CN110025621A - Tizoxanide and Nitazoxanide application in preparing anti-inflammatory drugs - Google Patents
Tizoxanide and Nitazoxanide application in preparing anti-inflammatory drugs Download PDFInfo
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- CN110025621A CN110025621A CN201910245961.1A CN201910245961A CN110025621A CN 110025621 A CN110025621 A CN 110025621A CN 201910245961 A CN201910245961 A CN 201910245961A CN 110025621 A CN110025621 A CN 110025621A
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/60—Salicylic acid; Derivatives thereof
- A61K31/625—Salicylic acid; Derivatives thereof having heterocyclic substituents, e.g. 4-salicycloylmorpholine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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Abstract
The invention discloses tizoxanide and/or the new medical usages of Nitazoxanide and its pharmaceutically acceptable salt, i.e., it is used for the purposes in anti-inflammatory drug in preparation, and for treating and inhibiting COX-2, NF- κ B, the purposes of the drug of the relevant disease of p65, I κ B α, ERK1/2 or p38MAPK.Described on the way, tizoxanide and/or Nitazoxanide and its pharmaceutically acceptable salt can be by inhibiting COX-2, and NF- κ B, p65, I κ B α, ERK1/2 or p38MAPK play related drug effect.
Description
Technical field
The present invention relates to a kind of anti-inflammatory drugs, and in particular to a kind of for activity and to be contained with tizoxanide and/or Nitazoxanide
There is the pharmaceutical composition of tizoxanide and/or Nitazoxanide, while designing the use in terms of above-mentioned substance is used to prepare anti-inflammatory drug
On the way.
Background technique
A kind of autoimmune response that inflammation, which is body, stimulates external perhaps allosome when this response is lacked of proper care or
Excessive response leads to when damaging certainly of body, to evolve into inflammation.Inflammation in people or is moved as a kind of important pathologic process
Very common in object body, most of disease is all along with the mediation of inflammation and generation, and inflammation makes disease pair
It is aggravated in the damage of body, such as rheumatic arthritis, diabetic complications, cancer, atherosclerosis, inflammatory bowel disease.?
During these, proinflammatory factor such as TNF-α (tumor necrosis factor-alpha), IL-6 (interleukin-6), IL-1 β (Interleukin -1β) with
And COX-2 (epoxidase) etc. plays an important role.
Lipopolysaccharides (LPS) is endotoxic main component, from the outer membrane of gram-negative bacteria cell wall.When body sense
After contaminating bacterium, a large amount of LPS can be discharged after bacterial death dissolves or destroys bacterium cell by artificial means and are come out, a large amount of LPS's
Stimulation can cause body that leukocytoreaction, exothermic reaction occurs, or even generate shock.Therefore, the inflammation that LPS induces is bacterium
One of main pathological symptoms of infection.In addition, growing on trees in the air, occupational dusts (grain dust etc.), cigarette of pollution
LPS can cause or aggravate a series of clinical diseases, such as asthma, Bronchopneumonia after sucking certain density LPS.Therefore,
LPS is the primary inducers for inducing inflammation to occur in inflammation research.But if LPS can be inhibited to stimulate induced COX-2 (ring
Oxygenase) activity raising, the cell factors such as TNF-α (tumor necrosis factor-alpha), IL-6 (interleukin-6), IL-1 β (Interleukin -1β)
Secretion is lowered, and can preferably be controlled the deterioration of inflammation, be played anti-inflammatory effect.
Tizoxanide (Tizoxanide, TIZ) and Nitazoxanide (nitazoxanide, NTZ) are all nitre thiophene salicylic acid amides
Derivative, wherein tizoxanide is the major active metabolite product of Nitazoxanide in vivo.Since the 1970's, Ren Menfa
Existing tizoxanide and Nitazoxanide have inhibiting effect to the anaerobic bacteria of infection humans and animals and a variety of worms, protozoon etc..Nitre azoles
Nit was approved by the fda in the United States for treatment children Giardia lamblia, hidden spore as antiparasitic agent in 2002
Disease caused by the Intestinal protozoans such as worm infect.The medicine Africa and South America it is multiple country use the result shows that, anti parasitic sense
It is good to contaminate effect, and has no adverse reaction.2011, the Nitazoxanide bulk pharmaceutical chemicals that China Agriculture Academe Shanghai Veterinary Institute develops
Object and dry suspensoid agent obtain national two class novel chiral synthon certificates.
Since the acetyl group in Nitazoxanide chemical structure is easy to hydrolyze, Nitazoxanide in animal or human body all
It is promptly metabolized into tizoxanide (Tizoxanide, TIZ), substantially can't detect Nitazoxanide in body.For azoles Buddhist nun
Effect of the spy in various inside and outside pharmacology and toxicologic study is consistent with Nitazoxanide, and therefore, tizoxanide is Nitazoxanide
Activity in vivo substance, Nitazoxanide is the prodrug forms of tizoxanide, in its activity research often with tizoxanide substitute nitre
Azoles nit.
It is generally acknowledged that the antiviral mechanism of Nitazoxanide is all its key enzyme for directly acting on pathogen itself, inhibit
The proliferation of pathogen, to reach its therapeutic effect, such as: the mechanism of Nitazoxanide anti parasitic and anaerobe is thought in research
Be to interfere its pyruvic acid: the electron transfer reaction that ferredoxin reductase (PFOR) is relied on, this is helminth and anaerobic bacteria
The important step of energetic supersession.Although the mechanism that Nitazoxanide resists certain virus infections not yet illustrates completely, existing reality
It tests result and is regarded as the proliferation that Nitazoxanide directly inhibits virus, such as: inhibiting the synthesis of influenza virus hemagglutinin and resist
The proliferation of influenza virus.And Nitazoxanide and tizoxanide can inhibit body cell inflammation related proteins COX-2's (epoxidase)
Activity, and inhibit
Summary of the invention
It is an object of the present invention to provide the new of tizoxanide and/or Nitazoxanide and its pharmaceutically acceptable salt
Medical usage, i.e., it is in preparation for the purposes in anti-inflammatory drug.
It is used on the way described, tizoxanide and/or Nitazoxanide and its pharmaceutically acceptable salt can inhibit COX-2,
NF- κ B, p65, I κ B α, ERK1/2 or p38MAPK.
The pharmaceutically acceptable salt, including inorganic acid salt, acylate, such as malate etc..
It is a further object to provide tizoxanide and/or Nitazoxanide and its pharmaceutically acceptable salt conducts
Inhibit COX-2, the small molecule compound of NF- κ B, p65, I κ B α, ERK1/2 or p38MAPK treat associated disease in preparation
Drug in purposes.
Described and inhibition COX-2, the disease of NF- κ B, p65, I κ B α, ERK1/2 or p38MAPK include but is not limited to that rouge is more
The diseases associated with inflammation that bacterial endotoxins such as sugared (LPS) induce.
It is a further object of the present invention to provide comprising tizoxanide and/or Nitazoxanide or its pharmaceutically acceptable salt
For pharmaceutical composition in preparation for the purposes in anti-inflammatory drug, described pharmaceutical composition includes tizoxanide and/or Nitazoxanide
Or its pharmaceutically acceptable salt and pharmaceutically acceptable various auxiliary materials.
The invention also discloses pharmaceutical composition purposes in preparing anti-inflammatory drugs.
Present invention finds tizoxanide and/or the new mechanism of action and drug effect of Nitazoxanide, have and inhibit COX-2,
The effect of NF- κ B, p65, I κ B α, ERK1/2 or p38MAPK.The compound can be used for treating correlative diseases.
Present invention finds, tizoxanide and/or Nitazoxanide are used for the mechanism of action in anti-inflammatory drug, present invention discover that
Tizoxanide and Nitazoxanide can be used as COX-2, and the inhibitor of NF- κ B, p65, I κ B α, ERK1/2 or p38MAPK protect cell
Because of the cellular inflammation effect caused by inflammation-induced object (such as lipopolysaccharides) stimulation, and inhibit various inflammatory factors such as on cell
The secretion of IL-1 β, IL-6, TNF-α etc., reduces the activity of cell COX-2 enzyme, to reach the anti-inflammatory work for inhibiting inflammatory development
With.
The invention further relates to the pharmaceutical compositions containing tizoxanide and Nitazoxanide and its pharmaceutically acceptable carrier.It should
Pharmaceutical composition can be applied through number of ways, such as oral tablet, capsule, pulvis, oral solution, injection and preparation capable of permeating skin.
According to the convention on conventional drug, pharmaceutically acceptable carrier includes diluent, filler, disintegrating agent, wetting agent, lubrication
Agent, colorant, flavoring agent etc..
Another aspect of the present invention is related to pharmaceutical composition, contains tizoxanide of the present invention and Nitazoxanide at least one
Kind pharmaceutically acceptable carrier.Described pharmaceutical composition can be prepared into various forms according to different way of administration.
Detailed description of the invention
Inhibit to Fig. 1 Nitazoxanide (NTZ) conspicuousness LPS induction RAW264.7 cell secretion (A) TNF-α, (B) IL-1 β,
(C)IL-6.Nitazoxanide (NTZ) concentration is respectively 50,100,150 μm of ol/L in figure, and action time is for 24 hours.Data are with averagely
Value ± variance indicates that # indicates LPS stimulation and blank control group significant difference (P < 0.01)；* respectively indicates Nitazoxanide
(NTZ) processing group and LPS stimulation group significant difference (P < 0.01).
Inhibit to Fig. 2 tizoxanide (TIZ) conspicuousness LPS induction RAW264.7 cell secretion (A) TNF-α, (B) IL-1 β,
(C)IL-6.Tizoxanide (TIZ) concentration is respectively 50,100,150 μm of ol/L in figure, and action time is for 24 hours.Data are with averagely
Value ± variance indicates that # indicates LPS stimulation and blank control group significant difference (P < 0.01)；* respectively indicates tizoxanide
(TIZ) processing group and LPS stimulation group significant difference (P < 0.01).
LPS is inhibited to Fig. 3 tizoxanide (TIZ) conspicuousness to induce RAW264.7 cell (A) TNF-α, (B) IL-1 β, (C)
The mRNA of IL-6 is transcribed.Tizoxanide (TIZ) concentration is respectively 50,100,150 μm of ol/L in figure, and action time is for 24 hours.Data
It is indicated with average value ± variance, # indicates LPS stimulation and blank control group significant difference (P < 0.01)；* respectively indicates for azoles
Nit (TIZ) processing group and LPS stimulation group significant difference (P < 0.01).
Inhibit to Fig. 4 tizoxanide (TIZ) conspicuousness the expressing quantity of LPS induction RAW264.7 cell COX-2 enzyme.Figure
Middle tizoxanide (TIZ) concentration is respectively 50,100,150 μm of ol/L, action time 6h.Data average value ± variance table
Show, # indicates LPS stimulation and blank control group significant difference (P < 0.01)；* respectively indicates tizoxanide (TIZ) processing group
With LPS stimulation group significant difference (P < 0.01).
LPS is inhibited to Fig. 5 tizoxanide (TIZ) conspicuousness to induce RAW264.7 cell NF- κ B p65, p-I κ B and p-IKK
The expressing quantity of α, but the expression quantity of I κ B can be increased.Tizoxanide (TIZ) concentration is respectively 50,100,150 μm of ol/ in figure
L, action time 6h.(A) western blot detects the protein hybridization figure of various NF- κ B signal accesses；(B) p65 in endochylema
Relative expression quantity；(C) in nucleus p65 p65 relative expression quantity；(D) phase of p-I κ B, p-IKK α and I kB protein expression quantity
Reduced value.Data indicate that # indicates LPS stimulation and blank control group significant difference (P < with average value ± variance
0.01)；*, * * respectively indicates tizoxanide (TIZ) processing group and LPS stimulation group significant difference (P < 0.05 or P < 0.01).
Inhibit to Fig. 6 tizoxanide (TIZ) conspicuousness the egg of LPS induction RAW264.7 cell MKK4, JNK, ERK and p38
White expression quantity.Tizoxanide (TIZ) concentration is respectively 50,100,150 μm of ol/L, action time 6h in figure.(A)
Westernblot detects the protein hybridization figure of various MAPKs signal paths；(B) ratio of p-JNK/JNK expressing quantity；(C)
The protein versus expression quantity of protein versus expression quantity (D) p-p38 of p38；(E) ratio of p-ERK/ERK expressing quantity；(F)
The ratio of p-MKK4/MKK4 expressing quantity.Data indicate that # indicates that LPS stimulation and blank control group are aobvious with average value ± variance
It writes sex differernce (P < 0.01)；*, * * respectively indicates tizoxanide (TIZ) processing group and LPS stimulation group significant difference (P <
0.05 or P < 0.01).
Specific embodiment
The present invention is further illustrated with embodiment below, but the present invention is not intended to be limited thereto.
Raw material used in embodiment or reagent are commercially available in addition to special instruction.
The release of embodiment 1, tizoxanide and Nitazoxanide anti-cell inflammatory factor
The RAW264.7 mouse macrophage that Nitazoxanide or tizoxanide are acted on to lipopolysaccharides (LSP) induction, passes through
IL-1 β, IL-6 and the secretion level of TNF-α detect its anti-inflammatory activity in detection RAW264.7 cell.
The preparation of drug solution:
(1) tizoxanide and Nitazoxanide are configured to 200mmol/L solution with DMSO respectively, are placed in -4 DEG C of preservations；
(2) lipopolysaccharides (lipopolysaccharide from Escherichia Coli E 0127:B8-purified
By phenol extraction, LPS), it is purchased from Sigma-Aldrich company, is configured to 1,0mg/mL solution with sterile water ,-
20 DEG C of preservations.
Cell strain:
RAW264.7 mouse macrophage, cell origin are purchased in ATCC by Shanghai Inst. of Life Science, CAS
Cell resource center.
MTT detects cytotoxicity:
RAW264.7 mouse macrophage is cleaned after abandoning old culture medium with 1 × PBS, then 0.25% trypsin digestion (2
~4min), it collects in cell and centrifuge tube, 1500rpm is centrifuged 5min.Supernatant is abandoned, culture medium is added, cell is resuspended, by cell
It is inoculated on 96 orifice plates, every hole cell number 5000~20000.After being incubated overnight, processing be grouped simultaneously stimulate, be administered: Con (in
The isometric DMSO of medicine group), the Nitazoxanide or tizoxanide of LPS (final concentration of 1 μ g/mL) and various concentration.20 as a child
Afterwards, MTT, 37 DEG C, 5%CO are added2It is incubated for 4h in incubator, discards culture medium, and 150 μ L DMSO are added into every hole, shakes
10~15min, and absorbance value (λ=490) are measured with microplate reader.Nitazoxanide of the cell survival rate greater than 80% replaces azoles Buddhist nun
Special concentration is used for anti-inflammatory experiment.
The foundation of cellular inflammation model:
Routinely recovery cell and secondary culture use the complete medium of DMEM+10%PBS, inoculating cell to 24
Porocyte culture plates, every hole cell suspension 1.0mL, cell number are 1.5 × 105It is a, it is placed in 5%CO2 incubator and is cultivated in 37 DEG C
Overnight.LPS (final concentration of 1 μ g/mL), which is added, stimulates cell, establishes cellular inflammation model.
Nitazoxanide or tizoxanide inhibit the release of the cellular inflammation factor:
Various concentration is added after LPS (final concentration of 1 μ g/mL) stimulation cell is added on the basis of cellular inflammation model
Nitazoxanide or tizoxanide (50,100,150 μm of ol/L).Take cell culture medium supernatant after 24 hours respectively, centrifugation removes
Degranulation object with ELISA detection IL-1 β, IL-6, TNF-α amount, it is for statistical analysis to detection data, to determine at drug
Whether inhibited manage the inflammatory reaction induced for LPS.
As a result:
As a result as depicted in figs. 1 and 2, from the graph, it is apparent that tizoxanide and its proto-drug nitre azoles Buddhist nun
The release for the cellular inflammation factor that spy can inhibit to conspicuousness LPS to induce, can be used for preparing new anti-inflammatory activity drug.
The transcription release of embodiment 2, tizoxanide anti-cell inflammatory factor
Tizoxanide is the active metabolite of the major metabolite and Nitazoxanide of Nitazoxanide in vivo and itself tool
There is the bioactivity as Nitazoxanide, therefore we have investigated tizoxanide and have acted on lipopolysaccharides (LSP) induction
RAW264.7 mouse macrophage transcribes water by the mRNA of IL-1 β, IL-6 and TNF-α mRNA in detection RAW264.7 cell
It puts down to detect its anti-inflammatory activity.
The preparation of drug solution:
(1) tizoxanide is configured to 200mmol/L solution with DMSO, is placed in -4 DEG C of preservations；
(2) lipopolysaccharides (lipopolysaccharide from Escherichia Coli E 0127:B8-purified
By phenol extraction, LPS), it is purchased from Sigma-Aldrich company, is configured to 1,0mg/mL solution with sterile water ,-
20 DEG C of preservations.
Cell strain:
RAW264.7 mouse macrophage, cell origin are purchased in ATCC by Shanghai Inst. of Life Science, CAS
Cell resource center.
The foundation of cellular inflammation model:
Routinely recovery cell and secondary culture use the complete medium of DMEM+10%PBS, inoculating cell to 6 holes
Tissue culture plate, every hole cell suspension 1.0mL, cell number are 1.5 × 105It is a, it is placed in 5%CO2 incubator and was cultivated in 37 DEG C
Night.LPS (final concentration of 1 μ g/mL), which is added, stimulates cell, establishes cellular inflammation model.
The mRNA transcriptional level of the tizoxanide inhibition cellular inflammation factor
Various concentration is added after LPS (final concentration of 1 μ g/mL) stimulation cell is added on the basis of cellular inflammation model
Tizoxanide (50,100,150 μm of ol/L).Respectively after 24 hours with QIAGEN companyMini Kit reagent
Box carries out the extraction of mRNA, with TaKaRa company kit PrimeScriptTMRT-reagent Kit(Perfect Real
Time the preparation for) carrying out CDNA, with TaKaRa company qRT-PCR kitPremix Ex TaqTMIt is fixed to carry out fluorescence
Measure PCR reaction.Carried out in 7500 type Real-time PCR instrument of Applied Biosystems company, the U.S. ABI detection IL-1 β,
The mRNA transcription amount inspection of IL-6, TNF-a.It is for statistical analysis to detection data, to determine what drug-treated induced LPS
Whether inflammatory reaction is inhibited (see Fig. 3).Confirm the cellular inflammation that tizoxanide can inhibit to conspicuousness LPS to induce
The mRNA transcription amount of the factor is to generate to inhibit these inflammatory factors, can be used for preparing new anti-inflammatory activity drug.
Embodiment 3, tizoxanide inhibit the expression of COX-2 zymoprotein
Tizoxanide is the active metabolite of the major metabolite and Nitazoxanide of Nitazoxanide in vivo and itself tool
There is the bioactivity as Nitazoxanide.It is enzyme necessary to prostaglandin (PGs) is synthesized, COX-2 enzyme can be catalyzed PGs synthesis
Participate in inflammation.Reaction therefore we investigated tizoxanide act on lipopolysaccharides (LSP) induction RAW264.7 mouse macrophage it is thin
Born of the same parents, the expression quantity by detecting protein hybridization (western blot) COX-2 zymoprotein detect its anti-inflammatory activity.
The preparation of drug solution:
(1) tizoxanide is configured to 200mmol/L solution with DMSO, is placed in -4 DEG C of preservations；
(2) lipopolysaccharides (lipopolysaccharide from Escherichia Coli E 0127:B8-purified
By phenol extraction, LPS), it is purchased from Sigma-Aldrich company, is configured to 1,0mg/mL solution with sterile water ,-
20 DEG C of preservations.
Cell strain:
RAW264.7 mouse macrophage, cell origin are purchased in ATCC by Shanghai Inst. of Life Science, CAS
Cell resource center.
The foundation of cellular inflammation model:
Routinely recovery cell and secondary culture use the complete medium of DMEM+10%PBS, inoculating cell to 6 holes
Tissue culture plate, every hole cell suspension 1.0mL, cell number are 1.5 × 105It is a, it is placed in 5%CO2 incubator and was cultivated in 37 DEG C
Night.LPS (final concentration of 1 μ g/mL), which is added, stimulates cell, establishes cellular inflammation model.
The detection of tizoxanide inhibition cell COX-2 enzyme protein expression amount
Various concentration is added after LPS (final concentration of 1 μ g/mL) stimulation cell is added on the basis of cellular inflammation model
Tizoxanide (50,100,150 μm of ol/L).It abandons old culture medium after 6 hours respectively, raffinate is blotted after 1 × PBS cleaning, by 6
Orifice plate is placed on ice, and 100 μ L protein lysates are added in every hole, and suitably being blown and beaten with liquid-transfering gun connects lysate sufficiently with cell
Touching.Cell is collected in EP pipe, 12000rpm is centrifuged 5min at 4 DEG C.Supernatant is drawn, BCA protein determination kit measures albumen
Concentration, remaining sample be stored in -80 DEG C it is spare.
Total protein sample is taken before experiment, and 5 × loading buffer, vortex of a quarter protein sample volume is added
Several seconds is uniformly mixed it, and boiling water bath 10min is spare.
Plastic plate appropriate is selected, sufficiently cleans and rear-inclined of hunting leak drains water in plate.Matched according to destination protein size
The SDS-PAGE separation gel of respective concentration is set, and sol solution is injected in plastic plate, distilled water flattens.Glue to be separated solidifies completely
Afterwards, inclination drains the water in plate, and configuration 5% is concentrated in glue injection plate, and is inserted into comb appropriate.It is calculated according to protein sample concentration
Volume needed for 30 μ g total protein of loading out, loading after glue to be concentrated solidifies completely.Deposition condition: concentration glue constant pressure 80V, marker
Voltage, constant pressure 120V are changed into separation gel and after separating.Transferring film condition, constant current 0.5A (it is unstable to have Current Voltage once in a while
The case where, the transferring film time is according to circumstances appropriately extended), the time determines according to destination protein size.Ponceaux pre-dyed determines transferring film
As a result.After 1 × PBS washes away Ponceaux dyeing, 5% skim milk closes 1h.1XPBS washes film 5min × 3, and primary antibody is incubated for, 4 DEG C of mistakes
Night.1 × PBST washes film 5min × 3, is protected from light is incubated for secondary antibody 1h under room temperature.1 × PBST washes film 5min × 3, infrared laser at
As system sweeps film.
As a result as shown in figure 4, the expression for the cell COX-2 protease that tizoxanide can inhibit to conspicuousness LPS to induce
Amount plays the effect for inhibiting cellular inflammation, can be used for preparing new anti-inflammatory activity medicine to inhibit the catalysis of COX-2
Object.
Embodiment 4, tizoxanide are the inhibitor of NF- κ two inflammatory signals accesses of B and MAPKs
Tizoxanide is the active metabolite of the major metabolite and Nitazoxanide of Nitazoxanide in vivo and itself tool
There is the bioactivity as Nitazoxanide.NF- κ B and MAPKs are two primary signal pathways for inducing inflammation.Therefore we
The RAW264.7 mouse macrophage that tizoxanide acts on lipopolysaccharides (LSP) induction has been investigated, NF- κ B and MAPKs two is detected
The expression quantity of key protein detects the anti-inflammatory activity of tizoxanide on inflammatory signals access.
The preparation of drug solution:
(1) tizoxanide is configured to 200mmol/L solution with DMSO, is placed in -4 DEG C of preservations；
(2) lipopolysaccharides (lipopolysaccharide from Escherichia Coli E 0127:B8-purified
By phenol extraction, LPS), it is purchased from Sigma-Aldrich company, is configured to 1,0mg/mL solution with sterile water ,-
20 DEG C of preservations.
Cell strain:
RAW264.7 mouse macrophage, cell origin are purchased in ATCC by Shanghai Inst. of Life Science, CAS
Cell resource center.
The foundation of cellular inflammation model:
Routinely recovery cell and secondary culture use the complete medium of DMEM+10%PBS, inoculating cell to 6 holes
Tissue culture plate, every hole cell suspension 1.0mL, cell number are 1.5 × 105It is a, it is placed in 5%CO2 incubator and was cultivated in 37 DEG C
Night.LPS (final concentration of 1 μ g/mL), which is added, stimulates cell, establishes cellular inflammation model.
The detection of tizoxanide inhibition cell NF- κ B and MAPKs signal path key expressing quantity
Various concentration is added after LPS (final concentration of 1 μ g/mL) stimulation cell is added on the basis of cellular inflammation model
Tizoxanide (50,100,150 μm of ol/L).It abandons old culture medium after 6 hours respectively, raffinate is blotted after 1 × PBS cleaning, by 6
Orifice plate is placed on ice, and 100 μ L protein lysates are added in every hole, and suitably being blown and beaten with liquid-transfering gun connects lysate sufficiently with cell
Touching.Cell is collected in EP pipe, 12000rpm is centrifuged 5min at 4 DEG C.Supernatant is drawn, BCA protein determination kit measures albumen
Concentration, remaining sample be stored in -80 DEG C it is spare.
Total protein sample is taken before experiment, and 5 × loading buffer, vortex of a quarter protein sample volume is added
Several seconds is uniformly mixed it, and boiling water bath 10min is spare.
Plastic plate appropriate is selected, sufficiently cleans and rear-inclined of hunting leak drains water in plate.Matched according to destination protein size
The SDS-PAGE separation gel of respective concentration is set, and sol solution is injected in plastic plate, distilled water flattens.Glue to be separated solidifies completely
Afterwards, inclination drains the water in plate, and configuration 5% is concentrated in glue injection plate, and is inserted into comb appropriate.It is calculated according to protein sample concentration
Volume needed for 30 μ g total protein of loading out, loading after glue to be concentrated solidifies completely.Deposition condition: concentration glue constant pressure 80V, marker
Voltage, constant pressure 120V are changed into separation gel and after separating.Transferring film condition, constant current 0.5A (it is unstable to have Current Voltage once in a while
The case where, the transferring film time is according to circumstances appropriately extended), the time determines according to destination protein size.Ponceaux pre-dyed determines transferring film
As a result.After 1 × PBS washes away Ponceaux dyeing, 5% skim milk closes 1h.1XPBS washes film 5min × 3, and primary antibody is incubated for, 4 DEG C of mistakes
Night.1 × PBST washes film 5min × 3, is protected from light is incubated for secondary antibody 1h under room temperature.1 × PBST washes film 5min × 3, infrared laser at
As system sweeps film.
As a result as shown in Figure 5 and Figure 6, cell NF- κ B and MAPKs that tizoxanide can inhibit to conspicuousness LPS to induce
The expression quantity of key protein on two inflammatory signals accesses, to inhibit these transcription factors to the transcriptional activation function of inflammatory factor
Can, play the effect for inhibiting cellular inflammation.Therefore, the inhibitor of two inflammatory signals accesses of tizoxanide NF- κ B and MAPKs,
It can be used for preparing new anti-inflammatory activity drug.
Claims (7)
1. tizoxanide and/or Nitazoxanide and its pharmaceutically acceptable salt are in preparation for the purposes in anti-inflammatory drug.
2. purposes according to claim 1, tizoxanide and/or Nitazoxanide and its pharmaceutically acceptable salt can press down
COX-2 processed, NF- κ B, p65, I κ B α, ERK1/2 or p38MAPK.
3. purposes according to claim 2, the pharmaceutically acceptable salt, including inorganic acid salt, acylate, such as
Malate etc..
4. a kind of pharmaceutical composition purposes in preparing anti-inflammatory drugs, described pharmaceutical composition includes tizoxanide and/or nitre
Azoles nit or its pharmaceutically acceptable salt and pharmaceutically acceptable auxiliary material.
5. a kind of pharmaceutical composition inhibits COX-2 in preparation, the use in NF- κ B, p65, I κ B α, ERK1/2 or p38MAPK drug
On the way, described pharmaceutical composition includes tizoxanide and/or Nitazoxanide or its pharmaceutically acceptable salt and pharmaceutically acceptable
Auxiliary material.
6. purposes according to claim 4 or 5, described pharmaceutical composition can be applied through number of ways, such as oral tablet
Agent, capsule, pulvis, oral solution, injection and preparation capable of permeating skin.
7. purposes according to claim 4 or 5, described pharmaceutical composition is solid pharmaceutical preparation, injection, external preparation, spray
Agent, liquid preparation or compound preparation.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109999045A (en) * | 2019-03-13 | 2019-07-12 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | The application of tizoxanide and Nitazoxanide in preparation novel autophagy derivant |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105793255A (en) * | 2013-10-04 | 2016-07-20 | 无限药品股份有限公司 | Heterocyclic compounds and uses thereof |
| CN107260695A (en) * | 2009-02-13 | 2017-10-20 | 罗马克实验室有限公司 | The controlled release drug formulation of Nitazoxanide |
| WO2018138352A1 (en) * | 2017-01-27 | 2018-08-02 | Genfit | Pharmaceutical compositions for combination therapy |
| WO2018215795A2 (en) * | 2017-05-26 | 2018-11-29 | United Kingdom Research And Innovation | Senolytic compounds |
| CN109999045A (en) * | 2019-03-13 | 2019-07-12 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | The application of tizoxanide and Nitazoxanide in preparation novel autophagy derivant |
-
2019
- 2019-03-13 CN CN201910245961.1A patent/CN110025621A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107260695A (en) * | 2009-02-13 | 2017-10-20 | 罗马克实验室有限公司 | The controlled release drug formulation of Nitazoxanide |
| CN105793255A (en) * | 2013-10-04 | 2016-07-20 | 无限药品股份有限公司 | Heterocyclic compounds and uses thereof |
| WO2018138352A1 (en) * | 2017-01-27 | 2018-08-02 | Genfit | Pharmaceutical compositions for combination therapy |
| WO2018215795A2 (en) * | 2017-05-26 | 2018-11-29 | United Kingdom Research And Innovation | Senolytic compounds |
| CN109999045A (en) * | 2019-03-13 | 2019-07-12 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | The application of tizoxanide and Nitazoxanide in preparation novel autophagy derivant |
Non-Patent Citations (7)
| Title |
|---|
| JIAOQIN SHOU ET AL: "Tizoxanide Inhibits Inflammation in LPS-Activated RAW264.7 Macrophages via the Suppression of NF-κB andMAPK Activation", 《INFLAMMATION》 * |
| KAREN K. Y. LAM ET AL: "Nitazoxanide Stimulates Autophagy and Inhibits mTORC1 Signaling and Intracellular Proliferation of Mycobacterium tuberculosis", 《PLOS PATHOGENS》 * |
| SEONG KEUN HONG ET AL.: "Nitazoxanide suppresses IL-6 production in LPS-stimulated mouse macrophages and TG-injected mice", 《INTERNATIONAL IMMUNOPHARMACOLOGY》 * |
| 余元勋等: "《中国疾病信号通路与靶向治疗学》", 31 May 2013, 安徽科学技术出版社 * |
| 巫善明主编: "《新发传染病与再发传染病》", 31 July 2010, 上海科技教育出版社 * |
| 陈临溪等: "《细胞信号转导药理与临床》", 31 October 2014, 人民军医出版社 * |
| 首姣琴等: "替唑尼特对脂多糖诱导 RAW264.7 细胞氧化应激及炎症因子的影响", 《中国动物传染病学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109999045A (en) * | 2019-03-13 | 2019-07-12 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | The application of tizoxanide and Nitazoxanide in preparation novel autophagy derivant |
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