CN105963302A

CN105963302A - Low-dosage medicine composition containing EGFR (epidermal growth factor receptor) tyrosine kinase and application thereof in preparation of anti-tumor transfer medicines

Info

Publication number: CN105963302A
Application number: CN201610519495.8A
Authority: CN
Inventors: 邵敬伟; 郑桂容
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2016-07-05
Filing date: 2016-07-05
Publication date: 2016-09-28
Anticipated expiration: 2036-07-05
Also published as: CN105963302B

Abstract

The invention relates to a low-dosage medicine composition containing EGFR (epidermal growth factor receptor) tyrosine kinase and an application thereof in preparation of anti-tumor transfer medicines. The low-dosage medicine composition with anti-tumor transfer function is characterized by comprising ursolic acid and gefitinib according to the weight ratio of 10:(1-5). The low-dosage medicine composition has the advantage that after the gefitinib with targeting function is jointly used together with the anti-tumor natural product of ursolic acid with high efficiency and low toxicity, the good anti-tumor transfer function on cancer cells is realized, and a safe and reliable novel candidate medicine is obtained.

Description

A kind of low dose pharmaceutical compositions containing EGFR tyrosine kinase and preparation prevention Application in tumor metastasis medicine

Technical field

The invention belongs to medicine for anti transfer of tumor field, in particular it relates to a kind of low dosage is used for having anti-swelling The pharmaceutical composition of tumor metastasis.

Background technology

Ursolic acid i.e. 3 beta-hydroxies-Folium Vaccinii vitis-idaeae-12-alkene-28-acid (3 β-hydroxy-urs-12-en-28-oic acid, letter Claim UA), have another name called maloic acid, belong to a-Amyrin (a-amyrin) type pentacyclic triterpenoid, its relative molecular weight is 456.68, molecular formula is C₃₀H₄₈O₃, structure is natural active compound distributed more widely in nature as shown in formula I, mainly with Dissociate or presented in glucosides, it be distributed widely in the multiple natural plants such as Folium Eriobotryae, Folium Vaccinii vitis-idaeae, Fructus Crataegi, Herba Hedyotidis Diffusae, Also be one of the main active of many Chinese medicines, there is pharmacological action widely, as anticancer, protect the liver, antiinflammatory, disease-resistant Poison, antioxidation etc. are wherein notable with active anticancer, not only have resistant function to multiple carcinogen, and thin to kinds of tumors Born of the same parents in vivo, outer all have inhibitory action.Because its side effect is little, toxicity is low, demonstrates bigger clinical practice potentiality.State in recent years The inside and outside antitumor to UA is studied the most deep, and find its at tumor prevention, treat and prevent the sides such as late recurrent transfer There are the advantage of uniqueness and potential application prospect in face.Patent N201510097801.9 discloses a kind of containing ursolic acid and ring The pharmaceutical composition of phosphamide, described compositions both can strengthen the antitumor drug effect of one pack system, also can reduce it to normal group The toxicity knitted, thus improve the effect of oncotherapy, have great application prospect at 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 Disorders of blood and people's Acute Lymphoblastic Leukemia, people's acute promyelocytic leukemic and human chronic myeloblastic leukemia have cell toxic effect Should.

Gefitinib (chemical name: N-(3-chloro-4-fluorophenyl)-7-methoxyl group-6-(3-morpholine propoxyl group) quinazoline-4- Amine), English name: Gifitinid, structure, as shown in formula II, is a kind of selectivity EGF-R ELISA (EGFR) tyrosine Inhibitors of kinases, it is adaptable to epidermal growth factor recipient tyrosine kinase (EGFR TK) gene has the local evening of sensitizing mutation Phase or three lines of Metastatic Nsclc (NSCLC) patient, two wires even first-line treatment.Gefitinib is slightly solubility medicine Thing, its dissolubility in aqueous is pH dependency, and i.e. in the aqueous solution that pH is the lowest, dissolubility is the biggest, at pH value about 7 Water in the most insoluble；, the patient that gefitinib exists sudden change to part EGFR has certain therapeutical effect, but, patient General can generation in 1-2 is recurred or transfer, or produces new sudden change and gefitinib is produced drug resistance, so, i.e. Use gefitinib (tyrosine kinase inhibitor) treatment still can not improve always existence in 5 years of Patients with Non-small-cell Lung Phase.Patent N201210566665.X discloses a kind of tablet containing gefitinib, it its can continue, steadily release effectively become Point, to previously accepting chemotherapeutical locally advanced lung cancer or Metastatic Nsclc reaches good therapeutic effect.

Using different carcinoma cell line as object of study, by by ursolic acid and gefitinib drug combination, thin to different carcinoma Born of the same parents system carries out Anticancer Activity in vitro test, and result shows, ursolic acid and gefitinib condition at low dosage is used in combination Under the proliferation apoptosis of cancerous cell particularly nonsmall-cell lung cancer, invasion and attack etc. are had significant inhibitory action, and in concentration and time Between the propagation of dependent anticancer.

Summary of the invention

It is an object of the invention to provide the pharmaceutical composition of the anti metastasis of a kind of low dosage, by having targeting The gefitinib of effect carries out drug combination with the Antitumor Natural Products ursolic acid of high-efficiency low-toxicity, right after investigating its drug combination The metastasis effect of cancerous cell, it is expected to obtain safer reliable novel drug candidate.

A kind of low dosage has the pharmaceutical composition of anti metastasis and the application in preparing medicine for anti transfer of tumor thereof, It is characterized in that amount that this pharmaceutical composition comprises material than for the ursolic acid of 10:1-5 and gefitinib, both combinations can rise Effect to Synergistic anti-cancer transfer.

Accompanying drawing explanation

Fig. 1. ursolic acid and gefitinib are used alone and are used in combination 24 h suppression result to A549 cell proliferation；

Fig. 2. ursolic acid and gefitinib are used alone and are used in combination 24 h suppression result to H1975 cell proliferation；

Fig. 3. ursolic acid and gefitinib are used alone and are used in combination 24 h suppression result to H1650 cell proliferation；

Fig. 4. ursolic acid and gefitinib are used alone and are used in combination 24 h suppression result to A549 cell invasion ability；

Fig. 5. ursolic acid and gefitinib are used alone and are used in combination 24 h suppression ratio to A549 cell invasion；

Fig. 6. ursolic acid and gefitinib are used alone and are used in combination 24 h suppression ratio to H1975 cell invasion；

Fig. 7. ursolic acid and gefitinib are used alone and are used in combination the suppression result after 24 h to A549 cell migration ability；

Fig. 8. ursolic acid and gefitinib are used alone and are used in combination 24 h suppression ratio to A549 cell migration；

Fig. 9. ursolic acid and gefitinib are used alone and are used in combination 24 h suppression ratio to 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 impact 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 situation of VCAM-1 protein expression.

Detailed description of the invention

In order to make content of the present invention easily facilitate understanding, below in conjunction with detailed description of the invention to of the present invention Technical scheme is described further, but the present invention is not limited only to this.

Embodiment 1

Ursolic acid and gefitinib are used alone and are used in combination 24 h inhibited proliferation to different lung cancer cell lines: use The Proliferation Ability of different lung cancer cell lines is lived by standard MTT colorimetric method for determining ursolic acid associating different with gefitinib ratio Property, result is as Figure 1-3.

As Figure 1-3, when ursolic acid (10 μMs) and gefitinib (1-5 μM) drug combination 24 in low strength range After h, it is to cancerous cell without had significant proliferation inhibitory action and little to Normocellular toxic and side effects, and follow-up we select to pacify The ursolic acid of complete effective low dosage concentration (without obvious killing functions of immunocytes) and gefitinib combination, continue to study it to pulmonary carcinoma Cell invasion and the rejection ability of transfer, to explore its potentiality in the application of anti metastasis field.

Embodiment 2

Ursolic acid and gefitinib are used alone and are used in combination the impact on A549 cell strain invasive ability

A549 cell first adds serum-free without phenol red medium overnight starvation, digests, the centrifugal cell concentration Han different pharmaceutical collected Blank cultures suspend.Take 12 orifice plates, the upper room of cell adds the 500 μ L cell suspending liquid containing different pharmaceutical concentration (about 5 × 10⁵/ hole), lower room adds the 500 μ l culture medium containing 10% calf serum, medicine effect 24 h, takes out cell, with 4% Paraformaldehyde fixes 20 min, wipes the non-migrating cell in upper strata with cotton swab, washes 3 times with PBS, by 0.1% violet staining 40 Min, washes 3 times with PBS, is taken off by the counterdie of cell, and mounting preserves, and takes pictures under inverted microscope under random 5 visuals field, and statistics is moved The cell number moved, result is as shown in Figure 4,5.

Experimental result ursolic acid as shown in Figure 4,5 and gefitinib are thin to A549 after being used alone and be used in combination 24 h The detection of born of the same parents' strain invasive ability, result shows, blank group, UA=10 μM and gefitinib (under the conditions of 5 μMs) individually make By group, through the cell number of microporous filter membrane apparently higher than ursolic acid and gefitinib drug combination group, the invasive ability of cell subtracts Weak, invasive ability is substantially inhibited.

Embodiment 3

Ursolic acid and gefitinib are used alone and are used in combination the suppression ratio to H1975 cell strain invasive ability, and step is same Embodiment 2, result is as shown in Figure 6.

Experimental result ursolic acid as shown in Figure 6 and gefitinib are thin to H1975 after being used alone and be used in combination 24 h The detection of born of the same parents' strain invasive ability suppression ratio, result shows, blank group, under the conditions of UA(10 μM) and gefitinib (5 μMs Under the conditions of) it being used alone group, the about H1975 cell invasion at percentage 70-80% passes through cell, when both administering drug combinations Waiting, only about the H1975 invasion and attack of percentage 35% are through cell, and both combinations significantly reduce the invasive ability of H1975 cell.

Embodiment 4

Ursolic acid and gefitinib are used alone and are used in combination the impact on A549 cell strain transfer ability

Take the logarithm the cell (about 3 × 10 of trophophase⁶/ hole) it is inoculated in this plate, it is placed in 37 DEG C, 5% CO₂Incubator in train Support 24 h, after the close fusion of cell, with stroke three parallel lines that white rifle head is vertical at 1/4,1/2, the 3/4 of orifice plate, use PBS cell 3 times, claps cut broadband under fluorescence microscope, records the coordinate axes of clapped picture.Add containing different pharmaceutical The culture medium of concentration, is placed in 37 DEG C, 5% CO₂Incubator in cultivate 24,48,72 h. again take pictures at same position, measure Migration distance, contrasts each group of migration distance taken pictures for twice, and result is as shown in Figure 7,8.

As shown in Figure 7,8, ursolic acid and gefitinib are used alone and are used in combination after 24 h A549 experimental result Cell strain cellular migration inhibition rate detects, and result shows: at 0 h, be administered intervention group and blank group contrast migration distance without Significantly affecting, after being administered 24 h, blank group cell there occurs that migration clearly, cut broadband narrow；When UA concentration It is that condition and gefitinib cell under conditions of 5 μMs of 10 μMs the most significantly there occurs migration；But make when both combine With rear, at gefitinib in the case of 5 μMs, cut broadband is substantially big than alone gefitinib.Therefore both drug combinations, Can the transfer ability of collaborative suppression cell, scratch width compared with blank group obvious difference and have statistical significance (P < 0.01).

Embodiment 5

Ursolic acid and gefitinib are used alone and are used in combination the suppression ratio to H1975 cell strain transfer ability, and step is same Embodiment 4, result is as shown in Figure 9.

Experimental result is as it is shown in figure 9, ursolic acid and gefitinib are thin to H1975 after being used alone and be used in combination 24 h Born of the same parents' strain cellular migration inhibition rate detects, and result shows: at 0 h, is administered intervention group with blank group contrast migration distance without bright Development rings, and after being administered 24 h, blank group cell cut broadband narrows, and cell significantly there occurs migration；When UA concentration It is that condition and gefitinib cell under conditions of 5 μMs of 10 μMs the most significantly there occurs migration, but migration distance does not has Blank group obvious；But after being used in combination both when, at gefitinib in the case of 5 μMs, cut broadband is substantially than list Big with gefitinib and ursolic acid.Therefore both drug combinations, can work in coordination with the transfer ability of suppression cell, and scratch width is with blank Matched group is compared obvious difference and has statistical significance (P < 0.01).

Embodiment 6

Ursolic acid and gefitinib are used alone and are used in combination H1975 cell RGD, ICAM-1, EGFR, VCAM-1 albumen The impact of expression

Cell is seeded to 6 orifice plates, reaches more than 80% until cell and add different pharmaceutical and intervene after cell 24 h, discard culture fluid, use The PBS rinsing cell of pre-cooling 2 times, the every hole in 6 orifice plates adds freshly prepared cell pyrolysis liquid RIPA (radioimmunopre-cipitation assay) 200 μ L [adds 10 μ L protease inhibitor, 10 μ in 1 mL RIPA L inhibitors of phosphatases and 5 μ L Phenylmethanesulfonyl fluoride (phenylmethanesulfonylfluoride, PMSF)], place on ice 30 min, every 5 min rock once, and 12000 × g 4 DEG C is centrifuged 15 min, takes supernatant, prepares total protein；Use two quinoline Formic acid (bicincho-ninic acid, BCA) determination of protein concentration kit measurement protein concentration, using β-actin level as Equal protein matter loading, takes 20 μ g protein and carries out sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electricity Swimming, on the protein delivery after then separating to polyvinylidene fluoride (polyvinylidene, PVDF) film, with containing under room temperature The TBST of 5% defatted milk powder closes 1 h, adds one and resists 4 DEG C of reactions overnight, washes 3 (10 min/ of film under secondary daily TBST room temperature Secondary), 1 h is at room temperature hatched in two anti-(the 1:5000 dilutions) adding HRP labelling, the most again washes film with TBST 3 times (10 min/ time), last electrochemiluminescence develops.Use image analysis software Image J that band is carried out gray value analysis, knot Fruit is such as Figure 10, shown in 11.

Experimental result is such as Figure 10, shown in 11, and blank group does not affect RGD, ICAM-1, EGFR, VCAM-1 protein level Expression；UA concentration 0 μM and gefitinib are under conditions of 5 μMs, to RGD, ICAM-1, EGFR, VCAM-1 protein expression water Flat impact is little；But when both administering drug combinations, the effect of Synergistic just can be played, it is possible to significantly suppress RGD, ICAM- 1, EGFR, VCAM-1 protein expression level, by lowering level and then the anticancer of RGD, ICAM-1, EGFR and VCAM-1 Propagation, suppression cell transfer.

Claims

1. a pharmaceutical composition with anti metastasis, it is characterised in that comprise the amount of material than the ursolic acid for 10:1-5 And gefitinib.

2. pharmaceutical composition application in preparing medicine for anti transfer of tumor as claimed in claim 1.