CN105963302B - A kind of low dose pharmaceutical compositions of the tyrosine kinase containing EGFR and its application in preparation prevention tumor metastasis medicine - Google Patents
A kind of low dose pharmaceutical compositions of the tyrosine kinase containing EGFR and its application in preparation prevention tumor metastasis medicine Download PDFInfo
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Classifications
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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
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5375—1,4-Oxazines, e.g. morpholine
- A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
-
- 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
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
A kind of low dose pharmaceutical compositions of the tyrosine kinase containing EGFR and its application in preparation prevention tumor metastasis medicine.The present invention provides a kind of pharmaceutical composition of the anti-tumor metastasis of low dosage, it is characterised in that the pharmaceutical composition includes the ursolic acid and Gefitinib that the mass ratio of the material is 10:1-5.By that will have the Antitumor Natural Products ursolic acid of the Gefitinib of targeting and high-efficiency low-toxicity to carry out drug combination, it was demonstrated that it has good inhibiting effect on tumor metastasis to cancer cell, it is expected to obtain safer reliable novel drug candidate.
Description
Technical field
The invention belongs to medicine for anti transfer of tumor fields, in particular it relates to which a kind of low dosage is used to have anti-swell
The pharmaceutical composition of tumor metastasis.
Background technique
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- amyrin (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
There are it to be distributed widely in a variety of natural plants such as loguat leaf, black bearberry, hawthorn, oldenlandia diffusa for free or glucosides form,
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 significant with anticancer activity, 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 biggish 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 contains ursolic acid and ring
The antitumor drug effect of one pack system had both can be enhanced in the pharmaceutical composition of phosphamide, the composition, 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.
Gefitinib (chemical name: N- (the chloro- 4- fluorophenyl of 3-) -7- methoxyl group -6- (3- morpholine propoxyl group) quinazoline -4-
Amine), English name: Gifitinid, structure are a kind of selective EGF-R ELISA (EGFR) tyrosine as shown in formula II
Kinase inhibitor is suitable for the part evening that epidermal growth factor recipient tyrosine kinase (EGFR TK) gene has sensitizing mutation
Three lines of phase or Metastatic Nsclc (NSCLC) patient, two wires even first-line treatment.Gefitinib is slightly solubility medicine
Object, dissolubility in aqueous solution is in pH dependence, i.e., solubility is bigger in the lower aqueous solution of pH, in pH value 7 or so
Water in it is almost insoluble；, Gefitinib, which has the patient being mutated to part EGFR, has certain therapeutic effect, still, patient
Recurrence can occur generally in 1-2 perhaps to shift or generate new mutation and generate drug resistance to Gefitinib, so, i.e.,
Total existence in 5 years of Patients with Non-small-cell Lung still cannot be improved using Gefitinib (tyrosine kinase inhibitor) treatment
Phase.Patent N201210566665.X discloses a kind of tablet containing Gefitinib, it its can continue, steadily discharge effectively at
Divide, good therapeutic effect is reached to the locally advanced lung cancer or Metastatic Nsclc that previously received chemotherapy.
It is thin to different carcinoma by by ursolic acid and Gefitinib 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, the condition of ursolic acid and Gefitinib being used in combination in low dosage
Under to proliferation apoptosis, invasion of cancer cell especially non-small cell lung cancer etc. have significant inhibiting effect, and in concentration and when
Between dependence inhibition cancer cell proliferation.
Summary of the invention
It is an object of the invention to provide a kind of pharmaceutical compositions of the anti-tumor metastasis of low dosage, by that will have targeting
The Antitumor Natural Products ursolic acid of the Gefitinib of effect and high-efficiency low-toxicity carries out drug combination, and it is right after its drug combination to investigate
The anti-transferance of cancer cell, it is expected to obtain safer reliable novel drug candidate.
A kind of low dosage has the pharmaceutical composition of anti-tumor metastasis and its is preparing the application in medicine for anti transfer of tumor,
It is characterized in that the pharmaceutical composition includes the ursolic acid and Gefitinib that the mass ratio of the material is 10:1-5, the two combination can be risen
The effect shifted to Synergistic anti-cancer.
Detailed description of the invention
Fig. 1 ursolic acid and Gefitinib are used alone and 24 h are used in combination to the suppression result of A549 cell Proliferation；
Fig. 2 ursolic acid and Gefitinib are used alone and 24 h are used in combination to the suppression result of H1975 cell Proliferation；
Fig. 3 ursolic acid and Gefitinib are used alone and 24 h are used in combination to the suppression result of H1650 cell Proliferation；
Fig. 4 ursolic acid and Gefitinib are used alone and 24 h are used in combination to the inhibition knot of A549 cell invasion ability
Fruit；
Fig. 5 ursolic acid and Gefitinib are used alone and 24 h are used in combination to the inhibiting rate of A549 cell invasion；
Fig. 6 ursolic acid and Gefitinib are used alone and 24 h are used in combination to the inhibiting rate of H1975 cell invasion；
Fig. 7 ursolic acid and Gefitinib are used alone and are used in combination the inhibition after 24 h to A549 cell migration ability
As a result；
Fig. 8 ursolic acid and Gefitinib are used alone and 24 h are used in combination to the inhibiting rate of A549 cell migration；
Fig. 9 ursolic acid and Gefitinib are used alone and 24 h are used in combination to the inhibiting rate of H1975 cell migration；
Figure 10 ursolic acid and Gefitinib be used alone and be used in combination to H1975 cell RGD, ICAM-1, EGFR,
The influence of VCAM-1 protein expression level；
Figure 11 ursolic acid and Gefitinib be used alone and be used in combination to H1975 cell RGD, ICAM-1, EGFR,
The case where VCAM-1 protein expression.
Specific embodiment
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 Gefitinib are used alone and 24 h are used in combination to the inhibited proliferation of different lung cancer cell lines:
Using standard MTT colorimetric method for determining ursolic acid with Gefitinib difference joint ratio to the Proliferation Ability of different lung cancer cell lines
Activity, as a result as shown in Figs. 1-3.
As shown in Figure 1-3, working as ursolic acid (10 μM) and Gefitinib (1-5 μM) drug combination 24 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 h, thus it is subsequent we select to pacify
The ursolic acid of complete effective low dosage concentration (no obvious killing functions of immunocytes) and Gefitinib combination, continue to study it to lung cancer
Cell invasion and the rejection ability of transfer, to explore it in the potentiality of anti-tumor metastasis field application.
Embodiment 2
Ursolic acid and Gefitinib are used alone and are used in combination the influence to A549 cell strain invasive ability
A549 cell first adds serum-free without phenol red medium overnight starvation, digests, the cell that is collected by centrifugation is containing different pharmaceutical
The blank cultures of concentration suspend.12 orifice plates are taken, the cell that 500 μ L concentration containing different pharmaceutical are added in the upper chamber of cell is outstanding
Supernatant liquid (about 5 × 105/ hole), lower room adds 500 μ l to contain the culture medium of 10% calf serum, and 24 h of drug effect, taking-up cell is used
4% paraformaldehyde fixes 20 min, wipes the non-migrating cell in upper layer with cotton swab, is washed 3 times with PBS, with 0.1% violet staining
40 min are washed 3 times with PBS, the counterdie of cell are removed, and mounting saves, and are taken pictures under random 5 visuals field under inverted microscope, are united
The cell number of migration is counted, as a result as shown in Figure 4,5.
Experimental result ursolic acid as shown in Figure 4,5 and Gefitinib are used alone and are used in combination thin to A549 after 24 h
The detection of born of the same parents' strain invasive ability, the results showed that, blank control group, UA=10 μM and Gefitinib (under the conditions of 5 μM) individually make
With group, it is apparently higher than ursolic acid and Gefitinib drug combination group across the cell number of miillpore filter, the invasive ability of cell subtracts
Weak, invasive ability is obviously inhibited.
Embodiment 3
Ursolic acid and Gefitinib are used alone and are used in combination the inhibiting rate to H1975 cell strain invasive ability, step
Suddenly with embodiment 2, as a result as shown in Figure 6.
Experimental result ursolic acid as shown in Figure 6 and Gefitinib are used alone and are used in combination thin to H1975 after 24 h
The detection of born of the same parents' strain invasive ability inhibiting rate, the results showed that, blank control group, under the conditions of UA(10 μM) and (5 μM of Gefitinib
Under the conditions of) exclusive use group, cell about is penetrated in the H1975 cell invasion of percentage 70-80%, when the two is administered in combination
It waits, only about the H1975 invasion of percentage 35% penetrate cell, and the two combination significantly reduces the invasive ability of H1975 cell.
Embodiment 4
Ursolic acid and Gefitinib are used alone and are used in combination the influence to A549 cell strain transfer ability
The cell (about 3 × 10 of logarithmic growth phase6/ hole) it is inoculated in the plate, 37 DEG C are placed in, 5% CO2Incubator in
24 h are cultivated, after cell close to after fusion, draw three parallel lines with white pipette tips are vertical at 1/4,1/2, the 3/4 of orifice plate,
It is cleaned cell 3 times with PBS, scratch 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 shown in Figure 7,8.
Experimental result is as shown in Figure 7,8, and ursolic acid and Gefitinib are used alone and are used in combination after 24 h to A549
The detection of cell strain cellular migration inhibition rate, the results showed that in 0 h, be administered intervention group and blank control group comparison migration distance without
It significantly affects, after 24 h are administered, apparent migration is had occurred in blank control group cell, and scratch broadband narrows；When UA concentration
For 10 μM of conditions and Gefitinib, cell is also all significantly migrated under conditions of 5 μM；But combining when the two makes
With rear, in Gefitinib in the case where 5 μM, scratch broadband is obviously bigger than Gefitinib is applied alone.Therefore the two drug combination,
Can collaboration inhibit the transfer ability of cell, scratch width difference with blank control group compared with it is obvious and with statistical significance (P <
0.01).
Embodiment 5
Ursolic acid and Gefitinib are used alone and are used in combination the inhibiting rate to H1975 cell strain transfer ability, step
Suddenly with embodiment 4, as a result as shown in Figure 9.
Experimental result as shown in figure 9, ursolic acid and Gefitinib be used alone and be used in combination it is thin to H1975 after 24 h
Born of the same parents' strain cellular migration inhibition rate detection, the results showed that in 0 h, intervention group is administered with blank control group comparison migration distance without bright
Development is rung, and after 24 h are administered, blank control group cell scratch broadband narrows, and cell is significantly migrated；When UA concentration
For 10 μM of conditions and Gefitinib, cell is also all significantly migrated under conditions of 5 μM, but migration distance does not have
Blank control group it is obvious；But after the two is used in combination, in Gefitinib in the case where 5 μM, list is obviously compared in scratch broadband
It is big with Gefitinib and ursolic acid.Therefore the two drug combination can cooperate with the transfer ability for inhibiting cell, scratch width and blank
Control group is obvious compared to difference and has statistical significance (P < 0.01).
Embodiment 6
Ursolic acid and Gefitinib are used alone and are used in combination to H1975 cell RGD, ICAM-1, EGFR, VCAM-1
The influence of protein expression level
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 is rinsed cell 2 times with the PBS of pre-cooling, and the cell pyrolysis liquid RIPA of Fresh is added in every hole into 6 orifice plates
[10 μ L protease inhibitors, 10 μ are added in (radioimmunopre-cipitation assay) 200 μ L in 1 mL RIPA
L inhibitors of phosphatases and 5 μ L phenylmethylsulfonyl fluorides (phenylmethanesulfonylfluoride, PMSF)], it places on ice
30 min, every 5 min rock once, and 4 DEG C of 12000 × g 15 min of centrifugation take supernatant, prepare total protein；Using two quinoline
Formic acid (bicincho-ninic acid, BCA) determination of protein concentration kit measurement protein concentration, using β-actin it is horizontal as
Equal protein matter loading takes 20 μ g proteins to carry out sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electricity
Swimming, then by the protein delivery after separation to polyvinylidene fluoride (polyvinylidene, PVDF) film, at room temperature with containing
The TBST of 5% skimmed milk power closes 1 h, 4 DEG C of primary antibody reactions is added overnight, secondary daily TBST washes 3 (10 min/ of film at room temperature
It is secondary), the secondary antibody (1:5000 dilution) for adding HRP label is incubated at room temperature 1 h, is washed again at room temperature with TBST film 3 times
(10 min/ times), last electrochemical luminescence development.Gray value analysis, knot are carried out to band using image analysis software Image J
Fruit is as shown in Figure 10,11.
For experimental result as shown in Figure 10,11, blank control group does not influence RGD, ICAM-1, EGFR, VCAM-1 protein level
Expression；0 μM of UA concentration and Gefitinib are under conditions of 5 μM, to RGD, ICAM-1, EGFR, VCAM-1 protein expression water
Flat influence is little；But when the two administering drug combinations, synergistic effect can be played, RGD, ICAM- can be significantly inhibited
1, EGFR, VCAM-1 protein expression level inhibits cancer cell by lowering the horizontal of RGD, ICAM-1, EGFR and VCAM-1
Proliferation, inhibit cell transfer.
Claims (1)
1. a kind of pharmaceutical composition is preparing the application in medicine for anti transfer of tumor, the pharmaceutical composition is it is characterized in that packet
The ursolic acid and Gefitinib for being 10:1-5 containing the mass ratio of the material.
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| CN101616671A (en) * | 2007-01-19 | 2009-12-30 | 卫材R&D管理有限公司 | Composition for treatment of pancreatic cancer |
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| CN106075387A (en) * | 2016-06-26 | 2016-11-09 | 张伟强 | A kind of oral formulations treating chronic leukemia and preparation method thereof |
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| 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;第842-848页，摘要、讨论 * |
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