquid: will be spare after 30 times of concentrated cleaning solutions, 30 times of distilled water dilutions；Washing: taking sealing plate film off, discard liquid, dries, Cleaning solution is filled it up in every hole, is discarded after standing 30 seconds, is so repeated 5 times, pats dry；Enzyme: the examination of enzyme mark is added in every hole in addition to blank well 50 μ l of agent；It incubates: washing；Colour developing: color developing agent A50 μ l is first added in every hole, adds color developing agent B50 μ l, and concussion mixes, and 37 DEG C are kept away Light develops the color 15 minutes；Terminate: every hole adds 50 μ l of terminate liquid, terminates reaction (blue is vertical at this time turns yellow)；Measurement: with blank air-conditioning Zero, 450nm wavelength sequentially measure the absorbance (OD value) in each hole, carry out in 15 minutes after measuring Ying Jia terminate liquid；

5) expression of real-time quantitative fluorescence PCR detection mouse ear tissue skin lesion inflammatory factor,

Total RNAs extraction: mouse ear tissue is put into 1ml Trizol, is cut with through the pretreated eye scissors of DEPC It is broken, 200 μ l chloroforms are added in EP pipe, in being placed at room temperature for 5min after oscillation, 4 DEG C of low-temperature centrifugations: 12000rpm/min, 15min；EP pipe is taken out after centrifugation, draws upper strata aqueous phase into another EP pipe, and 500 μ l isopropanols, room temperature after concussion mixes is added Place 10min；4 DEG C of 12000 rpm/min of low temperature are centrifuged 10min；Supernatant is abandoned, the DEPC water of 1ml pre-cooling is added in EP pipe Diluted 75% ethyl alcohol, oscillation EP pipe, which suspends, to be precipitated；In 4 DEG C of low-temperature centrifugations: 12000rpm/min, 10min abandon supernatant, room temperature It dries；It takes 10 μ l to dissolve without RNA enzyme water to precipitate；

RNA is quantified and reverse transcription: the RNA sample that will be dissolved in no RNA enzyme water dilutes 100 times, is read with ultraviolet specrophotometer Absorbance value A260 and the A260/A280 ratio at 260nm wavelength is taken, the RNA concentration of sample is then calculated according to absorbance value；

The reaction system of the reverse transcription is as shown in the table:

Reaction condition: 37 DEG C, 15min is reacted, 85 DEG C, reacts 5s, 4 DEG C of preservations；

RT-PCR:

Reaction condition: 95 DEG C, 30s；95℃,5s；60 DEG C, 30s, 72 DEG C, 45s, global cycle number be 30 times, 72 DEG C, 5min；

Statistical analysis, the table of experimental data are carried out to RT-PCR experimental result using 17.0 software of SPSS in the present invention Existing form is mean ± standard errorComparison between two groups carries out difference with Mann-Whitney t method of inspection It analyzes, the comparison between multiple groups carries out variance analysis with One-way ANOVA method, and data have system between P < 0.05 then thinks group Meter learns difference；The results show that the AD sample inflammation that the IAId can inhibit MC903 to induce, inhibits CD4 in corium⁺,Gr1⁺It is positive The infiltration of cell；Inhibit the expression of inflammatory factor (IL-4, IL-5, IL-6, IL-13, TSLP)；Skin is reduced to lose through the severe edema due to hypofunction of the spleen Degree；Mouse is inhibited to scratch behavior；Experimental result confirms that the IAId has the function of clearly anti-AD sample inflammation.

Claims (10)

1. the tryptophan metabolite indole -3-formaldehyde of structure as shown in Figure 7 treats or prevents inflammatory dermatoses in preparation Purposes in drug or skin care item, the inflammatory dermatoses are atopic dermatitis (eczema) or psoriasis or other are inflammatory Skin disease.

2. by purposes described in claim 1, which is characterized in that the tryptophan metabolite is selected from tryptophan metabolite IAId, IAA or/and TrA.

3. by purposes described in claim 1, which is characterized in that the drug is the drug with immunoregulation effect.

4. by purposes described in claim 1, which is characterized in that the treatment atopic dermatitis drug is external curing idiocrasy Dermatitis drug.

5. by purposes described in claim 1, which is characterized in that the indole -3-formaldehyde, which is used to prepare, inhibits MC903 induction The drug of atopic dermatitis sample inflammation.

6. by purposes described in claim 1, which is characterized in that the indole -3-formaldehyde inhibits CD4 in corium⁺,Gr1⁺It is positive The infiltration of cell；Inhibit the expression of inflammatory factor IL-4, IL-5, IL-6, IL-13, TSLP.

7. by purposes described in claim 1, which is characterized in that the indole -3-formaldehyde reduces the journey that skin is lost through the severe edema due to hypofunction of the spleen Degree；Inhibit scratching behavior.

8. by purposes described in claim 4, which is characterized in that external application system is made in the external curing atopic dermatitis drug Agent.

9. by purposes described in claim 4, which is characterized in that liniment or solidifying is made in the external curing atopic dermatitis drug Glue.

10. by purposes described in claim 4, which is characterized in that the external curing atopic dermatitis drug is used to prepare into External use skin care.