CN105579039A - Use of icaritin in preparation of drugs for treatment of diseases related to FLT-3 - Google Patents
Use of icaritin in preparation of drugs for treatment of diseases related to FLT-3 Download PDFInfo
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Abstract
Provided in the present invention is the use of icaritin in the preparation of drugs for the treatment of diseases related to FLT-3, in particular for acute myelogenous leukemia, acute lymphoblastic leukemia, or autoimmune diseases.
Description
A Kela, which is scheduled on to prepare to be used to treating FLT-3, purposes technical field in the medicine of related disorders
It is scheduled on to prepare to be used to treating FLT-3 the present invention relates to A Kela and has purposes in the medicine of related disorders, belongs to field of medicaments.
FLT-3 belongs to the family member of type III receptor tyrosine kinase, is a kind of signaling molecule.FLT-3 has expression in the various tissues such as liver, spleen, lymph, brain, placenta and gonad, also there is expression in normal medullary system and lymphoid lineage cell precursor simultaneously, there is expression in many hemopoietic system malignant diseases, its signal transduction path is relevant with numerous tumour pathways, therefore, FLT-3 turns into a preferable anti-tumor drug action target spot.Research shows there is expression in 70-90% acute myelocytic leukemia and acute lymphoblastic leukemia cell.
There are FLT-3 overexpression and abnormal activation in most of acute myelocytic leukemia patients.
FLT-3 easily undergos mutation as a kind of signaling molecule, and mutation includes：Nearly spanning domain internal series-connection is replicated, kinase domain is mutated and nearly spanning domain point mutation, is triggered the continuous activation of autoreceptor, and a series of downstream signaling pathways of sustained activation, is caused the abnormality proliferation of leukaemia.And FLT3-ITD mutation are to cause one of acute leukemia poor prognosis, recurrence rate height and the low major reason of survival rate.
A Kelading, also known as icariine, are that the new effective monomer that isolated main active icariin is obtained through enzymatic conversion is extracted from Herba Epimedii, its structural formula such as following formula（I shown in)：
The preparation method of the compound is disclosed in CN101302548B.This method is hydrolyzed using icariin as raw material with beta-glucosidase, and the precipitation acetone solution that hydrolysate centrifugation is obtained, centrifugal filtration obtains supernatant.The supernatant that centrifugation is obtained again is recrystallized with water, obtains icariine sterling.
Disclose icariine in the Chinese patent application of Application No. 200910180879.1 preparing prevention or treating the application that ERs ER-a36 is expressed as in the medicine of the chronic granulocytic leukemia of the positive, the disease being directed to has breast cancer, lung cancer, prostate cancer, cervical carcinoma and carcinoma of endometrium.
But in the prior art, the Jin mono- Walk of other target spots beyond ER-a36 are not studied for icariine.The content of the invention
The present inventor ^ t very have found that A Kela can treat the disease relevant with FLT-3 surely.
It is scheduled on to prepare to be used to treating FLT-3 it is an object of the invention to provide A Kela and has purposes in the medicine of related disorders.
One aspect of the present invention, which provides A Kela and is scheduled on to prepare to be used to treating FLT-3, purposes in the medicine of related disorders.
Preferably, described FLT-3 has related disorders to include acute myeloid leukaemia, ALL or autoimmune pathologies.
Preferably, described autoimmune pathologies include lupus erythematosus.
Preferably, described medicine is oral formulations or injection.
Preferably, described injection is intramuscular dose or intravenous injection.
The beneficial effects of the present invention are：The A Kela of the present invention is scheduled in treatment FLT-3 relevant disease and achieves extraordinary effect, and especially for acute myeloid leukaemia, acute lymphatic leukaemia or autoimmune pathologies, A Kela, which is established a capital, shows good therapeutic effect.Therefore, the present invention is applied there is provided wide prospect for the fixed Walk that enter of A Kela.Simultaneously as A Kela is derived from a monomer in Herba Epimedii surely, so raw material fixed A Kela is easy to get, toxic side effect is small, safe.Brief description of the drawings
Fig. 1 represents the fixed inhibiting rates to FLT-3 kinases of A Kela；
Fig. 2 represents the fixed acute myeloid leukemia cells in children MV-4-11 cell inhibitory effect curve maps to being overexpressed FLT-3 of A Kela；
Fig. 3 represents the fixed apoptotic effect figures to being overexpressed FLT-3 acute myeloid leukemia cells in children MV-4-11 of A Kela, Fig. 3 A, 3B, 3C, 3D, 3E and 3F are illustrated respectively in exposure 24 hours, the Apoptosis situation arrived by flow cytomery during 0 μ Μ, 10 μ Μ, 15 μ Μ, 20 μ Μ, 25 μ Μ, the A Kela of 30 μ Μ concentration determine；
Fig. 4 represents the fixed influence figures to the MV-4-11 cell cycles of A Kela, Fig. 4 Α, Fig. 4 Β, Fig. 4 C, Fig. 4 D, Fig. 4 E and Fig. 4 F represent MV-4-11 cells after exposure during the A Kela of 0 μ Μ, 0.625 μ Μ, 1.25 μ Μ, 2.5 μ Μ, 5 μ Μ, Ι Ο μ Μ concentration is fixed 48 hours respectively, flow cytomery cell cycle distribution situation；
Fig. 5 represents the fixed suppression curves for suppressing knurl to the subcutaneous allosome of MV-4-11 cell NOD/SCID mouse of A Kela.Wherein Fig. 5 A are that gross tumor volume reaches 300mm3During left and right, start administration, the fixed influences to tumour growth of A Kela；Fig. 5 B represent it is that gross tumor volume reaches 215mm3During left and right, start administration, the fixed influences to tumour growth of A Kela；
Fig. 6 represents the fixed acute lymphatic leukaemia cell RS4 being overexpressed to FLT-3 of A Kela;The suppression curve figure of 11 cells propagation.
Following examples are only used for, to of the invention illustrative, being not used in and limiting the present invention, modification, change, modification for being made in the scope of the present invention etc. are all within the scope of the present invention.
Term " FLT-3 has related disorders " refers to disease that is relevant or being related to FLT-3 activity, such as FLT-3 overexpressions and abnormal activation, and with the illness of these diseases.
Term " FLT-3 " overexpression refers to that 1. express FLT-3 in the cell for being generally not intended to express FLT-3;2. cause undesirable cell to be bred, so as to increase FLT-3 expression；3.FLT-3 cellular expression levels exceed normal level.
Term " disease of FLT-3 abnormal activations " refers to the disease caused by being mutated in the unusual a large amounts of FLT-3 or FLT-3.
Term " acute myeloid leukaemia " refers to that immature myelocyte is assembled in marrow, and the marrow hemopoiesis caused is suppressed disease.
Term " ALL " is referred to due to malignant hematologic disease undifferentiated or that the very poor lymphocyte of differentiation is caused by hematopoietic tissue infinite multiplication.
Term " autoimmune pathologies " refers to that body occurs immune response to autoantigen and causes the disease caused by damaged self tissue.Including but not limited to organ specific autoimmune's systemic disease and systemic autoimmune diseases.
Term " organ specific autoimmune's systemic disease " includes but is not limited to chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, chronic ulcerative colitis, the atrophic gastritis of pernicious anemia half, Goodpasture's syndrome, ordinary day Blisters sores, class day Blisters sores, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis etc..
Term " Systemic autoimmune systemic disease " includes but is not limited to systemic loupus erythematosus, rheumatoid arthritis, SV, chorionitis, pemphigus, dermatomyositis, mixing unpleasantness tissue disease, autoimmune hemolytic anemia, Autoimmune Thyroid Diseases, ulcerative colitis etc..
Acute myeloid leukemia cells in children MV-4-11 in the present invention is purchased from American Type Culture Collecti（ATCC) article number CRL-9591, the FLT-3 of the cell line is expressed as the positive.
FLT-3 kinases is purchased from Carna Biological Science Co., Ltd（Carna Bioscience Ltd.), product type is 08-154, lot number 07CBS-2350.
Acute lymphatic leukaemia cell RS4 in the present invention;11 are purchased from ATCC, and article number CRL-1873, the FLT-3 of the cell line is expressed as the positive.
Mouse is non-obese diabetes/severe combined immunodeficiency（Nod/SCID) mouse, 6 weeks, body weight 18.0-22.0g, male, purchased from Beijing Vital River Experimental Animals Technology Co., Ltd., was raised and sterile independent air-feeding cage（IVC), the sterilization feed configured exclusively for mouse, free drinking pure water are raised.
A Kelading is prepared by the method for embodiment 1.Embodiment
Embodiment 1
A Kelading preparation
A Kelading also known as icariine, are to extract isolated from Herba Epimedii.
The preparation method of barrenwort is disclosed in Publication No. CN 101302548 patent.This method is hydrolyzed using icariin as raw material with beta-glucosidase, and the precipitation acetone solution that hydrolysate centrifugation is obtained, centrifugal filtration obtains supernatant.The supernatant that centrifugation is obtained again is recrystallized with water, obtains barrenwort sterling.Icariin in the present invention is purchased from company of Shanxi Jiahe Plant Chemical Co., Ltd., purity 90%.Embodiment 2
Inhibitory action of the A Kelading to FLT-3 kinases
Experimental method：
1. configure 1 times of kinase buffer liquid
Buffer solution contains 50mM 4- hydroxyethyl piperazineethanesulfonic acids（HEPES pH=7.5), 0.0015% (m/v) (are purchased from An Naiji chemical companies, trade name 4- hydroxyethyl piperazineethanesulfonic acids, article number A010752;>, Brij30（0.0015%Bnj-35) (it is purchased from Shi get Jia Science and Technology Ltd.s of Shenzhen, trade name：Brij-35, goods number BRIJ-35), 10mM MgC12 and 2mM dithiothreitol (DTT)s（DTT) (Suo Laibao Science and Technology Ltd.s, trade name dithiothreitol (DTT), article number D8220-1 are purchased from).
2. configure terminate liquid
Terminate liquid contains lOOmM 4- hydroxyethyl piperazineethanesulfonic acids（HEPES pH=7.5 )、0.0015%(m/v)
(：Purchased from An Naiji chemical companies, trade name 4- hydroxyethyl piperazineethanesulfonic acids, article number A010752;>, Brij30（0.0015%Brij-35), 0.2% coating reagent（Coatingreagent #3 be purchased from caliper companies, article No. is PN760050) and 50mM ethylenediamine tetra-acetic acid chelating agent（EDTA) .
3. the configuration of A Kelading solution
A Kela is determined to be dissolved as 10mM solution with 100%DMSO, the concentration of solution is determined further according to the A Kela for needing to detect, 10mM A Kela is determined into solution corresponding concentration is diluted to 10%DMSO.
4. kinase reaction：FLT-3 kinases is added into 1 times of kinase buffer liquid, 2.5 times of enzyme solutions are configured to.Again by fluorescent dye（FAM) polypeptide of mark and ATP (are purchased from sigma companies, trade name ATP disodium hydrates, article number 213-579-1；) 1 times of kinase buffer liquid is added, it is configured to 2.5 times of substrate solutions.
No enzyme activity control group reacting hole, control group reacting hole and medicine group reacting hole are set respectively on 384 orifice plates.Without adding the dimethyl sulfoxide (DMSO)s of 5 L 10% in enzyme activity control group reacting hole（DMSO), 1 (1 times of kinase buffer solution of L；5 L 10% dimethyl sulfoxide (DMSO) is added in control group reacting hole（DMSO), 2.5 times of enzyme solutions of Ι Ο μ Ι ^；The dimethyl sulfoxide (DMSO)s of 5 L 10% are added in medicine group reacting hole（DMSO) A Kela of dissolving determines, 1 (2.5 times of enzyme solutions of ^L.Incubation at room temperature 10 minutes, then Ι Ο μ Ι 2.5 times of substrate solutions of ^ are added into each hole respectively.
28 °C are incubated 1 hour, add 25 L terminate liquid terminating reactions, pass through medicine sorting platform（Purchased from the EZ reader2.0 of Caliper companies) detect that obtaining no enzyme activity compares conversion ratio, control group conversion ratio and drug response group conversion ratio, conversion ratio=product amount/(product amount+amount of substrate） *100%.
5. the calculating of inhibiting rate：
According to formula：Inhibiting rate=(control group conversion ratio-medicine group conversion ratio）I (control group conversion ratios-compare conversion ratio without enzyme activity） *100%
By Fig. 1, A Kela is fixed to can be seen that A Kela is fixed inhibited to FLT-3 kinases to FLT-3 kinase inhibition curves, and its IC50 value is 28nM.Because FLT-3 has expression in acute myeloid leukaemia and acute lymphatic leukaemia, it is believed that A Kela is fixed to have therapeutic action for both diseases.Embodiment 3
Pass through MTT cell proliferation experiments（Colorimetric determination drug effect）Determine the fixed inhibitory action to acute myeloid leukemia cells in children MV-4-11 cells of A Kela
1. experiment material：RPMI IMDM culture mediums（Purchased from Gibco Life Technologies, Inc. of the U.S.）, hyclone（Purchased from Gibco Life Technologies, Inc. of the U.S.）, C02Cell culture medium congratulates Li Shi instrument companies purchased from Germany, and tetrazole is blue（MTT, purchased from Sigma Co., USA）, DMSO is (purchased from Sigma Co., USA）.
2. cell culture processes：Human muscle creatine kinase MV-4-11 cell culture is placed in 37 °C, 5%C0 in the RPMI IMDM culture mediums containing 10% hyclone2In cell culture incubator, pass on every other day, change liquid.
3. MTT cell proliferation experiments（Colorimetric determination drug effect）Method
After the MV-4-11 cells centrifugation of culture, 8.0*104 cells/ml cell suspensions are made into culture medium, are added to by every hole Ι Ο Ο μ Ι in 96 orifice plates.After culture 24 hours, the A Kela that Ι Ο Ο μ Ι various concentrations are added per hole determines, each 4 parallel holes of concentration.After culture 72 hours, 20 μ 1 50mg/ml Μ Τ Τ free serum culture based sols are added per hole.After culture 4 hours, culture medium is abandoned, 200W DMSO are added per hole, after slightly 10 minutes , formazans of concussion fully dissolve, cell absorbance value is determined under 570nm wavelength with ELIASA（OD values）.Set the fixed final concentration of 169.66 μ Μ of A Kela, 84.83 μ Μ, 42.41 μ Μ, 21.21 μ Μ, 10.6 μ Μ, 5.3 μ Μ, 2.65 μ Μ, 1.33 μ Μ, 0.66 μ Μ, calculate the MV-4-11 inhibitory rate of cell growth in each exposure concentrations, cell rise inhibiting rate curve is fitted by Origin5.0 softwares, and draws half effect inhibition concentration IC50.
4. experimental result
It is visible by Fig. 2, shown by MTT experiment result, MV-4-11 cells are in 169.66 μ Μ, exposure 72 hours in 84.83 μ Μ, 42.41 μ Μ, 21.21 μ Μ, 10.6 μ Μ, 5.3 μ Μ, 2.65 μ Μ, 1.33 μ Μ, 0.66 μ Μ A Kelading, inhibitory rate of cell growth is respectively 88.12%, 84.17%, 87.14%, 76.22%, 55.44%, 32.72%, 30.20%, 13.27%, 5.56%, IC50 be 7.48 μ Μ (see Fig. 2).Embodiment 4
A Kelading inducing cell apoptosis is acted on
Experimental principle：Phosphatidylserine（PS) it is distributed in the inner side of cell membrane lipid bilayer, and the PS in Apoptosis early stage, cell membrane is from turning on one's side in adipose membrane to side, Annexm V (Apoptosis detection agents is foretold sunset）It is Ca2+Dependence cardiolipin binding protein, has high affinity, therefore can mark viable apoptotic cell with Annexm V-FITC with PS；Propidium iodide（PI complete cell membrane) can not be passed through, but the cell of apoptosis middle and advanced stage and the cell membrane of dead cell can be passed through, and incarnadines nucleus, therefore PI can be used to mark middle and advanced stage apoptosis and dead cell.
Experiment material：Apoptosis kit（Purchased from Biolegend companies of the U.S.）, Annexin-V is (purchased from Biolegend companies of the U.S.）.
Experimental method：By MV-4-11 cells after exposure during 0 μ Μ, 10 μ Μ, 15 μ Μ, 20 μ Μ, 25 μ Μ and 30 μ Μ A Kela are fixed 24 hours, collect cell, centrifuge and remove culture medium, cleaned with phosphate buffer (PBS) twice, add Ι Ο Ο μ Ι combination buffers and the Apoptosis detection agents of 2 μ 1（Annexin-V), room temperature lucifuge 30min, adds the propidium iodides of 4 μ 1（Ρ Ι), lucifuge reaction 5min adds the combination buffers of 400 μ 1（Binding Buffer), detect apoptotic cell percentage with streaming instrument.
Experimental result：MV-4-11 cells can be observed after exposure during 0 μ Μ, 10 μ Μ, 15 μ Μ, 20 μ Μ, 25 μ Μ and 30 μ Μ A Kela are fixed 24 hours：As drug concentration is raised, live cell fraction is reduced,
The cell proportion increase of early apoptosis and middle and advanced stage apoptosis, and concentration dependent is presented（See Fig. 3).
Living cells B3 regions in Ο μ Μ A Kelading groups:) ratio be 88.2%, viable apoptotic cell（The regions of Β 4）Ratio is 8.2%, middle and advanced stage apoptotic cell（The regions of Β 2）Ratio is 3.3%, dead cell（B1 regions）Ratio is 0.2% (see Fig. 3 Α)；Living cells (regions of Β 3) ratio is 72.9%, viable apoptotic cell in Ι Ο μ Μ A Kelading groups（The regions of Β 4）Ratio is 17.3%, middle and advanced stage apoptotic cell（The regions of Β 2）Ratio is 9.5%, dead cell（B1 regions）Ratio is 0.2% (see Fig. 3 Β)；Living cells in 15 μ Μ A Kelading groups（The regions of Β 3）Ratio is 59.7%, viable apoptotic cell（The regions of Β 4）Ratio is 23.6%, middle and advanced stage apoptotic cell（The regions of Β 2）Ratio is 16.4%, dead cell（B1 regions）Ratio is 0.3% (see Fig. 3 C)；Living cells (the regions of Β 3 in 20 μ Μ A Kelading groups：) ratio be 39.0%, viable apoptotic cell（The regions of Β 4）Ratio is 34.0%, middle and advanced stage apoptotic cell（The regions of Β 2）Ratio is 26.8%, dead cell (B1 regions）Ratio is 0.2% (see Fig. 3 D)；Living cells (regions of Β 3) ratio is 13.8%, viable apoptotic cell in 25 μ Μ A Kelading groups（The regions of Β 4）Ratio is 46.6%, middle and advanced stage apoptotic cell（The regions of Β 2）Ratio is 39.5%, dead cell（B1 regions）Ratio is 0.0% (see Fig. 3 Ε)；Living cells (regions of Β 3) ratio is 10.3%, viable apoptotic cell in 30 μ Μ A Kelading groups（The regions of Β 4）Ratio is 53.1%, middle and advanced stage apoptotic cell（The regions of Β 2）Ratio is 36.5%, dead cell（B1 regions）Ratio is 0.0% (see Fig. 3 F).Embodiment 5
The influence of A Kelading cell cycles
Experimental principle：After cell is fixed through ethanol, membrane passage increase, DNA specific fluorescent dyes
Ρ Ι can be combined into intracellular with nucleic acid, and after RNase digestion RNA, the DNA content in the fluorescence volume and core of dyestuff is in ratio, therefore can reflect the ratio of cell DNA content by stream measuring PI fluorescence volumes.According to the feature of cell mitogen, the G0/G1 phases can be represented by single times of fluorescence volume of software analysis（I.e.：Resting stage and the cell proportion of DNA pre-synthesis phases）Cell proportion, double fluorescence volume represents the G2/M phases（S Jie：DNA has synthesized the cell with cell division start to finish before mitosis）Cell proportion, between the two for DNA synthesis the phase be the S phases.
Experiment material：Streaming instrument trade name is that Cytomics FC500 (are purchased from Beckman Coulter companies of the U.S.）, ribalgilase（RNaseA is purchased from Sigma Co., USA）, propidium iodide（PI, purchased from Sigma Co., USA）.
Experimental method：By MV-4-11 cells 0 μ Μ, 0.625 μ Μ, 1.25 μ Μ, 2.5 μ Μ, 5 μ Μ and
After being exposed 48 hours during 10 μ Μ A Kela is fixed, cell is collected, centrifuges and removes culture medium, use phosphate buffer（PBS) cleaning twice, adds the ethanol of lml 70% and is resuspended, -20 °C of fixations are stayed overnight, centrifugation is removed
After ethanol, phosphate buffer is used（PBS) cleaning twice, adds the phosphate buffers of 200 μ 1（PBS) it is resuspended, addition ribalgilase (RnaseA) 10 (g/ ml, 37 °C are incubated 30 minutes, add propidium iodide（PI) 5(^g/ ml, room temperature lucifuge 30 minutes is changed using the streaming instrument detection cell cycle.
Experimental result：Because MV-4-11 cells are higher in more than the Ι Ο μ Μ surely exposed 24 hours apoptosis ratios of A Kela, see embodiment 5, therefore reduction drug concentration, select Ο μ Μ, 0.625 μ Μ, 1.25 μ Μ, 2.5 μ Μ, 5 μ Μ, Ι Ο μ Μ A Kelading, MV-4-11 cells are after exposure during the A Kela of concentrations above is fixed 48 hours, flow cytomery cell cycle distribution situation, as a result shows to raise with drug concentration, apoptotic cell Apo ratios and 0()/01The increase of phase cell proportion（See Fig. 4).
In Ο μ Μ A Kelading groups, G0/G1 phases cell proportion is that 64.82%, G2/M phases cell proportion is that 12.25%, S phases cell proportion is 22.93%, and apoptotic cell ratio is 0% (see Fig. 4 Α)；In 0.625 μ Μ A Kelading groups, G0/G1 phases cell proportion is that 65.35%, G2/M phases cell proportion is that 13.49%, S phases cell proportion is 21.16%, and apoptotic cell ratio is 0% (see Fig. 4 Β)；In 1.25 μ Μ A Kelading groups, G0/G1 phases cell proportion is that 66.64%, G2/M phases cell proportion is that 11.92%, S phases cell proportion is 21.44%, and apoptotic cell ratio is 0% (see Fig. 4 C)；In 2.5 μ Μ A Kelading groups, G0/G1 phases cell proportion is that 67.30%, G2/M phases cell proportion is that 10.59%, S phases cell proportion is 22.11%, and apoptotic cell ratio is 5.11% (see Figure 40);5. (in ^1 A Kelading groups, 00/01 phase cell proportion is that 72.07%, G2/M phases cell proportion is that 8.89%, S phases cell proportion is 19.04%, and apoptotic cell ratio is 9.82% (see Fig. 4 Ε)；In Ι Ο Ο μ Μ A Kelading groups, G0/G1 phases cell proportion is that 84.64%, G2/M phases cell proportion is that 4.9%, S phases cell proportion is 10.46%, and apoptotic cell ratio is 11.46% (see Fig. 4 F).Embodiment 6
Zoopery
The foundation of animal experimental model
The MV-4-11 cells of culture are collected, centrifuges and removes culture medium, 4* 10 is configured to appropriate physiological saline7/ ml cell suspensions, with matrigel（Matrigel) with 1:1 ratio is mixed, and is inoculated in non-obese diabetes/severe combined immunodeficiency NOD/SCID mouse right fores armpit subcutaneously, every mouse 0.2ml (8* 106/ only）.Blank group is to give corn oil.
Experiment material：Matrigel（Trade name Matrigel is purchased from BD Biocoat companies of the U.S.）.
In NOD/SCID mouse hypodermic inoculation MV-4-11 cells, 2 weeks or so, knurl body reached 300mm3Left and right, it is grouped and starts administration, successive administration 18 days, the inhibiting rate that dosage is respectively 12.5mg/kg, 25mg/kg, 50mg/kg A Kelading tumour growths is respectively 20.4%, 37.4%, 47.2% (PO.001), is specifically shown in Fig. 5 A.
l00%;Vc is control group gross tumor volume,
Vt is tested group of gross tumor volume.Gross tumor volume=l/2*a*b a are the major diameter of tumor mass, and b is the minor axis of tumor mass：).Repeat experiment and reach 215mm in knurl volume3Left and right, is grouped and starts administration, dosage is respectively
12.5mg/kg, 25mg/kg, 50mg/kg A Kelading are respectively 44.6% (PO.05), 43.7% and 50.4% (PO.05) to the inhibiting rate of tumour growth, are specifically shown in Fig. 5 B.
Thus, it could be seen that A Kela is fixed to have good inhibiting rate for tumour caused by MV-4-11 cells, particularly when gross tumor volume is smaller, the inhibiting rate of medicine is higher.Embodiment 7
Pass through MTT cell proliferation experiments（Colorimetric determination drug effect）Determine A Kela fixed to acute lymphatic leukaemia RS4;The inhibitory action of 11 cells
1. experiment material：The culture mediums of RPMI 1640（Purchased from Gibco Life Technologies, Inc. of the U.S.）, hyclone（Purchased from Gibco Life Technologies, Inc. of the U.S.）, C02Cell culture medium congratulates Li Shi instrument companies purchased from Germany, and tetrazole is blue（MTT, purchased from Sigma Co., USA）, DMSO is (purchased from Sigma Co., USA）.
2. cell culture processes：People's acute lymphatic leukaemia RS4;11 cell culture are placed in 37 °C, 5%C0 in the culture mediums of RPMI 1640 containing 10% hyclone2In cell culture incubator, pass on every other day, change liquid.
3. MTT cell proliferation experiments（Colorimetric determination drug effect）Method
By the RS4 of culture;After the centrifugation of 11 cells, 8.0*10 is made into culture medium4Cell/ml cell suspensions, is added in 96 orifice plates by every hole Ι Ο Ο μ Ι.After culture 24 hours, the A Kela that Ι Ο Ο μ Ι various concentrations are added per hole determines, each 4 parallel holes of concentration.After culture 72 hours, 20 μ 1 50mg/ml MTT free serum culture based sols are added per hole.After culture 4 hours, culture medium is abandoned, 20 are added per hole, and (after slightly 10 minutes , formazans of concussion fully dissolve, cell absorbance value is determined with ELIASA under 570nm wavelength by ^1 DMSO（OD values）.Set the fixed final concentration of 88.2215 μ Μ of A Kela, 44.11075 μ Μ, 22.05538 μ Μ, 11.02769 μ Μ, 5.513844 μ Μ, 2.756922 μ Μ, 1.378461 μ Μ, 0.68923 μ Μ, calculate the RS4 in each exposure concentrations;11 inhibitory rate of cell growth, are fitted cell rise inhibiting rate curve, and draw half effect inhibition concentration IC by Origin5.0 softwares5Q。
4. experimental result
It is visible by Fig. 6, shown by MTT experiment result, RS4;11 cells are in 88.2215 μ Μ, exposure 72 hours during 44.11075 μ Μ, 22.05538 μ Μ, 11.02769 μ Μ, 5.513844 μ Μ, 2.756922 μ Μ, 1.378461 μ Μ, 0.68923 μ Μ concentration A Kela are fixed, inhibitory rate of cell growth is respectively 97.3637%, 98.00679%, 91.9099%, 77.55591%, 72.00618%, 26.09831%, 3.821215%, 1.618376%, IC5.For 3.82009 μ Μ.
Claims (5)
- Claims1. Ah can, which draw to be scheduled on to prepare to be used to treating FLT-3, purposes in the medicine of related disorders.2. purposes according to claim 1, it is characterised in that described FLT-3 has related disorders to include acute myeloid leukaemia, ALL or autoimmune pathologies.3. purposes according to claim 2, it is characterised in that described autoimmune pathologies include lupus erythematosus.4. purposes according to claim 1, it is characterised in that described medicine is oral formulations or injection.5. purposes according to claim 2, it is characterised in that described injection is intramuscular dose or intravenous injection.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201480039760.1A CN105579039A (en) | 2013-08-23 | 2014-08-20 | Use of icaritin in preparation of drugs for treatment of diseases related to FLT-3 |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310370640 | 2013-08-23 | ||
| CN2013103706407 | 2013-08-23 | ||
| CN201480039760.1A CN105579039A (en) | 2013-08-23 | 2014-08-20 | Use of icaritin in preparation of drugs for treatment of diseases related to FLT-3 |
| PCT/CN2014/084781 WO2015024512A1 (en) | 2013-08-23 | 2014-08-20 | Use of icaritin in preparation of drugs for treatment of diseases related to flt-3 |
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| CN105579039A true CN105579039A (en) | 2016-05-11 |
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|---|---|---|---|
| CN201480039760.1A Pending CN105579039A (en) | 2013-08-23 | 2014-08-20 | Use of icaritin in preparation of drugs for treatment of diseases related to FLT-3 |
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| CN (1) | CN105579039A (en) |
| WO (1) | WO2015024512A1 (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080214844A1 (en) * | 2007-01-23 | 2008-09-04 | Nan Zhang | Icaritin and desmethylicaritin as anti-cancer agents |
| CN100502911C (en) * | 2002-04-09 | 2009-06-24 | 四川省中药研究所 | An anti-rheumatism medicament and preparation method thereof |
| CN101843629A (en) * | 2010-06-11 | 2010-09-29 | 首都医科大学宣武医院 | New application of icariin and epimedium flavone containing icariin |
| CN102038673A (en) * | 2009-10-20 | 2011-05-04 | 北京盛诺基医药科技有限公司 | Application of hydroxyl benzopyrone compound for preparing medicament for treating leukemia |
-
2014
- 2014-08-20 CN CN201480039760.1A patent/CN105579039A/en active Pending
- 2014-08-20 WO PCT/CN2014/084781 patent/WO2015024512A1/en active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100502911C (en) * | 2002-04-09 | 2009-06-24 | 四川省中药研究所 | An anti-rheumatism medicament and preparation method thereof |
| US20080214844A1 (en) * | 2007-01-23 | 2008-09-04 | Nan Zhang | Icaritin and desmethylicaritin as anti-cancer agents |
| CN102038673A (en) * | 2009-10-20 | 2011-05-04 | 北京盛诺基医药科技有限公司 | Application of hydroxyl benzopyrone compound for preparing medicament for treating leukemia |
| CN101843629A (en) * | 2010-06-11 | 2010-09-29 | 首都医科大学宣武医院 | New application of icariin and epimedium flavone containing icariin |
Non-Patent Citations (2)
| Title |
|---|
| QIHUI LI等: "Icaritin induces AML cell apoptosis via the MAPK/ERK and PI3K/AKT signal pathways", 《INT. J. HEMATOL.》 * |
| SANG-HUN KANG等: "Icariside II Induces Apoptosis in U937 Acute Myeloid Leukemia Cells: Role of Inactivation of STAT3-Related Signaling", 《PLOS ONE》 * |
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| WO2015024512A1 (en) | 2015-02-26 |
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