CN105963305B - A kind of pharmaceutical composition containing tyrosine kinase inhibitor and its application in preparing medicine for anti transfer of tumor - Google Patents
A kind of pharmaceutical composition containing tyrosine kinase inhibitor and its application in preparing medicine for anti transfer of tumor Download PDFInfo
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- CN105963305B CN105963305B CN201610519494.3A CN201610519494A CN105963305B CN 105963305 B CN105963305 B CN 105963305B CN 201610519494 A CN201610519494 A CN 201610519494A CN 105963305 B CN105963305 B CN 105963305B
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
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Abstract
The object of the present invention is to provide a kind of pharmaceutical composition containing tyrosine kinase inhibitor and its application in anti-tumor metastasis, described in pharmaceutical composition include with the amount ratio of substance for 10:1 10 ursolic acid and Tarceva, the two combination can play the effect of synergistic antitumor transfer.It is combined has significant inhibiting effect to migration, invasion of malignant cell etc..
Description
Technical field
The present invention relates to a kind of pharmaceutical compositions, belong to field of medicinal compositions, in particular belong to the medicine of anti-tumor metastasis
Compositions field.
Background technology
Ursolic acid i.e. 3 beta-hydroxies-black bearberry -12- alkene -28- acid (3 β-hydroxy-urs-12-en-28-oic acid, letter
Claim UA) also known as malol, belong to a- amyrins (a-amyrin) type pentacyclic triterpenoid, relative molecular weight is
456.68 molecular formula C30H48O3, structure is natural active compound distributed more widely in nature as shown in formula I, mainly with
Free or glucosides form exists, and is distributed widely in a variety of natural plants such as loguat leaf, black bearberry, hawthorn, oldenlandia diffusa,
It is also one of the main active of many traditional Chinese medicines, there is extensive pharmacological action, such as anticancer, liver protection, anti-inflammatory, disease-resistant
It is malicious, anti-oxidant etc. wherein the most notable with active anticancer, not only there is resistant function to a variety of carcinogenic substances, but also thin to kinds of tumors
Born of the same parents in vivo, outer have inhibiting effect.Because of its Small side effects, toxicity is low, shows larger clinical application potentiality.State in recent years
The inside and outside antitumor research to UA is increasingly deep, and find its in tumor prevention, treat and prevent late recurrent transfer etc. side
There are unique advantage and potential application prospect in face.Patent N201510097801.9 discloses one kind and containing ursolic acid and ring
The pharmaceutical composition of phosphamide, the composition can both enhance the antitumor drug effect of one pack system, can also reduce it to normal group
The toxicity knitted has great application prospect to improve the effect of oncotherapy in therapeutic field of tumor.Patent
It is white to human liver cancer cell, human breast cancer cell, human lymphoma cell, people lymphoblast that N03150714.X discloses ursolic acid
Blood disease and people's Acute Lymphoblastic Leukemia, people's acute promyelocytic leukemic and human chronic myeloblastic leukemia have cell toxic effect
It answers.
Tarceva（Erlotinb, abbreviation El）, structure is that treatment late period for just listing in recent years is non-as shown in formula II
Small Cell Lung Cancer drug, Tarceva is listed in advanced Non-small cell lung second line treatment in international and domestic lung cancer is guided
One of drug, it is the targeted drug by oral medication non-small cell lung cancer.Epidermal growth factor recipient tyrosine kinase presses down
Preparation（the epidermal growth factor receptor tyrosine kinase inhibitor, EGFR-
TKI）It is important one of target therapeutic agent, is the small-molecule drug using EGFR as target spot, passes through competitive binding receptor
ATP or substrate-binding region block its signal transduction from source and play a role.Tarceva is as EGFR small molecule tyrosines
Kinase inhibitor, clinical application is extensive due to it has oral simplicity, Small side effects, is easy to be accepted by patients.Structure such as Fig. 2 institutes
Show.Patent N201510360703.X discloses the treatment of picropodophyllotoxin and Tarceva drug combination medication lung cancer, the two joint
It will produce collaboration effect of anti-lung cancer after use；Patent N 200680041064.X disclose suberoylanilide hyroxamic acid and
The treatment for kinds cancers such as lung cancer, breast cancer, cancers of pancreas is used in combination in Tarceva.
It is thin to different carcinoma by by ursolic acid and Tarceva drug combination using different cancerous cell lines as research object
Born of the same parents are to carry out Anticancer Activity in vitro test, the results showed that, being used in combination to malignant cell for ursolic acid and Tarceva is special
Not being proliferation apoptosis, invasion to non-small cell lung cancer etc. has significant inhibiting effect, is in the suppression of concentration and time dependence
The proliferation of non-small cell lung cancer processed.
Invention content
It is antitumor the object of the present invention is to provide a kind of pharmaceutical composition containing tyrosine kinase inhibitor and its preparing
Application in diversion medicaments, by by the Antitumor Natural Products black bearberry of tyrosine kinase inhibitor Tarceva and high-efficiency low-toxicity
Acid carries out drug combination, investigates the anti-transferance to cancer cell after its drug combination, is expected to obtain safer reliable
Inhibit the novel drug candidate of metastases.
A kind of pharmaceutical composition containing tyrosine kinase inhibitor and its application in anti-tumor metastasis, described in medicine
Compositions include with the amount ratio of substance for 10:The ursolic acid and Tarceva of 1-10, it is anti-swollen that the two combination can play collaboration
The effect of tumor metastasis.
Description of the drawings
Fig. 1 ursolic acid and Tarceva are used alone and are used in combination histamine results of 24 h to A549 cell Proliferations；
Fig. 2 ursolic acid and Tarceva are used alone and are used in combination histamine results of 24 h to H1975 cell Proliferations；
Fig. 3 ursolic acid and Tarceva are used alone and are used in combination histamine results of 24 h to H1299 cell Proliferations；
Fig. 4 ursolic acid and Tarceva are used alone and are used in combination inhibition knots of 24 h to A549 cell invasion abilities
Fruit；
Fig. 5 ursolic acid and Tarceva are used alone and are used in combination inhibiting rates of 24 h to A549 cell invasions；
Fig. 6 ursolic acid and Tarceva are used alone and are used in combination the inhibition after 24 h to A549 cell migration abilities
As a result；
Fig. 7 ursolic acid and Tarceva are used alone and are used in combination inhibiting rates of 24 h to A549 cell migrations；
Fig. 8 ursolic acid and Tarceva are used alone and are used in combination the inhibition after 48 h to A549 cell migration abilities
As a result；
Fig. 9 ursolic acid and Tarceva are used alone and are used in combination inhibiting rates of 48 h to A549 cell migrations；
Figure 10 ursolic acid and Tarceva are used alone and are used in combination the suppression after 72 h to A549 cell migration abilities
Result processed；
Figure 11 ursolic acid and Tarceva are used alone and are used in combination inhibiting rates of 72 h to A549 cell migrations；
Figure 12 ursolic acid and Tarceva are used alone and are used in combination in A549 cells to EGFR protein expression levels
It influences；
Figure 13 ursolic acid and Tarceva are used alone and are used in combination in H1975 cells to EGFR protein expression levels
Influence.
Specific implementation mode
In order to make content of the present invention easily facilitate understanding, With reference to embodiment to of the present invention
Technical solution is described further, but the present invention is not limited only to this.
Embodiment 1
Ursolic acid and Tarceva are used alone and are used in combination inhibited proliferations of 24 h to different lung cancer cell lines：
Proliferation Ability of the ratio to different lung cancer cell lines is combined using standard MTT colorimetric method for determining ursolic acid and Tarceva difference
Activity, as a result as shown in Figs. 1-3.
As shown in Figs. 1-3, work as ursolic acid（10 μM）And Tarceva（1-10 μM）Drug combination in the low concentration range
It is without had significant proliferation inhibiting effect and small to the toxic side effect of normal cell to cancer cell after 24 h.Therefore it is subsequently selected will be safe
Effective low dosage concentration（Without apparent killing functions of immunocytes）Ursolic acid and Tarceva combination, continue to study it thin to lung cancer
Born of the same parents invade and the rejection ability of transfer, to explore the potentiality that it is applied in anti-tumor metastasis field.
Embodiment 2
Ursolic acid and Tarceva are used alone and are used in combination the influence to A549 cell strain invasive abilities
A549 cells first add serum-free without phenol red medium overnight starvation, digest, the cell that is collected by centrifugation is used and contains different medicines
The blank cultures of object concentration suspend.12 orifice plates are taken, the cell of 500 μ L concentration containing different pharmaceutical is added in the upper chamber of cell
Suspension（About 5 × 105/ hole）, lower room add 500 μ L contain 10% calf serum culture medium, 24 h of drug effect, take out cell,
20 min are fixed with 4% paraformaldehyde, the non-migrating cell in upper layer is wiped with cotton swab, is washed 3 times with PBS, are contaminated with 0.1% crystal violet
40 min of color is washed 3 times with PBS, the counterdie of cell is removed, and mounting preserves, and is taken pictures under random 5 visuals field under inverted microscope,
The cell number of migration is counted, as a result as shown in Figure 4,5.
Experimental result is as shown in Figure 4,5, and ursolic acid and Tarceva are used alone and are used in combination after 24 h to A549
The detection of cell strain invasive ability, the results showed that, blank control group, UA（Concentration is under the conditions of 10 μM）And Tarceva（It is dense
Degree is under the conditions of 10-40 μM）Exclusive use group, the cell number across miillpore filter are apparently higher than ursolic acid and Tarceva（It is dense
Degree is under the conditions of 1-8 μM）Drug combination group；The two combination cell invasion ability is obviously inhibited, it is possible to reduce is replaced Lip river in distress
The dosage of Buddhist nun.
Embodiment 3
Ursolic acid and Tarceva are used alone and are used in combination the influence to A549 cell strain transfer abilities
The cell of logarithmic growth phase（About 3 × 106/ hole）It is inoculated in the plate, is placed in 37 DEG C, 5% CO2Incubator in
24 h are cultivated, after cell close to after fusion, three parallel lines are drawn with white pipette tips are vertical at 1/4,1/2, the 3/4 of orifice plate,
Cell is cleaned with PBS 3 times, and cut broadband is clapped under fluorescence microscope, records the reference axis of clapped picture.It is added and contains different medicines
The culture medium of object concentration is placed in 37 DEG C, 5% CO2Incubator in culture 24,48,72 h take pictures again at same position, survey
Determine migration distance, compares the migration distance that each group is taken pictures twice, as a result as illustrated in figs. 6-11.
Experimental result as illustrated in figs. 6-11, after ursolic acid and Tarceva are used alone and 24,48,72 h are used in combination
A549 cell strain cellular migration inhibition rates are detected, the results showed that：In 0 h, administration intervention group is migrated with blank control group comparison
Distance has no significant effect, and after 24 h are administered, apparent migration has occurred in blank control group cell, and cut broadband narrows；When
When UA is 10 μM a concentration of, cell is also significantly migrated；Tarceva is in high concentration（≥20 μM）Under conditions of,
The transfer ability of cell is inhibited, and cut healing rate slows down；But after the two is used in combination, in Tarceva >=4
In the case of μM, cut broadband is apparent wider than being applied alone Tarceva >=20 μM, and in the inhibition cell of concentration dependent
Transfer ability.After 48 h are administered, the cut of blank control group cell is closed；When UA is 10 μM a concentration of, cell
Apparent having occurred migrates, and scratch width becomes very narrow；Tarceva can significantly inhibit cell under conditions of >=20 μM
Transfer ability；But after the two is used in combination, under conditions of Tarceva is at 3 μM, it can significantly inhibit cell
Transfer ability, with the increase of Tarceva drug concentration, the transfer ability of cell can also become smaller.After 72 h are administered, blank
Under conditions of 10 μM, the cut of cell has all tended to be closed for control group, UA a concentration of 10 μM and Tarceva；But when two
After person is used in combination, under conditions of Tarceva is at 1 μM, it can significantly inhibit the transfer ability of cell.Both therefore
Drug combination can be in the transfer ability of the inhibition cell of concentration dependent and time dependence, scratch width and blank control group
Compared to difference obviously and with statistical significance（P<0.01）.
Embodiment 4
Ursolic acid and Tarceva are used alone and are used in combination to A549, H1975 cell EGFR protein expression levels
Influence
Cell inoculation to 6 orifice plates is discarded into culture after cell intervenes 24 h of cell up to 80% or more plus different pharmaceutical
Liquid rinses cell 2 times with the PBS of precooling, and the cell pyrolysis liquid RIPA of Fresh is added in every hole into 6 orifice plates
[10 μ L protease inhibitors, 10 are added in (radioimmunopre-cipitation assay) 200 μ L in 1 mL RIPA
μ L inhibitors of phosphatases and 5 μ L phenylmethylsulfonyl fluorides（Phenylmethanesulfonylfluoride, PMSF）], it puts on ice
30 min are set, every 5 min rocks once, and 12000 × g, 4 DEG C of 15 min of centrifugation take supernatant, prepare total protein；Using two quinolines
Quinoline formic acid（Bicincho-ninic acid, BCA）Determination of protein concentration kit measurement albumen concentration is made with β-actin levels
For equal protein matter loading, 20 μ g proteins is taken to carry out sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
Electrophoresis, then by the protein delivery after separation to polyvinylidene fluoride（polyvinylidene, PVDF）On film, use at room temperature
TBST containing 5% skimmed milk power closes 1 h, 4 DEG C of reactions of primary antibody is added overnight, secondary daily TBST washes film 3 times at room temperature（10
Min/ times）, add the secondary antibody of HRP labels（1:5000 dilutions）It is incubated at room temperature 1 h, film is washed again at room temperature with TBST
3 times（10 min/ times）, last electrochemical luminescence development.Gray value point is carried out to band using image analysis software Image J
Analysis, as a result as shown in Figure 12,13.
Experimental result is as shown in Figure 12,13, and blank control group does not influence the table of A549 and H1975 cell EGFR protein levels
It reaches；When a concentration of 10 μM of UA and Tarceva are under conditions of 20 μM, to the table of A549 and H1975 cell EGFR protein levels
It is little up to horizontal influence；But when the two administering drug combinations（Tarceva is under the conditions of 4 μM）, synergistic work can be played
With can significantly inhibit EGFR protein expression levels, by lowering the expression of EGFR albumen and then inhibition cancer cell
Proliferation, the transfer for inhibiting cell.
Claims (2)
1. a kind of pharmaceutical composition with activity of resisting tumor metastasis, it is characterised in that the amount ratio comprising substance is 10:1-10's
Ursolic acid and Tarceva.
2. application of the pharmaceutical composition as described in claim 1 in preparing medicine for anti transfer of tumor.
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| CN201610519494.3A CN105963305B (en) | 2016-07-05 | 2016-07-05 | A kind of pharmaceutical composition containing tyrosine kinase inhibitor and its application in preparing medicine for anti transfer of tumor |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101616671A (en) * | 2007-01-19 | 2009-12-30 | 卫材R&D管理有限公司 | Composition for treatment of pancreatic cancer |
| CN101896177A (en) * | 2007-12-13 | 2010-11-24 | 诺瓦提斯公司 | Combinations of therapeutic agents for treating cancer |
| CN102458466A (en) * | 2009-04-16 | 2012-05-16 | 默沙东公司 | Combination therapy using an anti-egfr agent(s) and igf-1r specific inhibitors |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013152313A1 (en) * | 2012-04-05 | 2013-10-10 | The Regents Of The University Of California | Compositions and methods for treating cancer and diseases and conditions responsive to growth factor inhibition |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101616671A (en) * | 2007-01-19 | 2009-12-30 | 卫材R&D管理有限公司 | Composition for treatment of pancreatic cancer |
| CN101896177A (en) * | 2007-12-13 | 2010-11-24 | 诺瓦提斯公司 | Combinations of therapeutic agents for treating cancer |
| CN102458466A (en) * | 2009-04-16 | 2012-05-16 | 默沙东公司 | Combination therapy using an anti-egfr agent(s) and igf-1r specific inhibitors |
Non-Patent Citations (1)
| Title |
|---|
| Oleanolic acid and ursolic acid induce apoptosis in four human liver cancer cell lines;Sheng-lei Yan等;《Toxicology in Vitro》;20091222;第24卷;摘要，讨论 * |
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