CN107582565A - A kind of medicine of targeted therapy of lung cancer and its application - Google Patents

Abstract

The invention discloses a kind of medicine of targeted therapy of lung cancer and its application.Inventor is by studying for a long period of time, find that aluminum sulfate can differentiate identification human normal tissue and cancerous tissue first, change cancer cell physiological characteristic, suppress cancer cells secrete multiple proteins hydrolase, suppress the metabolic function of cancer cell mitochondria, so as to realize antitumor action, the medicine can be incorporated into the DNA of cancer cell, promote its cracking, quickly strong convergence it can condense cell surface glycoprotein, effectively change the protein structure of cell surface, disabling signal path, change Cell microstructure, and combined with succinate dehydrogenase in cell mitochondrial, effectively control cancer cell multiplication and transfer, 99.8~99.9% or even 100% are up to the inhibiting rate of cancer cell.Aluminum sulfate Small side effects, safe, oral medication lung cancer has the advantages that dosage is micro-, rapid-action, short treating period, can reduce knurl body, apoptosis-induced, Inhibit proliferaton rapidly, final fragmentation comes off, and capturing lung cancer to the mankind has important practical significance.

Description

A kind of medicine of targeted therapy of lung cancer and its application

Technical field

The present invention relates to a kind of new opplication of compound, the medicine of more particularly to a kind of targeted therapy of lung cancer and its application.

Background technology

According to national tumour enrollment results analysis in 2013, the Cancer in China incidence of disease was 2,35/,100,000, lung cancer and breast cancer Man, women morbidity first place are occupied respectively, and the Cancer in China incidence of disease is in rising trend over 10 years.

2012 the whole nation 18 years old and above adult hypertension illness rate be 25.2%, diabetes prevalence 9.7%, with Compare within 2002, illness rate is in rising trend.40 years old and above crowd's chronic obstructive pulmonary disease illness rate are 9.9%.

The national residents chronic disease death rate is 5,33/,100,000 within 2012, accounts for the 86.6% of total death toll.Cardiovascular and cerebrovascular diseases, Cancer and chronic respiratory disease are underlying cause of death, account for the 79.4% of total death, and the wherein cardiovascular and cerebrovascular diseases death rate is 271.8/10 ten thousand, cancer mortality is that 144.3/10 ten thousand (first five position is lung cancer, liver cancer, stomach cancer, cancer of the esophagus, Colon and rectum respectively Cancer), the chronic respiratory disease death rate is 68/,100,000.After markization processing, except a few diseases such as coronary heart disease, lung cancer are dead Rate is died to have risen outside.

The treatment method of lung cancer mainly has following several：

(1) chemotherapy

Chemotherapy is the primary treatments of lung cancer, and more than 90% lung cancer needs to receive chemotherapeutic treatment.Chemotherapy is to cellule The effect of lung cancer, no matter early stage or late period relatively affirmed that or even the early stage ED-SCLC for having about 1% is cured by chemotherapy.Chemotherapy And the Main Means for the treatment of non-small cell lung cancer, the Tumor response rate of chemotherapeutic treatment non-small cell lung cancer is 40%~50%. Chemotherapy can not typically cure non-small cell lung cancer, can only extend survival of patients and quality of making the life better.Chemotherapy is divided into therapeuticization Treatment and adjuvant chemotherapy.Chemotherapy need to select different chemotherapeutics and different chemotherapy sides according to cancerous lung tissue type is different Case.Chemotherapy also has infringement to human normal cell, therefore chemotherapy needs to refer in cancer department doctor in addition to it can kill tumour cell Lead lower progress.In recent years effect of the chemotherapy in lung cancer has been no longer confined to inoperable Patients with Advanced Lung Cancer, and frequently as whole body Treatment is included in the comprehensive therapeutic plan of lung cancer.Chemotherapy can suppress medulla hematopoietic system, mainly leucocyte and hematoblastic decline, Granulocyte colony stimulating factor and blood platelet stimulating factor treating can be applied.Chemotherapy is divided into therapeutic chemotherapy and complementaryization Treat.

(2) radiotherapy

1. principle of reatment

Radiotherapy is optimal to ED-SCLC curative effect, and squamous cell carcinoma takes second place, and gland cancer is worst.Radiotherapy in lung cancer irradiation field should include Primary tumor, the mediastinum area of lymphatic metastasis.To be aided with drug therapy simultaneously.Squamous cell carcinoma sensitivity isocratic in having to ray Property, for lesion based on local Invasion, transfer is relatively slow, therefore multi-purpose radical cure treatment.Gland cancer is poor to radiation sensitive, and easy blood Road shifts, therefore less uses radiotherapy alone.Radiotherapy is a kind of local treatment, it is often necessary to combined chemotherapy.Radiotherapy and chemotherapy Joint can regard patient situation it is different, take Synchronous chemoradiotherapy or the alternately method of chemoradiotherapy.

2. the classification of radiotherapy

According to the purpose difference for the treatment of be divided into radical cure treatment, palliative treatment, preoperative neoadjuvant radiotherapy, postoperative adjuvant radiotherapy and Intracavitary radiotherapy etc..

3. the complication of radiotherapy

The complication of radiotherapy in lung cancer includes:Radiation pneumonitis, radiation esophagitis, radiation fibrosis of lung and radioactivity ridge Marrow is scorching.There is positive correlation in above-mentioned radiotherapy related complication, while there is also individual difference with Radiotherapy dosimetry.

(3) surgical intervention of lung cancer

Surgical intervention is the preferred and most important treatment method of lung cancer, and the only treatment method that can cure lung cancer. The purpose of surgical operation therapy lung cancer is：

Excision lung cancer primary lesion and metastatic lymph node completely, reach clinical cure；

The overwhelming majority of tumor resection, creates favorable conditions for other treatment, that is, subtracts knurl operation；

Palliative surgery：It is suitable for a few patients, such as intractable pleural cavity and hydropericardium, by cutting off pleura and pericardium kind Plant tubercle, cut-out pericardium and pleura, cure or alleviate clinical symptoms caused by pericardium and pleural effusion, extending life or Make the life better quality.Palliative surgery need to make locally and systemically chemotherapy simultaneously.Surgical operation therapy usually needs in the preoperative or postoperative work NACT, radiotherapy in the treatment, to improve the survival rate of the cure rate of surgical operation and patient.Survive 5 years of surgical treatment for lung cancer Rate is 30%~44%；The death rate 1%~2% of surgical operation therapy.The surgical intervention of operation will also be by various limitations.

Existing treatment method is with high costs, course for the treatment of length, less effective.Therefore, it is badly in need of a kind of medicine for treating lung cancer at present Thing is to solve the matter of great urgency of cancer patient.

Aluminum sulfate is a kind of common sulfate, the precipitating reagent in paper industry as sizing materials such as gum rosin, wax emulsions, Make flocculant in water process, can also make the interior of foam annihilator and stay agent, the white raw material of manufacture alum, aluminium, oil decolourizes, deodorization Agent, the auxiliary material etc. of some drugses.The the first big purposes of aluminum sulfate for constituting about total output 50% is to be used for papermaking, and second largest purposes is Flocculant is made in drinking water, industrial water and Industrial Wastewater Treatment, constitutes about aluminum sulfate total output 40%.When into this kind of water After adding aluminum sulfate, can generate glue, can adsorb and be settled out bacterium, the aluminium hydroxide of colloid and other suspensions wadding Piece, the color and taste of water are can control in drinking water treatment.

In terms of medicine, aluminum sulfate also has a small amount of application, and such as all states are burned, and Wu Shurong, Chen Jinyun, wait compound aluminum sulfates Prescription research [J] liberation medical academy journals of solution, 1981 (2) disclose intratumoral injection tumor of bladder achieve compared with The effect of good, to the cure rate of early stage tumor of bladder up to 91.1%.Later stage is further investigations have shown that compound aluminum sulfate injection There is certain therapeutic action to tumor of bladder, but subsequent 30 years for many years, have no that aluminum sulfate can be used for treating other Tumour.

The content of the invention

It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of medicine of targeted therapy of lung cancer and its answer With.

The technical solution used in the present invention is：

A kind of targeted drug for treating lung cancer, its active component include aluminum sulfate.

As the further improvement of said medicine, aluminum sulfate is aluminum sulfate more than injection stage, or pure with the following method Change obtains：

1) by aluminum sulfate and ultra-pure water mixed dissolution, aluminum sulfate solution is obtained；

2) refined filtration is carried out to aluminum sulfate solution, freeze-drying obtains aluminum sulfate.

As the further improvement of said medicine, medicine is ejection preparation.Further, ejection preparation is selected from injection powder pin Agent, parenteral solution.

As the further improvement of said medicine, the mass mixing ratio of aluminum sulfate and ultra-pure water is 1：(2~5), after dissolving The ultra-pure water purifying used.Further, the mass mixing ratio of aluminum sulfate and ultra-pure water is 1：3, the quality such as use after dissolving Ultra-pure water purifies.

As the further improvement of said medicine, refined filtration is carried out to aluminum sulfate solution using the filter membrane that aperture is 0.22 μm.

As the further improvement of said medicine, aluminum sulfate is selected from the hydrate of aluminum sulfate.

The present invention captures after the research of many decades, and finds " aluminum sulfate " first, can differentiate identification human normal tissue With cancerous tissue, its cancerous tissue is voluntarily come off from normal structure separation, while can differentiate and identify normal cell and cancer cell and select Selecting property dies of hunger the breakthrough first of cancer cell.It is that the most fast most definite and low toxicity of the treatment curative effect to malignant tumour is efficient so far, There is no inhibitory action substantially to normal cell, cancer cell can be killed rapidly and reduce knurl body generation primacy chemical drug, its curative effect is far excellent Traditional anti-cancer medicine at present, its birth can save ten hundreds of patient vitals, and the malignant tumour that is particularly suitable for use in solid carcinoma is controlled Treat.

Inventor is after many decades and puts into huge fund and is studied, and researches and develops world forefront primacy series anticancer of successfully having Special efficacy new drug, its curative effect are treatment of the century-old mankind so far for malignant tumour solid carcinoma, chemicotherapy, operation and modern any controlled What treatment means can not be realized, malignant tumour solid carcinoma can upon administration gradually voluntarily separated from human normal tissue and comes off.

Pharmacology：The critical path of Apoptosis is as follows：(1), cancer cell physiological characteristic can be changed.(2), cancer cells secrete is suppressed Multiple proteins hydrolase.(3), the metabolic function of cancer cell mitochondria is suppressed, so as to realize antitumor action.(4), the medicine energy It is attached in the DNA of cancer cell, promotes its cracking.(5), can quick strong convergence cohesion cell surface glycoprotein.(6), effectively control Cancer cell multiplication and transfer.(7), the medicine can effectively change the protein structure of cell surface.(8), disabling signal path.(9), change Become Cell microstructure.(10) and with succinate dehydrogenase in cell mitochondrial combined so as to change its biological effect.(11), because normal Otherwise cell sucks regular this irregular characteristic of cancer cell.(12), the compound can identify differentiate normal structure with it is improper Tissue, normal cell do not suppress normal cell with abnormal cell and selectively hungry to death and killing cancer cell so that malignant tumour The physianthropy important breakthrough that fragment comes off gradually is presented in entity cancerous tissue, 99.8~99.9% is up to tumor control rate, very To reaching 100%.

Toxicity：It is heavy dose of to have no obvious toxic-side effects through many decades research and test.

Drug effect：There are micro- dosage, rapid-action, short treating period, Small side effects, treating above-mentioned malignant tumour can contract rapidly Tubercle body, apoptosis-induced, Inhibit proliferaton.

Usage and dosage：Such as following table：

Sequence number	Gross tumor volume (cm3)	The sodium chloride injection of pharmaceutical quantities × 0.9%	Injecting method
				1	Solid carcinoma 2 × 2 × 2	1000mg×3mL	Local injection
2	Solid carcinoma 4 × 4 × 4	2000mg×6mL	Local injection
				3	Solid carcinoma 6 × 6 × 6	3000mg×9mL	Local injection
4	Solid carcinoma 8 × 8 × 8	4000mg×12mL	Local injection
				5	Solid carcinoma 10 × 10 × 10	5000mg×15mL	Local injection
6	Solid carcinoma 12 × 12 × 12	6000mg×18mL	Local injection

Note：The drug effect time is 3~5 minutes, and cancer cell starts tune occur to die, and prohibits after injection in 25~30 minutes Only walk about, avoid drug effect from losing.

Function is with curing mainly：Lung cancer.

Cell experiment as shown by data aluminum sulfate has obvious inhibitory action to 6 kinds of human lung carcinoma cell propagation, its IC₅₀Respectively For NCI-H446 (10.696mg/ml), NCI-H460 (6.822mg/ml), A549 (22.265mg/ml), NCI-H226 (7.500mg/ml), NCI-H596 (9.258mg/ml) and NCI-H292 (10.047mg/ml)；Have to 6 kinds of human lung carcinoma cells Obvious apoptosis-promoting effect, and there is concentration-effect relation；Can be by the cell-cycle arrest of 6 kinds of human lung carcinoma cells in G₀/G₁Phase, And there is concentration-effect relation.

By injecting aluminum sulfate preparation for treating lung cancer, there is micro- dosage, rapid-action, short treating period, Small side effects, energy It is rapid to reduce knurl body, apoptosis-induced, Inhibit proliferaton, as its malignant tumour volume of early detection 2cm × 2cm × 2cm to 3cm × 3cm × 3cm, can be achieved that only a pin need to be entered, 36h to 72h activity of tumor cells all disappears, the mankind are captured lung cancer have it is important Realistic meaning.

Brief description of the drawings

Fig. 1 is the HPLC chromatogram of aluminum sulfate after purification；

Fig. 2 is the pets of acute toxicity testing；

Fig. 3 is concentration-inhibiting rate curve of the aluminum sulfate to different lung carcinoma cells；A, B is respectively NCI-H446, NCI-H460 The concentration of cell-inhibiting rate curve；

Fig. 4 is concentration-inhibiting rate curve of the aluminum sulfate to different lung carcinoma cells；A, B is respectively A549, NCI-H226 cell Concentration-inhibiting rate curve；

Fig. 5 is concentration-inhibiting rate curve of the aluminum sulfate to different lung carcinoma cells；A, B is respectively NCI-H596, NCI-H292 The concentration of cell-inhibiting rate curve；

Fig. 6 and Fig. 7 is influence of the aluminum sulfate to proliferation of lung cancer cells, arrow instruction for non-viable non-apoptotic cell.Fig. 4 is aluminum sulfate Influence to proliferation of lung cancer cells, arrow represent non-viable non-apoptotic cell；

Fig. 8~Figure 13 is aluminum sulfate respectively to human lung carcinoma cell NCI-H446, NCI-H460, A549, NCI-H226, NCI- The influence of H596 and NCI-H292 apoptosis；

Figure 14 and Figure 15 is influence of the aluminum sulfate to Increase Apoptosis of Lung Cancer Cells, the nucleus shrinkage of arrow instruction, prompts for withering Die cell；

Figure 16~Figure 21 be aluminum sulfate respectively to human lung carcinoma cell NCI-H446, NCI-H460, A549, NCI-H226, The influence in NCI-H596 and NCI-H292 apoptosis cycles.

Embodiment

Confirm the testing data of chemical constitution

Experiment commission Hunan Province's Experimental Animal Center (Drug Safety Evaluation Center of Hunan Province's progress).

First, new drug title, molecular formula and molecular weight

Chinese name：Aluminum sulfate

Molecular formula：Al₂(SO₄)₃·18H₂O, molecular weight：666.4.

2nd, the method for confirming chemical constitution

1st, determination of moisture

1.1 test condition：

Instrument：V-30 cassette moisture tellers

Method：《Chinese Pharmacopoeia》The first method of aquametry of four general rules of version in 2015 0832.

1.2 measurement result

Moisture measurement result in table 1, aluminum sulfate

1.3rd, parse

Calculated according to sulfuric acid constructed of aluminium and moisture, the number containing the crystallization water is 18 in structure.

The measure of aluminium element

2.1st, test condition

Instrument：Agilent 240-DUO original absorption spectrometers

Method：Using graphite oven atomic absorption, aluminium element content in aluminum sulfate sample is determined.

2.2nd, test result

Aluminium element test result in table 2, aluminum sulfate sample

As a result show：The mean percent content of aluminium element is 8.15% in aluminum sulfate sample.

2.3rd, parse

According to above-mentioned determination of moisture, calculated by 18 crystallizations water, the ratio that aluminium element accounts for molecular weight is 8.11%, and above-mentioned The aluminium element content of atomic absorption detecting is consistent, further demonstrates that and contains aluminium element and 18 crystallizations water in sample.

The measure of sulfate ion

3.1st, test condition

Instrument：ICS900 ion chromatographs

Method：Using 25mM sodium hydroxides as leacheate, chromatographic column is Dionex IonpacTM As18 (4 × 250mm), Flow velocity is 1.0ml/min, sample size 25ul；Using anhydrous sodium sulfate as reference substance, calculated and contained with external standard method by peak area Amount.

3.2nd, test result

Sulfate radical content measurement result in table 3, aluminum sulfate

* it is actual concentrations.

After measured, it is 45.5% containing sulfate ion in sample.

3.3rd, parse

Sample chromatogram figure is consistent with reference substance chromatogram retention time, shows the sulfate radical containing high concentration in sample；Root According to moisture and aluminium element assay result, ion-chromatographic determination sulfate ion content is used as 43.4%, with molecule knot Sulfate ion molecular weight accounting is 43.2% basically identical in structure.

4th, conclusion

According to determination of moisture, aluminium element assay, sulfate radical content measurement result comprehensive analysis, this product molecular formula is Al₂(SO₄)₃·18H₂O。

, can be directly using the high-purity sulphuric acid aluminium (analysis of manufacturer production in order to thoroughly prevent three-waste free discharge to avoid purifying Pure or pharmaceutical grade), but have to carry out strict quality control from manufacturer source.

The purifying of aluminum sulfate

1) aluminum sulfate is pressed 1:3 mass ratio is dissolved in pure water；

2) with etc. the pure water of quality clean, refined filtration removal of impurities；

3) refined solution after refined filtration is freeze-dried to obtain the aluminum sulfate purified of fine white powder.

The HPLC chromatogram of aluminum sulfate is as shown in Figure 1 after purification, it is seen that the purity of aluminum sulfate after purification has before relatively purifying Larger raising.Being freeze-dried obtained aluminum sulfate has more preferable dissolubility.

With reference to experiment, technical scheme is further illustrated.

Safety experiment：

Experimental Animal Center experiment portion of safety experiment commission Zhongshan University is carried out, experimental design reference：

1. the tertiary cloud of Xu, the pharmacological experimental methodology third edition

2.2014 editions medicine safety pharmacology investigative technique guidelines.

Specific experiment method is as follows：

Experiment material

Aluminum sulfate, molecular formula：Al₂(SO₄)₃·18H₂O, molecular weight：666.4.

First, acute toxicity test

1. test objective：

Observe aluminum sulfate after single is given in certain time whether caused toxic reaction, for the preliminary poison for understanding medicine Property effect and its toxicity target organ, provide foundation for follow-up clinical test.

2nd, experimental animal and rearing conditions

SPF levels kunming mice 40,20 ± 2g, male and female half and half.Animal productiong supplying unit：In Zhongshan University experimental animal Heart production department, experimental animal production licence number are：SCXK (Guangdong) 2011-0029, animal quality verification of conformity： No.440085000 buys the date：On 08 15th, 2016；Animal identification method：With saturation picric acid by animal different parts Fur applies dye and represents different animals number, and different animal husbandry cages is made a distinction with animal feeding load card mark.Raising temperature 20~26 DEG C of degree；Humidity 40RH%~70RH%；Rate of ventilation：Receptacle is more than 15 times/hour；Stocking density：Group support, per cage Not more than 6.Use feed：The large and small mouse pellet of SPF levels, provided by Beijing Ke Ao Co., Ltds.Control group and administration group Pets are as shown in Figure 2.

3rd, experimental method：Mouse is randomly divided into control group and administration group, it is specific as follows：

Table 4, acute toxicity test packet and dosage

Group	Medicine	Dosage (mg/kg)	Capacity is administered	Size of animal
					Control group	Physiological saline	/	0.2ml/10g bw	10 female 10 heros
Administration group	Aluminum sulfate solution	2000	0.2ml/10g bw	10 female 10 heros

The compound method of aluminum sulfate solution is：Physiological saline 5ml, peace of the injection equipped with aluminum sulfate are accurately extracted with syringe In cuing open, mix 10~20min of standing and fully dissolve to obtain.

Method of administration：Gastric infusion.

Administration frequency and observing time：Gastric infusion 1 time, observation post administration 14 days.

Testing index：Clinical observation：The general symptom of observation animal daily；Measured body weight：In administration D0, D3, D7, D14 Weigh the weight of animals；Organ coefficient determines：In the 15th day put to death animal, anatomic observation main organs abnormal conditions, core, liver, 5 spleen, lung, kidney internal organs are weighed.

Result treatment and analysis：Handled with statistic software SPSS 24, calculate the average weight and organ coefficient of two groups of animals And make comparisons.

Experimental result：

During experiment, control group, administration group have no dead mouse and shown no obvious abnormalities.

Administration group the weight of animals is compared with control group, and there was no significant difference, refers to table 5.

The comparison of table 5, acute toxicity mouse weight

Organ coefficient statistical result refers to table 6.

The comparison of table 6, acute toxicity mice organs coefficient

* no significant difference (the P compared with control group<0.05) mouse all survives, and none is dead.

Conclusion：

Aluminum sulfate has preferable security, dosage 2000mg/kg, 0.2ml/10g bw0,2ml/10gbw physiology Salt solution, have no obvious toxic-side effects.

Lung cancer cell growth suppresses check experiment

Experiment commission Hunan Province's Experimental Animal Center (Drug Safety Evaluation Center of Hunan Province) is carried out.

In tests below, compound XL-012 refers to aluminum sulfate.

1st, experiment material

1.1 tested material：Compound is prepared：Aluminum sulfate 9g is weighed, adds 0.9% sodium chloride injection 15ml, and vibrate extremely All dissolvings, produce 600mg/ml aluminum sulfate solutions, and this is maximum concentration working solution, will be standby after mother liquor progress bacteria removing. Mother liquor is configured to 200 successively with 0.9% sodium chloride injection, 60,20,6,2,0.6mg/ml working solution.

1.2 positive control drug：Cis-platinum (DDP), lot number：SJJMI-IE, Tokyo HuaCheng Industry Co., Ltd；5 FU 5 fluorouracil (5-FU), lot number：HFBM160120325008, Amresco company.Positive control drug is prepared：DDP or 5-FU2mg is weighed, with new Fresh complete medium is configured to 100mM mother liquor, then mother liquor is configured to fresh complete medium 200 successively, 60,20,6, 2nd, 0.6 μM of working solution.

1.3 main material：

1.4 key instrument：

2nd, test method

2.1 cell culture

NCI-H446, NCI-H460, A549, NCI-H226, NCI-H596 and NCI-H292 cell covered with is taken, is used DMEM in high glucose complete medium containing 10%FBS, in 37 DEG C, 5%CO₂Cultivated in incubator, according to cell growth status, 1~ 2d is passed on or is changed liquid, standby to exponential phase.

2.2CCK-8 methods detect cell proliferation test

Take the logarithm the phase growth NCI-H446, NCI-H460, A549, NCI-H226, NCI-H596 and NCI-H292 cell With every hole 5 × 10³Individual cell is inoculated in 96 porocyte culture plates, after 12h cell attachments, sets vehicle control group, the positive Comparison medicine (DDP) group, positive control drug (5-FU) group, compound XL-012 groups (0.3-300mg/ml), every group of 5 multiple holes.It is molten Matchmaker's control group is with fresh DMEM culture medium incubated cells completely, and compound group is respectively with containing final concentration of 0.3-300mg/ml chemical combination The fresh DMEM incubated cells completely of thing, DDP and 5-FU groups are respectively with containing final concentration of 100,30,10,3,1,0.3 μM of compounds Fresh DMEM incubated cells completely.By the μ l of CCK-8 10 are added after above-mentioned processing mode incubated cell 72h in every hole, continue Measure the absorbance in each hole after culture 1h at 450nm using ELIASA.Using vehicle control group OD values as 100% cell viability, The ratio of remaining each group OD values and vehicle control group OD values is relative activity.Compound is evaluated to NCI- with cell proliferation inhibition rate The toxicity of H446, NCI-H460, A549 cell, if having cell proliferation inhibition rate ＞ 100%, the system for being judged to instrument is missed Difference, based on 100%.

The detection of 2.3 Apoptosis

2.3.1Annexin the double dye method detection Apoptosis of V-FITC and PI

Take NCI-H446, NCI-H460, A549, NCI-H226, NCI-H596 and NCI-H292 in exponential phase Cell, conventional digestion collects cell, with 5 × 10⁵The density in/hole is seeded in 6 orifice plates, after 12h cell attachments, sets solvent Control group, compound XL-012 groups (10,30,100mg/ml), every group of 5 multiple holes.Vehicle control group is trained with fresh DMEM completely Support base incubated cell, compound group respectively with containing final concentration of 10,30, the fresh DMEM completely of 100mg/ml compounds be incubated it is thin Born of the same parents.After 6h, conventional digestion collects cell, adds 500 μ l Binding Buffer buffer solutions and cell is resuspended, cell is transferred to In 1.5ml EP pipes, 5 μ l Annexin V-FITC, 5 μ l PI are added, 15min is incubated under the conditions of room temperature lucifuge, uses streaming Cell instrument detects apoptosis situation.

2.3.2 fluorescence colour detects Apoptosis

Cell is handled according to 2.3.1 methods, after compound handles 6h, adds Hoechst 33342 per hole in 6 orifice plates Dyeing liquor 1ml, fully covers cell, is placed in 37 DEG C of culture 20-30min.Dyeing liquor is discarded, glimmering after being washed 2-3 times with PBS Fluoroscopic examination is carried out under light microscope.

Influence of the 2.4 Flow cytometry compounds to the lung carcinoma cell cycle

Take NCI-H446, NCI-H460, A549, NCI-H226, NCI-H596 and NCI-H292 in exponential phase Cell, conventional digestion collects cell, with 5 × 10⁵The density in/hole is seeded in 6 orifice plates, after 12h cell attachments, sets solvent Control group, compound XL-012 groups (100,30,10mg/ml), every group of 5 multiple holes.Vehicle control group is trained with fresh DMEM completely Support base incubated cell, compound group respectively with containing final concentration of 10,30, the fresh DMEM completely of 100mg/ml compounds be incubated it is thin Born of the same parents.After 6h, conventional digestion collects cell, and the PI for overnight, adding 5 μ l is fixed with 70% cold ethanol, and room temperature lucifuge is incubated 30min, uses The flow cytomery cell cycle.

2.5 statistical analysis

Data are handled using the statistical softwares of SPSS 16.0, measurement data withRepresent, the ratio of two sample averages Examined compared with using Student T-Test, the comparison of mean is using One-way ANOVA inspections, P between multisample group<0.05 represents It is statistically significant, P<0.01 represents that examined difference is very significant.

3rd, evaluation of result

Influence of 3.1 aluminum sulfate to proliferation of lung cancer cells

After the compound processing cell of micro- Microscopic observation various concentrations, there is cell proliferation rate and slow down, cell fragment Increase, space between cells increases, phenomena such as shape of sand vacuole occurs in cell.Incubation time has clear and definite correlation to cell state together, About after 12h is co-cultured, being rounded occurs in cell, the phenomenon of shrinkage；After 24h is co-cultured, there is part cell and swell, cell is saturating Photosensitiveness is deteriorated, intercellular gap increase；After 48h is co-cultured, there is shape of sand vacuole in cell, the situations such as cell rupture occurs； After 72h is co-cultured, shape of sand vacuole like cell substantially completely ruptures, without complete under 300,100,30mg/ml concentration conditions The cell of cellular morphology, only see and be condensed into stain shape on a small quantity.Cell co-cultures with test sample or positive control drug DDP and 5-FU After 72h, cell propagation is substantially suppressed, and has concentration-effect relation, with significant difference compared with vehicle control group (P<0.01).Cell inhibitory effect is the results detailed in Table 7.

The influence of table 7, different compounds to proliferation of lung cancer cells

Note：* represent compared with vehicle control group, P<0.05, * * expressions are compared with vehicle control group, P<0.01.IC₅₀Represent Suppress the concentration of 50% tumour cell, Emax represents the maximal percentage inhibition to tumour cell.

Aluminum sulfate to concentration-inhibiting rate curve of lung carcinoma cell as seen in figures 3-5, Fig. 3 A, B be respectively NCI-H446 and NCI-H460 cells；Fig. 4 A, B are respectively A549 and NCI-H226 cells；Fig. 5 A, B are respectively NCI-H596 and NCI- H292 cells.

Influence of the aluminum sulfate to proliferation of lung cancer cells as shown in Figure 6 and Figure 7, arrow instruction for non-viable non-apoptotic cell.From figure It can be clearly seen that aluminum sulfate can promote lung carcinoma cell downright bad.

Influence of 3.2 aluminum sulfate to Increase Apoptosis of Lung Cancer Cells

Use concentration for 10,30, after 100mg/ml compound intervenes lung carcinoma cell 6h, collect cell, Annexin V- Apoptosis is detected after the double dyes of FITC/PI.Cell after double dyes can be divided into 4 groups by flow cytometer：Q1-UL represents mechanical damage Cell (Annexin V-/PI+)；Q1-UR non-viable apoptotic cells (Annexin V+/PI+)；Q1-LL survivaling cells (Annexin V-/PI-)；Q1-LR viable apoptotic cells (Annexin V+/PI-).Experimental result is as shown in table 8：

The influence of table 8, aluminum sulfate to human lung carcinoma cell apoptosis

Note：* represent compared with vehicle control group, P<0.05, * * expressions are compared with vehicle control group, P<0.01.

As a result show：Through compound XL-012 handle NCI-H446 cells late apoptic and non-viable non-apoptotic cell number apparently higher than Vehicle control group (P<0.05), and there is concentration-effect relation；The NCI-H460 cell late periods handled through compound XL-012 wither Die and non-viable non-apoptotic cell number is apparently higher than vehicle control group (P<0.05), and there is concentration-effect relation；At compound XL-012 The A549 early apoptosis of cells cell number of reason is apparently higher than vehicle control group (P<0.05), and there is concentration-effect relation；Through changing The NCI-H226 cells late apoptic and non-viable non-apoptotic cell number of compound XL-012 processing are apparently higher than vehicle control group (P<0.05), and With concentration-effect relation；The NCI-H596 cells late apoptic and non-viable non-apoptotic cell number handled through compound XL-012 is substantially high In vehicle control group (P<0.05), and there is concentration-effect relation；The NCI-H292 cells early stage handled through compound XL-012 Apoptosis cell is apparently higher than vehicle control group (P<0.05), and there is concentration-effect relation.Aluminum sulfate is to human lung carcinoma cell The influence of NCI-H446 apoptosis is as shown in Figure 5；In figure, A is vehicle control group, and B is aluminum sulfate 10mg/ml groups, and C is aluminum sulfate 30mg/ml groups, D are aluminum sulfate 100mg/ml groups；

Aluminum sulfate to high-transfer human lung carcinoma cell NCI-H446, NCI-H460, A549, NCI-H226, NCI-H596 and The influence of NCI-H292 apoptosis is respectively as shown in Fig. 8~Figure 13；In each figure, A is vehicle control group, and B is aluminum sulfate 10mg/ml Group, C are aluminum sulfate 30mg/ml groups, and D is aluminum sulfate 100mg/ml groups.

Influence of the aluminum sulfate to Increase Apoptosis of Lung Cancer Cells as shown in Figure 14 and Figure 15, the nucleus shrinkage of arrow instruction, is prompted For apoptotic cell.

Influence of 3.3 aluminum sulfate to the lung carcinoma cell cycle

Use concentration for 10,30, after 100mg/ml aluminum sulfate intervenes lung carcinoma cell 6h, cell is collected, with 70% cold second After alcohol is fixed, after PI dyeing, the flow cytometry analysis cell cycle, experimental result is as shown in table 9.

The influence of table 9, aluminum sulfate to the human lung carcinoma cell cell cycle

Note：* represent compared with vehicle control group, P<0.05, * * expressions are compared with vehicle control group, P<0.01.

As a result show：After aluminum sulfate processing, G₀/G₁Cell proportion substantially increases, G₂/ M phase ratios significantly reduce, and show it G mainly is arrested in by lung carcinoma cell to proliferation of lung cancer cells inhibitory action₀/G₁Phase, it is prevented to enter the S phases.

Aluminum sulfate is to NCI-H446, NCI-H460, A549, NCI-H226, NCI-H596 and NCI-H292 apoptosis cycle Influence respectively as shown in Figure 16~Figure 21；In each figure, A is vehicle control group, and B is aluminum sulfate 10mg/ml groups, and C is aluminum sulfate 30mg/ml groups, D are aluminum sulfate 100mg/ml groups.

In summary, under this experiment condition, aluminum sulfate can significantly inhibit 6 kinds of proliferation of lung cancer cells, and with concentration- Effect relation, under a high concentration condition, with the extension of time, can kill cancer cell completely, it can be by cell-cycle arrest In G₀/G₁Phase, inducing cell apoptosis.This compound performance suppression cancer cell concentration is higher, is mg/ml levels, may be distinctive with it Mechanism of action is related.The compound may directly act on tumor cell surface, can change the physiological characteristic of cancer cell, in cell After surface aggregation, it can effectively change the protein structure of cell surface, cause the albumen precipitation of cell surface and cytoplasm, lead Cause cell permeability degradation, space between cells is shunk, and reduces the splitting ability of tumour cell, effectively control the propagation of cell with And transfer.After compound enters into the cell, cancer cells secrete multiple proteins hydrolase can be suppressed, it is thin cancer can be bonded directly to In the DNA of born of the same parents, cause DNA cracking, and effectively suppress the metabolic function of cancer cell mitochondria, with reference to the amber in mitochondria Acidohydrogenase, change its biological effect, cause Cell microstructure to change, disabling signal path, disturb the life of tumour cell Long metabolism, induced tumor Apoptosis, realizes the effect for killing cancer cell.

It is recommended that：

Finished as zoopery mouse tumor transplanting maturation is injected into pin administration, answer embolism to enter needle passageway or closing inserting needle Passage, while nursing must be singly only isolated, there are certain smell and the small bulge of appearance because drug injection finishes more or less, mouse is such as The nursing of fruit colony, which occurs, mutually baits and causes liquid medicine outflow to influence drug effect.As human tumor inserting needle does not need embolism inserting needle to lead to Road is closed into needle passageway.

Described above is only the preferred embodiment of the present invention, it is noted that for the common skill of the art For art personnel, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications Also within protection scope of the present invention.

Claims (10)

1. application of the aluminum sulfate in targeted therapy of lung cancer medicine is prepared.

2. application according to claim 1, it is characterised in that：The formulation of medicine is ejection preparation.

3. application according to claim 2, it is characterised in that：Ejection preparation is selected from injection powder injection, parenteral solution.

4. application according to claim 1, it is characterised in that：Aluminum sulfate is selected from the hydrate of aluminum sulfate.

A kind of 5. targeted drug for treating lung cancer, it is characterised in that：Its active component includes aluminum sulfate.

6. targeted drug according to claim 5, it is characterised in that：Aluminum sulfate is aluminum sulfate more than injection stage, or is made Purifying obtains with the following method：

1) by aluminum sulfate and ultra-pure water mixed dissolution, aluminum sulfate solution is obtained；

2) refined filtration is carried out to aluminum sulfate solution, freeze-drying obtains aluminum sulfate freeze-dried powder.

7. targeted drug according to claim 5, it is characterised in that：Medicine is ejection preparation.

8. targeted drug according to claim 7, it is characterised in that：Ejection preparation is selected from injection powder injection, parenteral solution.

9. targeted drug according to claim 6, it is characterised in that：The mass mixing ratio of aluminum sulfate and ultra-pure water is 1：(2 ~5) the ultra-pure water purifying, used after dissolving.

10. the targeted drug according to claim 6 or 9, it is characterised in that：Using the filter membrane that aperture is 0.22 μm to sulfuric acid Aluminum solutions carry out refined filtration.