CN104398509A

CN104398509A - Application of CFTR (cystic fibrosis transmembrane conductance regulator) inhibitor CFTRinh-172 in preparation of drug for prevention and treatment of leukemia cell mediated diseases

Info

Publication number: CN104398509A
Application number: CN201410638399.6A
Authority: CN
Inventors: 孙华钦; 许文明; 陈小章
Original assignee: West China Second University Hospital of Sichuan University
Current assignee: West China Second University Hospital of Sichuan University
Priority date: 2014-11-13
Filing date: 2014-11-13
Publication date: 2015-03-11
Anticipated expiration: 2034-11-13
Also published as: CN104398509B

Abstract

The invention provides application of CFTR (cystic fibrosis transmembrane conductance regulator) inhibitor CFTRinh-172 in preparation of a drug for prevention and treatment of leukemia cell mediated diseases, the CFTRinh-172 can effectively suppress leukemia cell proliferation through the inhibition of CFTR high expression in karyocyte, and provides a new way for preparation of the drug for prevention and treatment of leukemic cell mediated diseases.

Description

The application of CFTR inhibitor C FTRinh-172 in the medicine of the disease of preparation control leukaemia mediation

Technical field

The invention belongs to medical art, be specifically related to the application of CFTR inhibitor C FTRinh-172 in the medicine of the disease of preparation control leukaemia mediation.

Background technology

The encoding gene of cystic fibrosis transmembrane conductance regulator (CFTR) is the principal element causing cystic fibrosis (CF), within 1989, is found first.The site of CFTR the 7th to autosomal long-armed on (7q31), the chloride channel that a kind of cAMP that coded product is made up of 1480 aminoacid regulates is a member of ABC (ATP-blndingeasoette) protein family.Containing two hydrophobic domains, these two structures all have complicated transferring film part, two ATP binding structural domains and an intracellular domain.Cftr gene is Cystic fibrosis transmembrane conductance regulator, and it can regulate and promote the transhipment of epithelial cell membrane electrolyte ion.

Cystic fibrosis is the autosomal recessive hereditary diseases caused by cftr gene suddenlys change, and major lesions is eccrine dysfunction, and respiratory system, liver and gall, male reproductive system, sweat gland and pancreas can be made to sustain damage.Chrome lung symptom adds that the not much CF that points out of intestinal absorption diagnose.At pancreas, CFTR is distributed on face, the chamber cell membrane of conduit system, is the chloride channel of cAMP regulation and control, makes chloride ion enter alveolar lumen or catheter lumen by pancreatic ductal cells, express in the many epithelial tissue of human body.Up to now, found that CFTR has sudden change and more than the 200 kind of pleomorphism site of more than 1 000 kinds.These sudden changes and polymorphism can cause the different phenotypes of many typical cases and atypical cystic fibrosis.At present, to CFTR relevant disease spectrum understanding and understanding be mainly pulmonary disease and pancreas relevant disease.

In physiological conditions, the propagation of hemocyte, differentiation, apoptosis are in dynamic equilibrium, and leukemia is Hematopoietic Malignancies, and it is characterized in that hematopoetic cell differentiation anacmesis, undifferentiated cell is bred in a large number.In recent years, much research confirms that leukaemia can some cytokines of spontaneous generation, is acted on the propagation of cell itself by autocrine link.

, all there is nonspecific shortcoming in the treatment leukemia generally used in prior art or suppress leukaemia value-added chemotherapeutics, do not have special for target gene albumen, such as daunorubicins etc., often with toxic and side effects.Therefore, develop that to have specific target drug very urgent.

The relevant report that cftr gene and leukaemia to be rised in value is yet there are no in prior art.

Summary of the invention

The invention provides one cFTRthe application of inhibitor C FTRinh-172 in the medicine of the disease of preparation control leukaemia mediation.

Its structural formula is as follows:

CFTRinh-172 structural formula

CFTRinh-172 is the not dependent selectivity of electric potential difference cFTRinhibitor, k _ibe 300 nM, effect is not had for MDR1, ATP sensitive potassium-channel or a series of transport protein.The propagation of CFTRinh-172 by suppressing the high expressed of CFTR in nucleus effectively to suppress leukaemia, for the disease for the treatment of leukaemia mediation provides new way.

CFTR is to the regulating and controlling effect of hemopoietic in using forestland of the present invention biological Brachydanio rerio research.Find that the expression of the marker gene of the suppressed rear hemopoietic forebody cell of expression of CFTR in the embryo of early development obviously weakens; Signal path research shows that CFTR is positioned at Wnt3(wingless-type MMTV integration site family, member 3A, the aptery signal part factor 3,) downstream and positive correlation regulation and control Wnt(Wingless, aptery) signal key gene Dvl(Dishevelled, albumen at random) expression; Further molecular studies show that CFTR protects Dvl albumen from lysosomal degraded in conjunction with Dvl albumen, thus maintain the transduction of wnt signal.

Because the unconventionality expression of wnt signal and leukemic morbidity are closely related.So the present invention finds according to the molecule mechanism in zebra fish model, have detected CFTR and act in leukemia.The unconventionality expression of result display CFTR is closely related in leukemic morbidity, and in leukaemia, CFTR exists the expression of higher level.Use RNA interfering technology or the process of CFTR inhibitor to leukaemia, cell all occurs that propagation significantly weakens and adjoint apoptotic phenomenon.

Being reported in mice according to SONAWANE et al (journal of pharmaceutical sciences, vol.94, No.1) uses the CFTRinh-172 of high dose not find toxic reaction; Thiagarajah(Clinical pharmacology & Therapeutics, vol.92, No.3) report that CFTRinh-172 creates very low toxicity because of the metabolic secretion of kidney minimum level.Comprehensively have been reported now, consider that CFTRinh-172 is that this medicine causes the toxic reaction of animal hardly for the special micromolecular inhibitor of cftr gene, there is very high targeting.

The reported first of the present invention molecular regulation mechanism of CFTR to hemopoietic, and provide new approach for leukemic treatment.CFTRinh-172 is the special inhibitor for cftr gene albumen in body that screening obtains, and does not have target spot in vivo on other gene protein, and in long-term use procedure, demonstrated its effectiveness suppressed cftr gene protein function.The special medicine for cftr gene protein function, and the molecule mechanism of effect is clear.

Accompanying drawing explanation

Fig. 1 is the immunoblot results figure of the CFTR Protein Detection of embodiment 1.

Fig. 2 is the immunofluorescence results figure of the CFTR Protein Detection of embodiment 1.

Fig. 3 is the result figure of leukaemia's experiment of embodiment 2.

Fig. 4 is the result data figure of the LC of embodiment 2.

Detailed description of the invention

Below in conjunction with detailed description of the invention, essentiality content of the present invention is described in further detail.

Embodiment 1

CFTR Protein Detection blood cell proliferation

Concrete steps are as follows:

1. the peripheral blood 1ml extracting leukemia people (sample 1) and non-leukemia people (sample 2) respectively preserves in the purple head or green head tube of anticoagulant, carries out the separation of mononuclearcell the same day;

2. use people's mononuclearcell separating medium of Tianjin Hao ocean biological product company limited to be separated the mononuclearcell of peripheral blood, concrete operations are according to product description;

3. a part of cell adds non-denatured protein lysate cracking mononuclearcell, the 15ug total protein extracting cell uses western blotting method to detect CFTR protein expression level (beta-tubulin albumen is as reference), another part is smear on microscope slide, uses Immunofluorescence test CFTR protein expression level;

4. contrast cftr gene protein expression level, immunoblot assay result (Fig. 1) shows: the CFTR albumen of sample 2 does not occur Band signal, shows that hemocyte is in lower propagation level; There is obvious signal in sample 1, shows that hemocyte has abnormality proliferation.

5. immunofluorescence results (Fig. 2) display: CFTR protein expression appears in the part-blood cell of sample 1, shows that hemocyte is in low-level propagation level; All there is CFTR high expressed in whole hemocytees of sample 2, shows that hemocyte is in hyperproliferation state.

Immunoblotting and Immunofluorescence test result all show: in the sample 1 of leukemia people, high expressed has appearred in CFTR albumen, demonstrate CFTR albumen and hemocyte abnormality proliferation is closely related.

Embodiment 2

Cell culture

The 1640 culture medium 37 degree Celsius cultivation of leukaemia CCRF-CEM containing 10% calf serum.

Cell experiment

Toward being 50 μm containing adding CFTRinh-172 in the culture medium of cell to final concentration, matched group adds isopyknic dimethyl sulfoxide (DMSO); Process and after 72 hours, cell is counted, found that CFTRinh-172 processed group cell quantity significantly reduces (Fig. 3 and Fig. 4), show that CFTRinh-172 exists significant inhibitory action to leukaemia.

Claims

The application of 1.CFTR inhibitor C FTRinh-172 in the medicine of the disease of preparation control leukaemia mediation.
2. the application of CFTR inhibitor C FTRinh-172 according to claim 1 in the medicine of the disease of preparation control leukaemia mediation, is characterized in that: described disease is leukemia.