CN103948598B - The combination medicine for the treatment of chronic myelocytic leukemia - Google Patents

Abstract

The invention discloses a kind of combination medicine treating chronic myelocytic leukemia, described medicine includes imatinib and atabrine, the mass concentration ratio of described imatinib and atabrine is 1:8 1:12, and provides atabrine application in preparation treatment chronic myelocytic leukemia medicine.Present invention discover that atabrine can reduce CML cell K562, mdr cell K562 R and 32D Bcr/abl^T315IBcr/abl in cell and phosphorylated protein level thereof, reduce the Bcr/abl albumen in patient CML leukaemia, combination with imatinib can be worked in coordination with and be caused the lethal effect to CML cell with relatively low-dose, and effective equally to the mdr cell of chronic myelocytic leukemia, and to PBMC of healthy people without substantially poison seondary effect.Atabrine and imatinib are the medicine that clinical practice is ripe, have the advantages such as taking convenience, low cost, provide new more efficiently therapeutic scheme for clinical treatment CML.

Description

The combination medicine for the treatment of chronic myelocytic leukemia

Technical field

The present invention relates to field of medicaments, more particularly, relate to a kind of drug combination treating chronic myelocytic leukemia Thing.

Background technology

Chronic myelocytic leukemia (CML) is a kind of Malignancy originating from hematopoietic stem cell, in China Annual morbidity be about 0.36/10 ten thousand, middle position age of onset is 40-50 year.In CML shows as Bone Marrow of Patients, myeloid cell is not subject to Limit a large amount of propagation in ground, assemble, suppress bone marrow normal hematopoiesis, and pernicious granulocyte is spread to whole body by blood, cause suffering from There is anemia, hemorrhage, infection and organ infiltration in person, thus serious harm patient body health even results in death.There are about 95% The t(9 of CML patient's existing characteristics；22) chromosome translocation, this transposition forms Bcr/abl fusion gene, the Bcr/abl of coding Fusion protein has lasting protein tyrosine kinase activity, it is possible to many bars such as activation downstream STAT5, AKT, NF B are led to Road, promotes propagation and the survival of cell, is the molecular basis of CML morbidity.Based on to Bcr/abl protein conformation and cheese ammonia thereof The research of acid kinase function, becomes the important focus of association area research using it as the specific medicament of target searching treatment CML. The nineties, Zimmermann seminar finds through screening, micromolecular compound imatinib (Imatinib, IM, Gleevec, CGP-57148B, STI571) this fusion protein of specific blockage can be combined with ATP by competitive inhibition Bcr/abl albumen Tyrosine kinase activity, thus suppress CML cell proliferation and promote its apoptosis.Calendar year 2001 U.S. FDA ratifies it for clinic Treatment CML, and it is increasingly becoming the first-line treatment measure of CML.The discovery of Imatinib, is the revolutionary breakthrough in CML treatment history, The treatment making CML enters the Imatinib epoch.The development and application of Imatinib and the second filial generation and third generation medicine makes The life span of chronic myelocytic leukemia patient is extended to 10-20 by 4-5.But answering of tyrosine kinase inhibitor With still not effecting a radical cure CML.

While Imatinib curative effect obtains certainly, its defect the most gradually shows especially.Such as, due to Imatinib not Have and really act on CML stem cell, cause CML patient must take this medicine, once disease after drug withdrawal the most all the life The probability of recurrence or progression of disease rises；Expensive expenses for medicine allows patient can't bear the heavy load；Imatinib suffers from early days mainly for CML Person, centering patients with terminal complete remission rate is less than 30%, and life-time service there will be a series of toxic reaction；Especially part is suffered from Person may occur in which that Imatinib drug resistance and ratio rise year by year, and this main sudden change with Bcr/abl of a large amount of report displays causes it right Imatinib sensitivity reduces relevant；Additionally, Bcr/abl gene amplification causes this expressing fusion protein increase to be also its drug resistance Important mechanisms.For these problems, some new tyrosine kinase inhibitors such as Nilotinib and Dasatinib is continuously available Development and application, but still cannot treat the CML patient that there is T315I sudden change smoothly.Three generations's tyrosine that new development is got up Inhibitors of kinases Ponatinib, effective to the patient of T315I sudden change, but the serious toxic and side effects occurred due to it and face and remove City, its future prospect is the most uncertain.Above-mentioned strategy, is all conceived to the tyrosine kinase activity site for change, designs new Molecule, thus overcome the drug resistance brought of suddenling change.But it is contemplated that, while overcoming old mutational site to bring drug resistance, new The combination in mutational site or mutational site can constantly produce, and accordingly, overcoming of resistance problems is still shouldered heavy responsibilities.

A get Ping (Quinacrine, QC) has another name called malaria silk ribbon to be put down, and mepacrine belongs to acridine derivatives, is a heterocyclic three Cycle compound.Extracted by the bark of Peruvian bark tree, be once extensively used as dyestuff, antimicrobial drug and antimalarial.Quinacrine is Early being widely used during the Second World War as antimalarial agent, it has the advantage that toxic and side effects is little, after gradually by chloroquine (CQ) generation Replace.Recent study find QC on oncotherapy, have great biological effect, it have simultaneously suppression NF-κ B path and Activate P53 path, it is possible in the cyton that the proteasome inhibitors such as blocking-up MG-132 cause, the exception of heat shock response is sharp Live, the function such as key protein synthesis in suppression resistance mechanism.QC Graft Versus Tumor mechanism and spectrum of effect gradually draw attention.In recent years Carry out Quinacrine and there is on oncotherapy the effectiveness of its uniqueness, it combine with some tumour medicines can be used for treat hepatocarcinoma, Breast carcinoma, cervical cancer, carcinoma of prostate, colorectal cancer and P388 leukaemia etc..But whether Quinacrine can be used to Treatment CML, current and unclear.

Summary of the invention

First technical problem to be solved by this invention be to provide a kind of treat chronic myelocytic leukemia combine use Medicine.

Second technical problem to be solved by this invention is to provide imatinib and atabrine drug combination is controlled in preparation Treat the application in chronic myelocytic leukemia medicine.

3rd technical problem to be solved by this invention is to provide atabrine at preparation treatment chronic myelocytic leukemia Application in medicine.

In order to solve above-mentioned first technical problem, the invention provides the drug combination for the treatment of chronic myelocytic leukemia Thing, it is characterised in that described medicine includes that imatinib and atabrine, the mass concentration ratio of described imatinib and atabrine are 1:8 1:12.

As a preferred version, described imatinib and atabrine mass concentration ratio be 1:10.

In order to solve above-mentioned second technical problem, the invention provides imatinib and atabrine drug combination in preparation Application in treatment chronic myelocytic leukemia medicine.

As a preferred version, the mass concentration ratio of described imatinib and atabrine is 1:8 1:12.

As a preferred version, the mass concentration ratio of described imatinib and atabrine is 1:10.

In order to solve above-mentioned 3rd technical problem, the invention provides atabrine at the preparation treatment white blood of chronic granulocyte Application in medicine.

As a preferred version, described chronic myelocytic leukemia is containing Bcr/abl^T315IThe chronic granulocyte of sudden change is white Disorders of blood.

It is an advantage of the current invention that and present invention discover that Quinacrine can reduce CML cell K562, mdr cell K562-R and 32D-Bcr/abl^T315IBcr/abl in cell and the degraded of phosphorylated protein thereof, it is possible to reduce patient CML white The Bcr/abl albumen of disorders of blood cell, the combination with Imatinib can work in coordination with the lethal effect caused CML cell, and to chronic The mdr cell of granulocyte leukemia is effective equally, and to PBMC of healthy people without substantially poison seondary effect. Quinacrine and Imatinib is the medicine that clinical practice is ripe, has the advantages such as taking convenience, low cost, for clinic Upper treatment CML provides new more efficiently therapeutic scheme.

Accompanying drawing explanation

Fig. 1 .Quinacrine causes the intracellular Bcr/abl of K562 and the degraded of phosphorylated protein thereof.(A) Quinacrine Chemical structural drawing.(B) processing K562 cell respectively 12,24,36 hours with 4 μMs of Quinacrine, cell is collected cracking After, the cell pyrolysis liquid Western blot of equivalent detect Bcr/abl, phospho-Bcr/abl and phospho-STAT5, Caspase-3 level.

Fig. 2 .Quinacrine causes 32D-Bcr/abl, 32D-Bcr/abl^T315I, K562 drug-resistant cell strain-K562R, CML Bcr/abl and/or the degraded of phosphorylated protein in patient leukaemia.

Fig. 3 .Quinacrine causes the intracellular Bcr/abl of K562 and phosphoric acid thereof not by mRNA and proteasome pathway The degraded of change form albumen.

The rising of autophagy mark P62, LC3 level that Fig. 4 .Quinacrine causes is degraded with it Bcr/abl and phosphorus thereof The effect of acidic form albumen is unrelated.

Fig. 5 .Quinacrine Yu Imatinib Synergistic killing effect to K562 cell.

Fig. 6 .Quinacrine can substantially strengthen the Imatinib lethal effect to CML cell.

Fig. 7 .Quinacrine and Imatinib closes and adds the effect to CML morphocytology.

The apoptosis of Fig. 8 .Quinacrine and Imatinib induction CML cell.

Fig. 9 .Quinacrine and Imatinib closes the Bcr/abl pathway associated protein adding suppression CML cell.

Figure 10 .Quinacrine and Imatinib closes the Clone formation effect adding suppression patient CML leukaemia, and right The nontoxic seondary effect of PBMC of healthy people.

Detailed description of the invention

Below in conjunction with specific embodiment, the present invention is expanded on further.Experimental technique used in following embodiment such as without Specified otherwise, is conventional method.Material used in following embodiment, reagent etc., if no special instructions, all can be from business way Footpath obtains.Should be understood that these embodiments are merely to illustrate the present invention rather than limit the scope of the present invention.In the following example The experimental technique of unreceipted actual conditions, generally according to normal condition, or according to the condition proposed by manufacturer.

Cell processes

Human chronic polymorpho nuclear leukemia cells K562 and persister K562R and 32D-Bcr/abl thereof^T315ICell is all containing 10% heat-inactivated fetal bovine serum (FBS, Gibco BRL, Gaithersburg, ML), penicillin (100U/ml) and streptomycin (100 μ G/ml) RPMI1640 culture medium (Sigma-Aldrich, St Louis, MO) is cultivated, is positioned over 37 DEG C, containing 5%CO₂'s In constant incubator.K562R persister cell every other day adds 1 μM of Imatinib and maintains the long period.32D-Vector Cell needs the W3 cell conditioned medium (containing IL3) adding 10% to cultivate.In an experiment, cell is all with 2 × 10⁵/ ml inoculates, and uses phase Answer the Quinacrine of concentration, Imatinib process.Imatinib is dissolved in DMSO, Quinacrine and is dissolved in sterilized water, and it stores Concentration is respectively 10mM and 20mM.Cell viability trypan-blue exclusion method of testing detects.Morphologic observation is by collecting cell also Get rid of to microscope slide with cytospin (Shandon, Runcorn, UK), and with Rui Shi Giemsa (BASO Diagnostic Inc., Shanghai, China) dyeing, at optical microscope (Olympus BX-51, Olympus Optical, Japan) under observe.

Western blot analysis

Quinacrine (4 μMs, unless stated otherwise), Imatinib (0.4 μM, unless stated otherwise) process cell specific Time after, by K562, K562R, 32D-Bcr/ablT315I cell collects and cracks extraction albumen by centrifugal.By egg White equivalent loading, to 6-12%SDS-PAGE glue, carries out electrophoresis, and shift trace to NC film (Amersham Biosciences, Piscataway,NJ,USA).After 5% skim milk closing membrane, 4 DEG C hatch the anti-c-abl in rabbit source one (Santa Cruz, CA)、phospho-Bcr(Cell Signaling,Beverly,MA)、STAT5(Santa Cruz,CA)、phospho-STAT5 (Cell Signaling,Beverly,MA)、phosphor-Crkl(Cell Signaling,Beverly,MA)、Total- Caspase-3(Cell Signaling,Beverly,MA)、Cleaved-Caspase-3(Cell Signaling, Beverly, MA) or the anti-PARP-1 in Mus source one (Cell Signaling, Beverly, MA), Crkl (CellSignaling, Beverly,MA).Then two anti-(Cell Signaling, Beverly, the MA) that horseradish peroxidase connects are hatched.And adopt With chemical substrate luminous (Cell Signaling) detection.β-actin antibody (Merck, Darmstadt, Germany) is used for examining Survey the applied sample amount of albumen.

RNA extracts analysis

Quinacrine (4 μMs) and Imatinib (0.4 μM) singly adds or closes to add and processes the different time point of cell.By K562 Cell is collected by centrifugal.The method provided according to Invitrogen company's T RIzol test kit extracts the total serum IgE of cell, RT-PCR kit (Promega, Madison, WI, USA) reverse transcription is used to become cDNA.Real-time quantitative PCR is at ABI Carrying out in PRISM7900 real-time PCR (Perkin-Elmer, Torrance, CA), 10 μ l reaction systems are as follows: 2 × SYBR Green PCR Master Mixture(Applied Biosystems,Foster City,CA,USA)5μl,β- Actin or each 0.05 μ l of the forward and reverse primer of genes of interest product, cDNA1 μ l, ddH₂O3.8μl.Response procedures is as follows: 50 DEG C 2 Minute (UNG is hatched), 95 DEG C 10 minutes (heat start PCR), be repeated below circulation 40 times: 95 DEG C 30 seconds, 60 DEG C 60 seconds, finally exist Set up solubility curve for 60 DEG C.Above all of reaction and detection are all covering the MicroAmp optical96 of transparent sealed membrane Carry out on the Sptting plate of hole.All PCR reaction all uses 3 samples, and calculates the standard deviation of reaction experiment error.All of number According to all using ABIPRISM SDS2.0 software analysis.The threshold value threshold cycle that the supporting computed in software of this instrument goes out (Ct) period needed for reaching the fluorescence intensity (florescence intensity) of certain numerical value is represented.Application " Δ Ct Method ", β-actin is in order to correct the amount of all of RNA in differential responses.

RNA interference and virus are packed and transfection

The complementary nucleotide sequence disturbed for the RNA that P62 and ATG5 design is special, complementary nucleotide sequence is by giving birth to work Biological (Shanghai, China) synthesis.The nucleotide sequence of synthesis is annealed, is connected to pSIREN-RetroQ Vector (Clontech Laboratories, Mountain View, CA) retrovirus interference carrier, after order-checking 100% coupling, enters Row virus packaging.Virus is packed: 1) 293T cell tryptase enzymic digestion is centrifuged rear overhang in complete medium；2) 2.0 × 10 are taken⁶Cell connects Plant in 10cm culture dish and add 10mL complete medium；3) after 24h, transfection prepares, and 293T cell degrees of fusion controls 40% ～60% be advisable；4) in the eppendorf pipe of a 1.5mL, add 3.75 μ g Pumvc3-gag-pol plasmids, 2.5 μ GpCMV-VSV-G plasmid and 3.75 μ gMSCV retroviral vectors.Mix gently.5) the DMEM470 μ l without antibiotic is taken in separately One sterilizing EP pipe, adds 30 μ l FuGene6 and mixes gently, incubated at room 5 minutes.6) by the mixing of DMEM, FuGene6 Thing adds mixing, incubated at room 20 minutes in ready plasmid.7) DMEM, FuGene6, DNA mixture is dripped gently in treating Transfection 293T cell in and rotate mixing gently.8) 293T cell changes to 10mL fresh training liquid after cultivating 24h.9) receive after 48h Take virus.

Virus target cell infection: 1) infect the previous day, choose non-TC treated culture dish (6 holes: #351146).2) Being coated in incubator with 10 μ g/mL people source fibronectin, the 6 every holes of hole culture dish add 2mL.3) culture dish every hole in hole adds Enter 1mL, after 24 hours, exhaust fibronectin；4) count cell and be made into 4 × 10⁵Every milliliter of suspension, inoculating cell is in being coated In the culture dish crossed, 0.5ml/ hole (2 × 10⁵).5) add viral supernatants 2ml/ hole, add polybrene immediately and make final concentration It is 8 μ g/ml.6) in 22 DEG C～32 DEG C centrifugal infection, 1500g, 90min.7) every hole adds the dilution of 2.5ml complete medium Polybrene, puts back to incubator；8), after infecting 48h, add 0.75 μ g/ml puromycin (Sigma-Aldrich) screening and obtain Positive must surely turn cell line.

Mononuclearcell separates

The specimen blood gathered, is mixed its proportional diluted by RPMI1640 culture fluid.Add Ficoll(Ficoll-Paque Plas, GE Healthy) in 15ml sterile centrifugation tube, tilt centrifuge tube, the blood after dilution is slowly added to, keeps blood Forming interface clearly with Ficoll, it is to avoid mixing, Ficoll is 3:4 with the volume ratio of blood, needs to be placed in room temperature and put down before using Weighing apparatus.400g, room temperature is centrifuged 30 minutes, and owing to the density of Ficoll is 1.077, the erythrocyte density of people is 1.093, and granulocyte is close Degree is 1.092, mononuclearcell between 1.076～1.090, separated after, blood plasma and platelet are positioned at upper strata, erythrocyte because of Meet Ficoll coagulation bunchiness money shape and be deposited on event and granulocyte at the bottom of pipe and be all deposited on lower floor.Middle level is lymphocyte separation medium, Mononuclearcell then because be layered liquid-tight degree suitable and intensive plasma layer and be layered liquid interface on, white membranaceous Thing.Draw the cell of layering liquid liquid level, mononuclearcell can be separated to from peripheral blood.Carefully mononuclearcell is drawn, Moving to, in another aseptic 15ml centrifuge tube, add 5 times and wash twice with the RPMI1640 of upper volume, 1500rpm room temperature is centrifuged 10 Minute.After final centrifugation, if gained cell is mixed with erythrocyte, then add 1ml erythrocyte cracked liquid re-suspended cell, fully after mixing Incubated at room 10 minutes, splitting erythrocyte.Add appropriate RPMI1640 and wash away erythrocyte cracked liquid, i.e. obtain mononuclearcell Stand-by.

Clone forming Test

The methylcellulose medium of factor-containing is purchased from Stemcell Technologies.Operate to specifications, By 1 × 10⁵Patient's BMNC of left and right and training liquid, methylcellulose medium and Quinacrine (4 μMs), After Imatinib (0.4 μM) and Quinacrine (4 μMs) and Imatinib (0.4 μM) two medicine combination group are sufficiently mixed, absorption contains Culture medium 1ml having cell and medicine is placed in 30mm culture dish, and each process group respectively sets 3 multiple holes.Culture dish is placed in incubator Middle cultivation, observes each group of colony number after 10-14 days, more than 50 cells being that a colony number counts.

Statistical analysis

For statistics data both from each 3 samples totally 3 times test result, Student ' s t-test is used for Evaluating the difference between two groups, p < 0.05 is considered to have notable statistics difference.Two medicines close add index of cooperation by CompuSyn computed in software, CI < 1 is considered to have cooperative effect.

Embodiment 1.Quinacrine can cause CML cell and the 32D-Bcr/abl containing Bcr/abl-T315I sudden change^T315I Intracellular Bcr/abl and the minimizing of phosphorylated protein thereof.

We have found that the Quinacrine of 4 μMs can cause classical CML cell type-K562 cell by a series of screening Interior Bcr/abl and/or the minimizing of phosphorylated protein, and the mode (Fig. 1 .B) that presentative time relies on.With 4 μMs of Quinacrine Processing K562 cell 12,24,36 hours respectively, after cell is collected cracking, the cell pyrolysis liquid Western blot of equivalent detects Bcr/abl, phospho-Bcr/abl and phospho-STAT5, caspase-3 level.All results have carried out at least repeating three Secondary independent trials.

Fig. 2 shows that Quinacrine causes 32D-Bcr/abl, 32D-Bcr/abl^T315I, K562 drug-resistant cell strain-K562R, Bcr/abl and/or the degraded of phosphorylated protein in patient CML leukaemia.Quinacrine process with same concentration K562 persister cell K562R(Fig. 2 .A), stably express the 32D-Bcr/abl cell of Bcr/abl and containing Bcr/abl- The 32D-Bcr/abl of T315I sudden change^T315ICell (Fig. 2 .B), patient's CML BMNC (Fig. 2 .C), all find same Phenomenon.In Fig. 2, (A) processes 32D-Vector, 32D-Bcr/abl, 32D-Bcr/abl respectively with 4 μMs of Quinacrine^T315I Cell 12,24,36 hours, after cell is collected cracking, the cell pyrolysis liquid Western blot of equivalent detect Bcr/abl, Phospho-Bcr/abl level.(B) processing K562R cell respectively 12,24,36 hours with 4 μMs of Quinacrine, cell is received After collection cracking, the cell pyrolysis liquid Western blot of equivalent detects Bcr/abl, phospho-Bcr/abl level.(C) with 4 μMs Quinacrine processes patient CML leukaemia 24 hours, after cell is collected cracking, and the cell pyrolysis liquid albumen of equivalent Trace detection Bcr/abl level.All results have carried out independent trials the most in triplicate.

Bcr/abl and phosphorylated protein degradation effect thereof in the cells such as the K562 caused by embodiment 2.Quinacrine Mechanism

Owing to there being document to report, Quinacrine has the ability that DNA and RNA combines, and we guess that Quinacrine is The no mRNA level in-site that have impact on Bcr/abl albumen, is inverted and Real-by the RNA after collecting Quinacrine process K562 Time PCR etc. test, it has been found that Quinacrine can not reduce the mRNA level in-site (Fig. 3 .A) of Bcr/abl.Fig. 3 shows Quinacrine causes the intracellular Bcr/abl of K562 and phosphorylation form albumen thereof not by mRNA and proteasome pathway Degraded.(A) process K562 cell respectively 12,24,36 hours with the Quinacrine of 4 μMs, after cell is collected, utilize Trizol Method extracts intracellular total serum IgE, after being inverted to cDNA, by the mRNA level in-site of Real-time PCR detection Bcr/abl.(B) 4 μ are used The Quinacrine of M processes K562 cell 6,12 hours respectively, accompanies or does not accompany at 1 μM of proteasome inhibitor MG132 of 3 hours Reason, after cell is collected cracking, the cell pyrolysis liquid Western blot of equivalent detect Bcr/abl, phospho-Bcr/abl and Ubiquitin level.All results have carried out independent trials the most in triplicate.Thus it is presumed that Quinacrine may The degraded being caused Bcr/abl by protein level, then we use proteasome inhibitor MG132-1 μM of 3h and 6h, The Quinacrine-4 μM of conjunction of 12h adds, and finds that MG132 can not suppress the degraded (figure of the Bcr/abl caused by Quinacrine 3.B), the Quinacrine probability by proteasome pathway degraded Bcr/abl can substantially be got rid of with regard to this.

After getting rid of above-mentioned all mechanisms, we have suggested autophagy approach, first we have detected autophagy correlating markings Thing P62 and LC3 albumen and the change of mRNA level in-site, pleasantly surprised be Quinacrine-4 μM raises in time dependent mode simultaneously Both mRNA and protein level (Fig. 4 .A).In order to study further these phenomenons whether with Quinacrine caused by Bcr/abl degradation effect is relevant, and we have knocked out P62 and autophagy key gene ATG5 in K562 cell, afterwards and K562-NC Cell with the Quinacrine process of 4 μMs, observes Bcr/abl and phosphorylation egg thereof after collecting above-mentioned different time points albumen simultaneously White level changes, and finds that these key genes controlling autophagy can not reverse the Bcr/ caused by Quinacrine after knocking out Abl signs of degradation (Fig. 4 .B).

Fig. 4 show the rising of autophagy mark P62, LC3 level that Quinacrine causes degrade with it Bcr/abl and The effect of phosphorylation form albumen is unrelated.(A) K562 cell is processed respectively 12,24,36 hours, carefully with the Quinacrine of 4 μMs Born of the same parents are collected, and after part cracking, the cell pyrolysis liquid Western blot of equivalent detects the level of P62, LC3；A part is RNA Extracting and reversion process, and finally utilize the mRNA level in-site of Real-time PCR detection P62 and LC3.(B) shRNA technology is utilized to strike Except P62 and ATG5 in K562 cell, with the Quinacrine of 4 μMs process respectively K562-NC, K562-shRNA-P62 and Each 12,24,36 hours of K562-shRNA-ATG5 cell, after cell is collected cracking, the cell pyrolysis liquid Western blot of equivalent The level of detection Bcr/abl, phospho-Bcr/abl, P62, ATG5.All results have carried out independence examination the most in triplicate Test.

Due to the lethal effect of Quinacrine individual processing K562 inconspicuous, we attempt Quinacrine afterwards Share with classical CML medicine Imatinib, in the hope of obtaining preferable coordinating effect in the case of low concentration, exist simultaneously Reverse the drug resistance situation of some patient to a certain extent.

Embodiment 3.Quinacrine and Imatinib share to work in coordination with and cause CML cell killing effect

We with the Quinacrine of (2,2.5,3,2.5,4,4.5,5 μMs) and (0.2,0.25,0.3,0.35,0.4, 0.45,0.5 μM) Imatinib the most singly add and close and add process K562 cell, expect that blue counting calculates cell and kills by cell platform Hinder effect, utilize CompuSyn computed in software two medicine collaborative finger in the case of the definite proportion of Quinacrine:Imatinib=10:1 Number, finds that the two has good cooperative effect (Fig. 5).These data are at least repeated the independent trials of three times, each three samples This.

We process K562, K562R and 32D-Bcr/abl, 32D-with 4 μMs of-Quinacrine, 0.4 μM of Imatinib By cell platform, Vector cell, expects that blue counting calculates motility rate, processes the experiments such as cell rejection tablet Wright's staining observation, finds both Share the death that can substantially cause K562, K562R and 32D-Bcr/abl cell, and to normal 32D-Vector cell (Fig. 6, 7) and Normal human peripheral's hemocyte is without obvious effect (Fig. 9 .A).

Fig. 6 shows that Quinacrine can substantially strengthen the Imatinib lethal effect to CML cell.(A) with 4 μMs The Imatinib of Quinacrine and 0.4 μM the most singly adds and closes and adds process K562 cell 24,48 hours, and motility rate is all expected by platform Blue repulsion testing inspection.(B) the most singly add with the Quinacrine of 3.5 μMs and the Imatinib of 3 μMs and close add process K562R thin Born of the same parents 24,48 hours, motility rate all expects blue repulsion testing inspection by platform.(C) with the Quinacrine of 4 μMs and 0.4 μM Imatinib the most singly adds and closes and adds process 32D-Vector and 32D-Bcr/abl cell 24 hours, and motility rate all expects orchid by platform Repel testing inspection.All of value is all average with standard deviation.These data are at least repeated the independent trials of three times, every time Three samples.

Fig. 7 shows that Quinacrine and Imatinib closes and adds the effect to CML morphocytology.(A) with 4 μMs The Imatinib of Quinacrine and 0.4 μM the most singly adds and closes and adds process K562 cell 48 hours, cell be collected and get rid of to Microscope slide, dyes with Rui Shi Giemsa, and observes with the Olympus BX60 of 100 ×/1.3 object lens.Literary composition shows generation The image of table.(B) the most singly add with the Quinacrine of 3.5 μMs and the Imatinib of 3 μMs and close add process K562R cell 48 Hour, cell is collected and gets rid of to microscope slide, dyes with Rui Shi Giemsa, and with the Olympus of 100 ×/1.3 object lens BX60 observes.Literary composition shows representational image.(C) the most singly add with the Quinacrine of 4 μMs and the Imatinib of 0.4 μM And conjunction adds process 32D-Vector and 32D-Bcr/abl cell 24 hours, cell is collected and gets rid of to microscope slide, with Rui Shi Ji's nurse Sa dyeing, and observe with the Olympus BX60 of 100 ×/1.3 object lens.Literary composition shows representational image.All knots Fruit has carried out independent trials the most in triplicate, each three samples.

It is real by apoptosis pathway that embodiment 4.Quinacrine and Imatinib work in coordination with the CML cell killing effect caused Existing

The change that we add, to two medicines conjunctions, the cellular morphology caused by Wright's staining is analyzed, thus it is speculated that two medicines are probably By the apoptosis-induced cell killing effect caused, then utilize Western blot technology for detection apoptosis mark PARP-1 and The change of the albumen such as Caspase-3, finds that two medicines conjunctions occur in that when adding process and significantly shears band, point out apoptotic Raw, and singly add and matched group occurs without or occurs and unconspicuous shearing band.

Fig. 8 shows the apoptosis of Quinacrine and Imatinib induction CML cell.(A) with the Quinacrine of 4 μMs and The Imatinib of 0.4 μM the most singly adds and closes and adds process K562 cell 48 hours, and cell is collected and cracks, and the cell of equivalent splits Solve the Quinacrine and 3 that the levels (B) such as liquid Western blot detection apoptosis-related protein PARP-1, Caspase-3 are with 3.5 μMs μM Imatinib the most singly add and close and add process K562R cell 48 hours, cell is collected and cracks, the cell cracking of equivalent The liquid levels such as Western blot detection apoptosis-related protein PARP-1, Caspase-3.(C) with the Quinacrine and 0.4 μ of 4 μMs The Imatinib of M the most singly adds and closes and adds process 32D-Vector and 32D-Bcr/abl cell 24 hours, and cell is collected and splits Solve, the cell pyrolysis liquid of the equivalent level of Western blot detection apoptosis-related protein Caspase-3.All results have been carried out extremely Few independent trials in triplicate.

Embodiment 5.Quinacrine is closed with Imatinib and is added the fall causing CML intracellular Bcr/abl pathway associated protein Solve and activity reduces

We detect simultaneously Quinacrine Yu Imatinib mono-add and close add process K562, K562R, 32D-Vector and After 32D-Bcr/abl cell, intracellular Bcr/abl pathway associated protein: Bcr/abl, phospho-Bcr/abl, STAT5, The change of phospho-STAT5 and downstream anti-apoptotic proteins Bcl-xl, finds that two medicines close to add and causes the obvious of above-mentioned albumen Reduce, demonstrate above-mentioned two medicines conjunctions further and add the obvious effect to CML cell.

Fig. 9 shows that Quinacrine and Imatinib closes the Bcr/abl pathway associated protein adding suppression CML cell.(A) use The Imatinib of the Quinacrine of 4 μMs and 0.4 μM the most singly adds and closes and adds process K562 cell 48 hours, and cell is collected also Cracking, the cell pyrolysis liquid of equivalent Western blot detects Bcr/abl pathway associated protein such as Bcr/abl, phospho-Bcr/ The levels such as abl, STAT5, phospho-STAT5, Bcl-xl (B) Quinacrine of 3.5 μMs and the Imatinib of 3 μMs distinguish Singly adding and close and add process K562R cell 48 hours, cell is collected and cracks, and the cell pyrolysis liquid Western blot of equivalent detects The water such as Bcr/abl pathway associated protein such as Bcr/abl, phospho-Bcr/abl, STAT5, phospho-STAT5, Bcl-xl Flat.(C) the most singly add with the Quinacrine of 4 μMs and the Imatinib of 0.4 μM and close add process 32D-Vector and 32D- Bcr/abl cell 24 hours, cell is collected and cracks, and the cell pyrolysis liquid of equivalent Western blot detects Bcr/abl path The levels such as associated protein such as Bcr/abl, phospho-Bcr/abl, STAT5, phospho-STAT5, Bcl-xl.All results are entered Go independent trials the most in triplicate.

Embodiment 6.Quinacrine and Imatinib close and add the impact of the Clone formation on patient's CML BMNC

We obtain the BMNC of an example CML patient, detected by colony formation Quinacrine Yu Imatinib granulocytic impact slow on patient.Find that two medicines close to add to patient CML equally effectively, be two medicines Conjunction adds the Clinical feasibility of scheme and lays a good foundation (Figure 10 .B).

Figure 10 shows that Quinacrine and Imatinib closes the Clone formation effect adding suppression patient CML leukaemia, And seondary effect nontoxic to PBMC of healthy people.Collect normal human peripheral blood five example and patient's CML marrow blood specimen One example, utilizes Ficoll density－gradient centrifuga－tion method separation mononuclearcell.(A) with the Quinacrine of 4 μMs and 0.4 μM Imatinib the most singly adds and closes and adds process PBMC of healthy people 72 hours, by platform, motility rate all expects that blue the repulsion tries Test detection.(B) the most singly add with the Quinacrine of 4 μMs and the Imatinib of 0.4 μM and close patient CML adding process separator well BMNC, processes 10 days with Clone formation gel test, and clone's number by Microscopic observation and counts.All tests are the most every Three samples of secondary repetition.

To sum up, the clinical CML case to Imatinib drug resistance gradually increases, almost all of CML acute transformation phase and 15%～ Drug resistance is there is in 20% patient recurred after Imatinib treats to Imatinib.Increase the dosage of Imatinib or change the Secondary signal transduction inhibitor such as AMN107 (Nilotinib) and Dasatinib (Dasatinib) etc. are to most of saltant types CML is effective, but to having Bcr/abl^T315IThe CML of sudden change is the most invalid.In view of resistance problems present on Present clinical, the present invention The combination medicine of Quinacrine Yu Imatinib related to, can not only be special at low concentration (only needing a small amount of Imatinib) The CML cells such as opposite sex Synergistic killing K562, simultaneously the most effective to K562 mdr cell K562R.It addition, Quinacrine can lure Lead containing Bcr/abl^T315IBcr/abl and the degraded of phosphorylated protein thereof in the 32D cell of sudden change, for for containing Bcr/abl^T315I The CML treatment of sudden change provides scheme.Quinacrine is easily prepared, and two medicines share and can obviously reduce medical expense, reduce simultaneously Side effects of pharmaceutical drugs, have good clinical generalization value.

The molecular basis of CML morbidity is Bcr/abl fusion gene, expresses Bcr/abl fusion protein, and Quinacrine can drop Low Bcr/abl and the content of phosphorylated protein thereof, and Imatinib is mainly risen by the PTK activity of Selective depression Bcr/abl Effect, the target spot of two medicine effects is different.The present invention treats the composition of medicine of chronic myelocytic leukemia and passes through Quinacrine Combine use with Imatinib two medicine, suppression CML cell can be worked in coordination with and grow, breed, promote CML apoptosis, promote that Bcr/abl melts Hop protein degraded and suppression tyrosine kinase (PTK) activity etc..According to experimental result, at 0.3-0.5 μM of Imatinib and 3-5 μ In the range of MQuinacrine combination group, two medicines are obvious to CML cell killing effect, and the quality of Imatinib with Quinacrine is dense Degree ratio is at the preferred 1:10 of 1:8-1:12() ratio under K562 cell had Synergistic killing effect, wherein 4 μMs of Quinacrine Best to K562 cell effect with 0.4 μM of Imatinib combination group.It addition, 4 μMs of Quinacrine can individually cause K562 etc. The minimizing of Bcr/abl and its phosphorylated protein in CML cell, and the combination of two medicines to PBMC of healthy people without substantially Toxic and side effects, and the medical expense of patient can be greatly reduced, and for reducing patient CML, the drug resistance of Imatinib is had reality Using value.

The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art Member, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should be regarded as Protection scope of the present invention.

Claims (1)

1. atabrine application in preparation treatment chronic myelocytic leukemia medicine, it is characterised in that described chronic granulocyte Leukemia is containing Bcr/abl^T315IThe chronic myelocytic leukemia of sudden change.