CN109568299A

CN109568299A - Ambroxol purposes in preparing tumor chemotherapeutic drug Synergistic preparations

Info

Publication number: CN109568299A
Application number: CN201710897337.0A
Authority: CN
Inventors: 沙先谊; 张秀磊
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2017-09-28
Filing date: 2017-09-28
Publication date: 2019-04-05

Abstract

The invention belongs to biomedicine technical fields, it is related to new application of the ambroxol in pharmacy, more particularly to ambroxol in preparing tumor chemotherapeutic drug Synergistic preparations purposes, in the present invention, ambroxol can enhance the Treated with Chemotherapeutic Drugs such as taxol, docetaxel, adriamycin, cis-platinum, camptothecine object for the lethal effect of the kinds of tumor cells such as A549, MCF-7, B16F10；Nude mice A549 subcutaneous tumors and A549-luciferase pulmonary metastases model are established in the present invention, using ambroxol and paclitaxel plus therapeutic scheme, the more effective expansion for inhibiting tumor focus extends the life span of nude mice, and ambroxol dosage more high effect is better；Tumour cell autophagy is adjusted in ambroxol, and the lethal effect of ambroxol enhancing taxol may be related with its regulating cell autophagy.Common drug of the ambroxol as the acute and chronic bronchitis of clinical treatment and bronchial asthma, toxic side effect is small, curative for effect, and ambroxol and paclitaxel plus are had good prospects using the clinical conversion for the treatment of tumour especially lung tumors.

Description

Ambroxol purposes in preparing tumor chemotherapeutic drug Synergistic preparations

Technical field

The invention belongs to biomedicine technical fields, are related to new application of the ambroxol in pharmacy, and in particular to ambroxol exists Prepare purposes in tumor chemotherapeutic drug Synergistic preparations

Background technique

It shows according to statistics, as aging of population aggravation, environmental pollution aggravate, the morbidity and mortality one of malignant tumour It is straight in rising trend, seriously threaten human life and health." the Third National population cause of the death tune issued according to hygiene department Look into " data show, urban and rural residents, China mortality of malignant tumors is in world's higher level, and is in lasting growth trend. The conventional method of clinical practice treatment tumour has operation excision, chemotherapy, radiotherapy etc., and each of them therapy all has certain control Treatment advantage, but there is also there is its limitation, such as: operation excision has stringent indication and adaptation population；Radiation and chemotherapy lacks Few selectivity, side effect is larger and can cause the drug resistance of tumour cell, often eventually leads to the failure of clinically oncotherapy. Currently, lung cancer death rate in malignant tumour occupy the first, much higher than breast cancer, cancer of pancreas and colon cancer etc., and only 15% patient survival can exceed that wherein non-small cell lung cancer (NSCLC) accounts for the 75~80% of lung cancer sum 5 years；Patient exists It has been mostly advanced stage when diagnosis, has often needed to carry out chemotherapy, but the drug resistance and toxic side effect of traditional front-line chemotherapeutic agents, cause clinic Therapeutic effect is poor；Molecular targeted agents are only preferable to patient's curative effect of target gene mutation, resistance to there is also being also easy to produce after medication The problems such as medicine.Therefore, development selectivity is good, toxic side effect is small, inhibits drug resistance and the wide chemotherapy of tumors strategy of adaptation population and changes Treatment method is the hot spot of tumour related fields basis and clinical research.

Practice display, during some malignant tumor chemotherapies (as shown in Figure 9), chemotherapeutics withers in induction tumour cell Also it can induce tumour cell autophagy (autophagy) while dying.The generation of tumour cell autophagy on the one hand can be swollen as protection The survival mechanism of oncocyte inhibition apoptosis；The continuous activation of another aspect autophagy makes cell be unable to maintain that its basic structure, causes Death of neoplastic cells, i.e. autophagy are dead.Such as, fluorouracil acts on the inducible tumour cell of human colon cancer cell and protects Property autophagy, reduce tumour cell to the sensibility or even drug resistance of drug；Taxol (Paclitaxel, PTX) is thin in induction tumour While born of the same parents' apoptosis, lower drug concentration can promote the mTOR and its stream substrates of tumour cell autophagy GAP-associated protein GAP The phosphorylation degree of p70s6k weakens, and Beclin1 and Atg5 are raised, and taxol is prompted to start tumour cell by Beclin1 The generation of autophagy leads to drug resistance and reduces chemotherapeutic treatment effect.

It is usually used in alleviating the postoperative clinical symptoms of patients with lung cancer row lobectomy of lungs in ambroxol clinical practice, reduces lung's damage Wound prevents the generation of pulmonary complication after radical resection of pulmonary carcinoma.It is bright in the Consensus of experts of Chinese thoracic surgery peri-operation period lung preversation It really points out that ambroxol can be clinically used for postoperative lung preversation, improves patient's lung function.Since ambroxol and classical autophagy inhibit The chemical structure of agent chloroquine is similar, and related with the regulation of lysosome during cell autophagy；

Basis based on the prior art, the quasi- new application for providing ambroxol in pharmacy of present inventor, specifically relates to And ambroxol purposes in preparing tumor chemotherapeutic drug Synergistic preparations, the ambroxol generate apoptosis of tumor cells in chemotherapeutics It can be further improved the tumor sensitivity and antitumous effect of chemotherapeutics on exposure basis.

Summary of the invention

It is an object of the invention to the bases based on the prior art, provide new application of the ambroxol in pharmacy, specifically relate to And ambroxol purposes in preparing tumor chemotherapeutic drug Synergistic preparations.

Its chemical name is trans- -4- [(2- amino -3,5- dibromo-benzyl) amino] hexamethylenes for ambroxol of the present invention Alcohol, the entitled Ambroxol of English, general entitled ambroxol.

May have the function of the regulation of tumour cell autophagy according to ambroxol drug in the present invention, using chemotherapeutics from Inducing action is bitten, the biological function for adjusting autophagy with ambroxol generates good response, raises the formation of autophagosome while blocking The degradation of end autophagosome, so that intracellular autophagosome enrichment, maintains autophagy process continuous activation, to activate tumour thin Born of the same parents' autophagy death pathway generates the tumour that chemotherapeutics is further increased on apoptosis of tumor cells exposure basis in chemotherapeutics Sensibility and antitumous effect.

In the present invention, ambroxol can enhance the chemotherapeutics pair such as taxol, docetaxel, adriamycin, cis-platinum, camptothecine In the lethal effect of the kinds cancers cell such as non-small cell lung cancer, breast cancer, gastric cancer, liver cancer, melanoma；

In the present invention, nude mice A549 non-small cell lung cancer subcutaneous tumors model is established, is controlled using ambroxol and paclitaxel plus Treatment scheme can become apparent from inhibition tumour growth, have preferable antitumous effect, and the higher inhibitory effect of ambroxol dosage is better； A549-luciferase pulmonary metastases model is established, using ambroxol and paclitaxel plus therapeutic scheme, can obviously inhibit lung The expansion of portion's tumor focus extends the life span of nude mice；Tumour cell autophagy is adjusted in ambroxol, and ambroxol enhances Japanese yew The lethal effect of alcohol may be related with its regulating cell autophagy function；The ambroxol is as the acute and chronic branch gas of clinical treatment The common drug of Guan Yan and bronchial asthma, moreover it is possible to alleviate the postoperative clinical symptoms of patients with lung cancer row lobectomy of lungs, toxic side effect It is small, it is curative for effect, ambroxol and paclitaxel plus are had good prospects using the clinical conversion for the treatment of tumour.

In the present invention, the taxol of the ambroxol and various concentration of investigating various concentration using mtt assay is incubated for tumour jointly Toxicity after cell, as shown in Figure 1, taxol gradually increases the lethal effect of tumour cell with the increase of ambroxol concentration By force, the ambroxol (100 μM) of high concentration makes the IC of taxol in 48h₅₀Value reduces by 82.79 times, the results show that ambroxol improves The sensibility of taxol killing tumor cell, and show concentration dependent and time dependence.

Table 1 is the IC that various concentration ambroxol and taxol are incubated for taxol after A549 cell 24,48,72h jointly₅₀Value, The result shows that ambroxol can obviously reduce the IC of taxol₅₀IC after value, 100 μM of ambroxols and taxol are incubated for for 24 hours altogether₅₀Value from 107.00 ± 4.38 are down to 1.49 ± 0.13ng/mL, and the ambroxol of high concentration makes IC₅₀Value reduction becomes apparent from.

Table 1

In the present invention, with Annexin V-FITC/PI apoptosis detection kit detection various concentration ambroxol from it is different Level of Apoptosis after the taxol of concentration is incubated for jointly evaluates influence of the ambroxol to taxol induced apoptosis, such as Fig. 2 institute Show, 50 μM of ambroxol makes the taxol apoptosis rate of 0.01 μ g/mL increase to 54.7% from 27.4%, as a result with MTT experiment compared with It is identical, the results show that ambroxol can improve the ability of taxol induced apoptosis of tumor cells.

In the present invention, establishing lotus to have the nude mice of A549 subcutaneous tumors is animal model, gives ambroxol, Japanese yew by vein The different dosings groups such as alcohol, ambroxol joint taxol, using gross tumor volume, nude mice weight, inhibition rate of tumor growth as metrics evaluation The antitumor curative effect that ambroxol and paclitaxel plus use, carries out internal pharmacodynamic evaluation, Fig. 3,4 show the ammonia of 20mg/kg The taxol of bromine rope and 3mg/kg can enhance subcutaneous tumors inhibitory effect after sharing, and the purple of the ambroxol of 100mg/kg and 3mg/kg China fir alcohol can significantly increase subcutaneous tumors inhibitory effect after sharing, and antitumous effect is better than the taxol of 10mg/kg；Table shown in table 2 Bright Ax 100mg/kg group inhibition rate of tumor growth is that 3.54%, PTX 3mg/kg is 33.79%, PTX 3mg/kg+Ax 20mg/ It is 68.66%, PTX 3mg/kg+Ax 100mg/kg group is 75.48% that kg group, which is 42.23%, PTX 10mg/kg group, as a result table Bright, ambroxol can enhance the ability that taxol inhibits subcutaneous tumors growth, improve antitumor curative effect, and dosage more high effect is better.

Table 2 is weight, tumor weight and the Tumor growth inhibition of the nude mice of different dosing group in A549 subcutaneous tumors model Rate.The inhibitory effect of 3mg/kg PTX+100mg/kg Ax is better than 10mg/kg PTX, and toxicity is smaller.

Table 2

In the present invention, establishing lotus to have the nude mice of A549 lung tumors is animal model, by nude mice tail vein injection A549- Luciferase cell constructs A549-luciferase Pulmonary metastases model, and skill is imaged using small animal living body after administration Art evaluates different time points lung tumor growth situation.Using nude mice weight, life cycle as index, ambroxol and taxol are studied Be used in combination the therapeutic effect for lung tumors, Fig. 5 show living imaging as a result, show the ambroxol of 20mg/kg with The taxol of 3mg/kg can reduce the expansion of lung tumors lesion after sharing, and the Japanese yew of the ambroxol of 100mg/kg and 3mg/kg Alcohol is significantly reduced the expansion of lung tumors lesion after sharing, and antitumous effect is better than the taxol of 10mg/kg；Fig. 6 is shown Each group median survival interval is 25,27.5,30,33,37.5,44 days, shows that ambroxol can be obviously prolonged lotus lung tumors nude mice Life cycle, and high dose effect becomes apparent, the results showed that, ambroxol can enhance the energy that taxol inhibits lung tumor growth Power, improves antitumor curative effect, and dosage more high effect is better.

The present invention shows that ambroxol can have with the expression quantity of the western bolt detection significant albumen LC3 of autophagy, Fig. 7 Effect regulating cell autophagy as a result, and show concentration dependent and time dependence, 50 μM, 8h can effectively influence autophagy, when Concentration is 300 μM, and the time is that effect is the most obvious when for 24 hours, shows 50 μM of ambroxol and the Japanese yew of 0.01 μ g/mL shown in Fig. 8 Autophagy level is further up after alcohol combination, and LC3 expression quantity is further up.

Ambroxol of the present invention can be used for preparing in tumor chemotherapeutic drug Synergistic preparations；The chemotherapeutics includes Taxol, docetaxel, adriamycin, cis-platinum, camptothecine etc., the tumour include non-small cell lung cancer, breast cancer, gastric cancer, Liver cancer, melanoma etc..

Detailed description of the invention

Fig. 1 is that cell is deposited after various concentration ambroxol and various concentration taxol are incubated for A549 cell 24,48,72h jointly The ability of taxol killing tumor cell is remarkably reinforced in motility rate, display ambroxol, and the ambroxol reinforcing effect of high concentration becomes apparent from.

Fig. 2 is that ambroxol and taxol are incubated for Level of Apoptosis after A549 cell, 100 μM of ambroxols and taxol jointly Apoptosis rate rises to 54.7% from 27.4% after being incubated for for 24 hours altogether.

Fig. 3 is different dosing group tumor volume change curve in A549 subcutaneous tumors model.Ambroxol can enhance taxol suppression The ability of subcutaneous tumors growth processed, and the effect that dosage is 100mg/kg becomes apparent.

Fig. 4 is in A549 subcutaneous tumors model, and the 14th day tumour is stripped rear photo after administration.

Fig. 5 is lung tumors size variation after the treatment of different dosing group in A549-luciferase lung tumors model. The ability that taxol inhibits lung tumor growth can be remarkably reinforced in ambroxol, and the effect that dosage is 100mg/kg becomes apparent.

Fig. 6 is the life span ratio of nude mice after the treatment of different dosing group in A549-luciferase lung tumors model Compared with.Ambroxol joint taxol can be obviously prolonged the life span of lotus lung tumors nude mice, and dosage is the effect of 100mg/kg More preferably.

Fig. 7 is the influence (A: the influence of concentration that ambroxol expresses autophagy GAP-associated protein GAP LC3；B: the influence of time), ammonia Bromine rope can be obviously promoted the rising of LC3 expressing quantity, show time-and concentration-dependent, 50 μM, 8h can Effective Regulation Autophagy；When concentration is 300 μM, the time is that effect is the most obvious when for 24 hours.

Fig. 8 is the influence after taxol and ambroxol combination to A549 cell autophagy albumen LC3 expression.Ambroxol and Japanese yew After alcohol is total to incubated cell, LC3 expression quantity is further up.

Fig. 9 is cell autophagy access and intervention schematic diagram.

Specific embodiment

It elaborates below with reference to embodiment to the present invention, but protection scope of the present invention is not limited to following implementations Example.

Embodiment 1:

The toxicity of A549 cell is incubated for using the paclitaxel plus ambroxol of mtt assay evaluation various concentration:

Taxol is configured to 0.001,0.01,0.05,0.1 μ g/mL with 1640 culture mediums containing 10% serum before experiment Series of concentrations, suitable ambroxol is added in the paclitaxel solution of 0.001,0.01,0.05,0.1 μ g/mL, makes final concentration Respectively 0,20,50,100 μM；

The cell of logarithmic growth phase is inoculated in 96 orifice plates with the density of 8000cells/well, after cell is adherent, It gives by the above experimental concentration containing drug solns, every 100 μ L of hole, each concentration is arranged three multiple holes, is placed in cell plates after administration It is cultivated for 24 hours in 37 DEG C of incubators, after cultivating, culture solution is gently sucked out, the MTT solution (PBS of 0.5 μ g/mL is added in every hole Prepare) 100 μ L, it is placed in 37 DEG C of incubators and cultivates 4h, culture medium is discarded after incubation, 200 μ LDMSO solution are added in every hole, It is protected from light in 37 DEG C and is incubated for 15min, measure absorbance at 570nm with microplate reader.Using the groups of cells of not dosing as control, with nothing The hole of cell but same treatment calculates cell survival rate as blank group as follows:

It calculates half using Logistic models fitting dose-effect relationship by 5.0 software of Graphpad Prism and inhibits Concentration IC₅₀Value.

Embodiment 2:

It is incubated for using the paclitaxel plus ambroxol of Annexin V-FITC/PI apoptosis detection kit evaluation various concentration The level of apoptosis of A549 cell: taxol is configured to the dense of 0.01 μ g/mL with 1640 culture mediums containing 10% serum before experiment Degree, is added suitable ambroxol in the paclitaxel solution of 0.01 μ g/mL, makes final concentration of 50 μM.By the thin of logarithmic growth phase Born of the same parents are inoculated in 6 orifice plates with the density of 50000cells/well, after cell is adherent, give blank training by the above experimental concentration Base, 0.01 μ g/mLPTX, 50 μM of Ax, 0.01 μ g/mLPTX+50 μM Ax solution, every hole 2mL are supported, each concentration is arranged three again Hole.Cell plates are placed in 37 DEG C of incubators after administration and are cultivated for 24 hours, after cultivating, culture supernatants is collected, is washed with PBS Cleaning solution is collected after washing 3 times, and 0.25% pancreatin without EDTA in right amount is added and digests, and closes with culture supernatants and cleaning solution And cell is collected by centrifugation, with PBS respin 2 times.Take 0.5-1 × 10⁵The Annexin V-FITC/PI of 200 μ L is added in a cell Dispel mixing cell in conjunction with liquid, be protected from light be added 5 μ L Annexin V-FITC and 5 μ L propidium iodides (Propidium Iodide, PI it) dyes, carries out flow cytomery after being placed at room temperature for 15min.

Embodiment 3:

Using the nude mice of lotus A549 subcutaneous tumors as model, using gross tumor volume, tumor weight, life cycle as index, evaluation PTX with The therapeutic effect of anticancer drug can be improved after Ax combination:

By A549 cell routinely CMC model, when the degrees of fusion to be grown to 80%-90%, digestion centrifugation is simultaneously with PBS weight It is outstanding, preparation 2 × 10⁷The single cell suspension of/mL, the right axillary for being seeded to BALB/c nude mice is subcutaneous, every 200 μ L.To tumour growth To 50-100mm³When (be denoted as 0 day), tumor-bearing mice is randomly divided into 6 groups, every group 6, sets physiological saline group respectively (saline), high dose ambroxol group (Ax:100mg/kg), low doses of paclitaxel group (PTX:3mg/kg), low doses of paclitaxel+ Low dosage ambroxol group (PTX:3mg/kg+Ax:20mg/kg), high dose taxol group (PTX:10mg/kg), low dosage Japanese yew Alcohol+high dose ambroxol group (PTX:3mg/kg+Ax:100mg/kg) was administered in the 0th, 4,8,12 day, and evaluation index is as follows:

(1) tumor growth curve: every two days measurement tumour major diameter a and minor axis b, according to formula V=0.5 × a × b²The volume of tumour is calculated, and draws tumor growth curve；

(2) changes of weight curve: every two days weighing nude mice weight, and draw changes of weight curve.If mouse weight drops It is low more than 15%, then it is assumed that dosage regimen is more toxic；

(3) inhibition rate of tumor growth (IRT%): nude mice is put to death in fortnight, tumour is removed, takes pictures and weigh tumour weight Amount calculates Tumor growth inhibition percentage as follows:

Wc is the knurl weight of saline control group, and Wt is the knurl weight of each treatment group；

(4) tumor tissues LC3 expression quantity: the expression quantity of immunohistochemistry measurement tumor tissues LC3 albumen is utilized；

(5) tumor tissues Tunel dyeing detection apoptosis: the apoptosis water of the method measurement tumor tissues of Tunel dyeing is utilized It is flat.

Embodiment 4:

By A549-luciferase cell routinely CMC model, when the degrees of fusion to be grown to 80%-90%, digest from The heart is simultaneously resuspended with PBS, preparation 2 × 10⁷The single cell suspension of/mL, tail vein injection is seeded to BALB/c nude mice, every 200 μ L.Lung tumor growth situation is observed using living imaging, (is denoted as 0 day) when lung tumor growth suitable size, lotus knurl is small Mouse is randomly divided into 6 groups, every group 6, sets physiological saline group (saline), high dose ambroxol group (Ax:100mg/kg), low respectively Dosage taxol group (PTX:3mg/kg), low doses of paclitaxel+low dosage ambroxol group (PTX:3mg/kg+Ax:20mg/kg), High dose taxol group (PTX:10mg/kg), low doses of paclitaxel+high dose ambroxol group (PTX:3mg/kg+Ax:100mg/ Kg), it was administered in the 0th, 4,8,12 day, evaluation index is as follows:

(1) tumor growth curve: utilizing bioluminescence imaging technology, lower observation lung tumors is imaged in small animal living body within every four days Size, and draw tumor growth curve；

(3) life cycle: the death time of record each group tumor-bearing mice, 5.0 Software on Drawing of GraphPad Prism is utilized Survivorship curve calculates and compares the mean survival time (MST) of each group.

Embodiment 5:

The significant albumen of LC3 autophagy after being co-cultured for 24 hours with the ambroxol and A549 of western bolt detection various concentration Expression quantity, detection ambroxol and A549 co-culture different time after LC3 albumen expression quantity, detect 50 μM ambroxol with Being incubated for A549 cell after the taxol combination of 0.01 μ g/mL for 24 hours influences the expression quantity of LC3 albumen.

Claims

1. ambroxol purposes in preparing tumor chemotherapeutic drug Synergistic preparations, its chemical name is trans- -4- for the ambroxol [(2- amino -3,5- dibromo-benzyl) amino] cyclohexanol, the entitled Ambroxol of English, general entitled ambroxol.

2. purposes according to claim 1, which is characterized in that the chemotherapeutics is selected from taxol, docetaxel, Ah mould Element, cis-platinum or camptothecine.

3. purposes according to claim 1, which is characterized in that the tumour be non-small cell lung cancer, breast cancer, gastric cancer, Liver cancer or melanoma.

4. by purposes described in claims 1 or 2 or 3, which is characterized in that the ambroxol and taxol, docetaxel, Ah Mycin, cis-platinum or camptothecine are used in combination, for enhancing killing tumor cell such as, Lines A549, human milk The effect of gland cell system MCF-7 or mouse melanin tumor cell system B16F10.

5. by purposes described in claims 1 or 2 or 3, which is characterized in that the ambroxol enhancing taxol suppression of the low concentration The ability of subcutaneous tumors growth processed, the ambroxol enhancing taxol of high concentration reduce tumour growth to the lethal effect of tumour cell Speed.

6. by purposes described in claims 1 or 2 or 3, which is characterized in that the ambroxol of the low concentration makes tumor bearing nude mice Pulmonary lesions become smaller, and the ambroxol of high concentration inhibits the growth of the pulmonary lesions of tumor bearing nude mice, extend the existence of Lung metastases nude mice Time reduces chemical therapy toxic side effect, improves life quality.

7. by purposes described in claims 1 or 2 or 3, which is characterized in that the ambroxol is used for modulate tumor cell A549, MCF-7, B16F10 autophagy.