CN103933046B - A kind of anti-pancreatic cancer medicament composition and its application, medicine box and package - Google Patents
A kind of anti-pancreatic cancer medicament composition and its application, medicine box and package Download PDFInfo
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- CN103933046B CN103933046B CN201310753515.4A CN201310753515A CN103933046B CN 103933046 B CN103933046 B CN 103933046B CN 201310753515 A CN201310753515 A CN 201310753515A CN 103933046 B CN103933046 B CN 103933046B
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- 230000002946 anti-pancreatic effect Effects 0.000 title claims abstract description 10
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- 239000000203 mixture Substances 0.000 title abstract description 11
- 229960002576 amiloride Drugs 0.000 claims abstract description 49
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- 239000005551 L01XE03 - Erlotinib Substances 0.000 claims abstract description 39
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Abstract
The invention belongs to biomedicine technical field, it is related to a kind of anti-pancreatic cancer pharmaceutical composition and application, medicine box and package.Described pharmaceutical composition, medicine box and package contain the epidermal growth factor tyrosine kinase inhibitor Tarceva for the amiloride and therapeutically effective amount of the therapeutically effective amount simultaneously, separately or sequentially used as combination formulations, wherein, the molar ratio of amiloride and Tarceva is 500:1~1:50.Pharmaceutical composition, medicine box and the package of the present invention is for that when treating tumour, can obtain the drug effect being used alone better than Tarceva, improve the effect of clinical chemotherapy.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a medicinal composition for resisting pancreatic cancer, and application, a medicinal box and a packaging piece thereof.
Background
According to statistical reports, the incidence of pancreatic cancer is rising year by year, in the fourth place of common malignant tumors in Europe and America and in the ninth place of death of malignant tumors in China. Pancreatic cancer is difficult to diagnose at early stage, 80% of patients are in middle and late stage when diagnosis is confirmed, only 10% -15% of patients can be subjected to radical operation, and the survival rate of 5 years after operation is only 15% -20%. Generally, patients with advanced pancreatic cancer have a survival time of less than 3 months and a 5-year survival rate of less than 1%.
Currently, drug therapy (including chemotherapy, molecular targeted therapy, traditional Chinese medicine and supportive symptomatic therapy) is an important treatment for advanced pancreatic cancer. Gemcitabine (GEM) is still the gold standard drug for clinical first-line chemotherapy for advanced pancreatic cancer, but its efficacy is far from satisfactory. With the rapid development of molecular biology and genomics, researches show that the occurrence, development and metastasis of pancreatic cancer are closely related to multiple gene mutations and the abnormality of cell signaling pathways, so that molecular targeted drugs alone or in combination with chemotherapy become a new research hotspot for treating pancreatic cancer. Currently, erlotinib (Tartarib) in EGFR-TKI is the only molecular targeted drug approved by FDA for treating advanced pancreatic cancer, but the survival benefit is still very limited, and large-scale clinical trials are needed to evaluate the curative effect.
Amiloride hydrochloride is a long-term clinical applicationThe oral potassium-protecting diuretic has the advantages of strong potassium-protecting and sodium-discharging effects, quick response, long action duration, light side effect, small dosage and the like, and has the effects of reducing blood pressure and improving cardiac function. Amiloride hydrochloride is combined with hydrochlorothiazide and furosemide, so that the amiloride hydrochloride can still be applied to patients with liver function damage due to no liver metabolism, and the clinical application safety is better. The amiloride has other target point inhibiting effects including Na for regulating pH value in tumor cells+-H+Exchanger1 (Na)+-H+exchanger1, NHE 1), the urokinase-type plasminogen activator (uPA) that mediates tumor migration, invasion and metastasis, thus having a certain anti-tumor effect; but the concentration of the in vitro anti-tumor effect is in the range of several millimoles, which is difficult to achieve in vivo; therefore, whether amiloride has the application prospect of resisting tumors in vivo and the deep mechanism description still need to be further researched.
To date, no report has been found on the combination of amiloride hydrochloride and erlotinib hydrochloride for the treatment of pancreatic cancer.
Disclosure of Invention
The invention aims to provide a new medicinal application of amiloride and derivatives thereof, and simultaneously provides a new medicinal composition for enhancing the curative effect and application of erlotinib serving as an anti-tumor medicament; in particular to a medicine composition for resisting pancreatic cancer, application thereof, a medicine box and a package.
The anti-pancreatic cancer pharmaceutical composition of the present invention comprises a therapeutically effective amount of amiloride and epidermal growth factor tyrosine kinase inhibitor (EGFR-TKI) Erlotinib as a combined preparation for simultaneous, separate or sequential use; the composition contains amiloride and erlotinib, and the molar ratio of the amiloride to the erlotinib is 500: 1-1: 50;
in the above pharmaceutical composition, amiloride may exist in the form of a base or a salt, including but not limited to hydrochloride, sulfate, etc.;
in the pharmaceutical composition, the derivatives having amiloride as the parent nucleus include, but are not limited to, amiloride Dimethyl (DMA), 5-N-ethyl-N-isopropyl amiloride (5-ethylisopropropylamiloride, EIPA), Methylisobutylamide (MIBA), and 5-N, N-cyclohexane amiloride (5-N, N-hexamethyl amide, HMA);
in the pharmaceutical composition, erlotinib may exist in the form of a base or a salt, including but not limited to hydrochloride, sulfate, and the like.
The pharmaceutical composition of amiloride and erlotinib can be used for preparing an anti-pancreatic cancer drug;
the pharmaceutical composition of amiloride and erlotinib contains amiloride and erlotinib in a molar ratio of 500: 1-1: 50; the amiloride in the pharmaceutical composition can exist in a basic form or a salt form, including but not limited to hydrochloride, sulfate and the like;
in the pharmaceutical combination of amiloride and erlotinib, amiloride as a parent nucleus derivative includes, but is not limited to, amiloride Dimethylamiloride (DMA), 5-N-ethyl-N-isopropyl amiloride (5-ethylisopropamide, EIPA), methyl isobutyl amiloride (MIBA) and 5-N, N-cyclohexane amiloride (5-N, N-hexamethylene amide, HMA) and the like;
the erlotinib may exist in base form or salt form including but not limited to hydrochloride, sulfate, and the like.
The invention also provides a kit for resisting pancreatic cancer, which comprises a first kit unit containing amiloride hydrochloride and a second kit unit containing erlotinib;
wherein the first kit unit containing amiloride hydrochloride comprises a clinically effective dose of amiloride hydrochloride and the second kit containing erlotinib comprises a clinically effective dose of erlotinib hydrochloride.
The invention also provides a package for packaging the anti-pancreatic cancer medicine, which comprises a first package unit containing amiloride and a second package unit containing erlotinib, wherein the package spaces of the first package unit and the second package unit are independent;
wherein the first packaging unit containing amiloride hydrochloride comprises a clinically effective dose of amiloride hydrochloride and the second packaging unit containing EGFR-TKI comprises a clinically effective dose of erlotinib hydrochloride.
In the present invention, the kit or the package further comprises instructions for providing the unit containing the clinically effective dose of amiloride hydrochloride and the unit containing the clinically effective dose of erlotinib hydrochloride as a preparation for combined use in anti-pancreatic cancer therapy.
The invention performs a cytotoxicity test of amiloride hydrochloride sensitization Erlotinib hydrochloride (Erlotinib) on human pancreatic cancer Bxpc-3 cells, an influence test of the combination of amiloride hydrochloride and Erlotinib hydrochloride on the cell cycle, and a Western Blot method for detecting the influence of amiloride hydrochloride and Erlotinib hydrochloride on EGFR and downstream STAT3, Akt and Erk phosphorylation thereof; results show that the amiloride hydrochloride and erlotinib hydrochloride can be combined to be applied to obviously increase the effect of resisting pancreatic cancer;
in the test, the composition of amiloride hydrochloride and erlotinib is used for the Bxpc-3 cells of the human pancreatic cancer, and the result shows that the cell inhibition effect is more obvious than that of the erlotinib used alone; when the composition achieves the same anti-tumor effect as that of single use of erlotinib, the use amount of erlotinib is obviously reduced, and the synergistic effect of erlotinib is increased along with the increase of the use amount of amiloride;
the test result of the invention also shows that the combination of amiloride hydrochloride and erlotinib significantly increases the effect of erlotinib on cell G1 retardation; meanwhile, amiloride hydrochloride can obviously increase the inhibition effect of erlotinib on EGFR downstream signal molecules of the target point, including inhibiting the phosphorylation levels of downstream STAT3, Akt and Erk of EGFR.
The invention provides a new pharmaceutical application of amiloride hydrochloride, namely the amiloride hydrochloride is used as a tumor chemotherapy sensitizer and is combined with erlotinib to increase the pancreatic cancer resistance of the amiloride hydrochloride, so that the clinical chemotherapy effect is improved; the pharmaceutical compositions, kits and packages contain a therapeutically effective amount of amiloride and a therapeutically effective amount of the epidermal growth factor tyrosine kinase inhibitor (EGFR-TKI) Erlotinib (Erlotinib) as a combined preparation for simultaneous, separate or sequential use; when the pharmaceutical composition, the medicine box and the package are used for treating tumors, the drug effect superior to that of single use of erlotinib can be obtained.
Drawings
FIG. 1 shows the cytotoxic effect of amiloride hydrochloride-enhanced erlotinib hydrochloride on human pancreatic cancer Bxpc-3 cells, wherein cell viability was determined using the MTT method, p <0.05VS amiloride 0 group, p <0.01VS amiloride 0 group;
the cytotoxicity of the amiloride hydrochloride-sensitized erlotinib hydrochloride on human pancreatic cancer cells PANC-1, Aspc-1 and CFPAC-1 is also shown, wherein the cell survival rate is determined by an MTT method, and p is less than 0.05VS amiloride 0 group and p is less than 0.01VS amiloride 0 group.
FIG. 2 shows the effect of amiloride hydrochloride in combination with erlotinib hydrochloride on the cell cycle, wherein,
p <0.05VS Control group, #, p <0.01VS Control group, #, p <0.05VS Erlotinib group;
wherein, the long-term growth inhibition effect of the amiloride hydrochloride-sensitized erlotinib hydrochloride on human pancreatic cancer Bxpc-3, PANC-1, Aspc-1 and CFPAC-1 cells is also shown, wherein the cells are stained by giemsa staining solution and the clone number, x, p is counted under a microscope<0.05VS controlGroup, p<The control group of 0.01VS was used,#，p<the 0.05VS amiloride group,##，p<0.05VS amiloride group.
FIG. 3 shows a Western Blot method for detecting the effect of amiloride hydrochloride in combination with erlotinib hydrochloride on EGFR and downstream STAT3, Akt and Erk phosphorylation.
Detailed Description
The present invention is further illustrated by the following specific examples. It is not intended that the invention be limited thereto.
Example 1 cytotoxic Effect of Amylori hydrochloride-sensitizing Erlotinib hydrochloride (Erlotinib) on human pancreatic cancer Bxpc-3 cells
Experimental materials:
human pancreatic cancer Bxpc-3 cells (Shanghai cell Bank of Chinese academy of sciences) at 37 ℃ with 5% CO2The culture was carried out routinely under the conditions of RPMI-1640 (Gibco) containing 10% fetal bovine serum (Hyclone). Amiloride hydrochloride standards were purchased from the chinese drug biologies institute, and the stock solution 50mM was prepared from DMSO. The erlotinib hydrochloride raw material medicine is provided by Xuanhong medicine (Jinan), and mother liquor is prepared by 10 mMDMSO. MTT was purchased from Sigma (USA).
The experimental method comprises the following steps:
1. well-differentiated cells were trypsinized, suspended in conventional medium and adjusted to a cell density of 5X 104Cells/ml. 100. mu.l/well was inoculated into a 96-well plate and cultured overnight in an incubator.
2. The culture medium is replaced the next day, working solution is added into 10 mul/hole, the final concentration of the drug group amiloride hydrochloride is respectively 20, 50 and 100 mul (the added series working solution is diluted by PBS, the concentration of DMSO is less than 0.2%), the final concentration of erlotinib hydrochloride is respectively 3, 10 and 30 mul, and the working solution with the concentration is respectively added into 10 mul/hole of the drug combination. Blank control contained DMSO 0.5%.
3. The cells after administration were incubated at 37 ℃ with 5% CO2Incubated under conditions for 48 hours.
4. Mu.l of MTT (5mg/ml) solution was added to each well and incubation in the incubator was continued for 4 hours.
5. The medium was washed off, 100. mu.l of DMSO was added, and the mixture was placed on a shaker at 37 ℃ to dissolve it sufficiently, and the value of absorbance was measured at a detection wavelength of 570 nm. The growth inhibition rate of the cells was calculated. N =4, the experiments were performed in triplicate.
The results of the experiments show that amiloride hydrochloride increases the cytotoxic effect of erlotinib hydrochloride on Bxpc-3 cells (as shown in FIG. 1).
Example 2 cytotoxic Effect of amiloride hydrochloride-sensitized erlotinib hydrochloride on human pancreatic cancer PANC-1, Aspc-1 and CFPAC-1 cells
Experimental materials:
human pancreatic cancer PANC-1, Aspc-1 and CFPAC-1 cells (Shanghai cell Bank of Chinese academy of sciences) at 37 ℃ with 5% CO2The culture was carried out routinely under the conditions of DMEM containing 10% fetal bovine serum (Hyclone), RPMI-1640 and IMDM (Gibco). Amiloride hydrochloride standards were purchased from the chinese drug biologies institute, and the stock solution 50mM was prepared from DMSO. The erlotinib hydrochloride raw material medicine is provided by Xuanhong medicine (Jinan), and mother liquor is prepared from 10mM DMSO. MTT was purchased from Sigma (USA).
The experimental method comprises the following steps:
1. well-differentiated cells were trypsinized, suspended in conventional medium and adjusted to a cell density of 5X 104Cells/ml. 100. mu.l/well was inoculated into a 96-well plate and cultured overnight in an incubator.
2. The culture medium is replaced the next day, working solution is added into 10 mul/hole, the final concentration of the drug group amiloride hydrochloride is respectively 20, 50 and 100 mul (the added series working solution is diluted by PBS, the concentration of DMSO is less than 0.2%), the final concentration of erlotinib hydrochloride is respectively 3, 10 and 30 mul, and the working solution with the concentration is respectively added into 10 mul/hole of the drug combination. Blank control contained DMSO 0.5%.
3. The cells after administration were incubated at 37 ℃ with 5% CO2Incubated under conditions for 48 hours.
4. Mu.l of MTT (5mg/ml) solution was added to each well and incubation in the incubator was continued for 4 hours.
5. The medium was washed off, 100. mu.l of DMSO was added, and the mixture was placed on a shaker at 37 ℃ to dissolve it sufficiently, and the value of absorbance was measured at a detection wavelength of 570 nm. The growth inhibition rate of the cells was calculated. N =4, the experiments were performed in triplicate.
The experimental results show that: amiloride hydrochloride on PANC-1, Aspc-1 and CFPAC-1 cells increased the cytotoxic effect of erlotinib hydrochloride (as shown in FIG. 1).
Example 3 Long-term growth inhibition of human pancreatic cancer Bxpc-3, PANC-1, Aspc-1 and CFPAC-1 cells by Aminolide hydrochloride in combination with erlotinib hydrochloride
Experimental materials:
human pancreatic cancer Bxpc-3, PANC-1, Aspc-1 and CFPAC-1 cells (Shanghai cell Bank of Chinese academy of sciences) at 37 ℃ with 5% CO2The culture medium is RPMI-1640 containing 10% fetal bovine serum (Hyclone), DMEM, RPMI-1640 and IMDM (Gibco) by a conventional method. Amiloride hydrochloride standards were purchased from the chinese drug biologies institute, and the stock solution 50mM was prepared from DMSO. The erlotinib hydrochloride raw material medicine is provided by Xuanhong medicine (Jinan), and mother liquor is prepared from 10mM DMSO. Giemsa stain was purchased from Sigma.
The experimental method comprises the following steps:
1. well differentiated cells were trypsinized, suspended in conventional medium, plated at 3000/well in 6-well plates and cultured overnight in an incubator.
After 2.48h, the culture medium was changed and the drug was added to make the final concentrations of amiloride hydrochloride and erlotinib hydrochloride 30,3 μ M, respectively. Blank control contained DMSO 0.5%.
3. The cells after administration were incubated at 37 ℃ with 5% CO2Cultured under the conditions for 14 days.
4. The medium was washed off, the Giemsa methanol stain was added, photographs were taken and the number of clones counted under the microscope.
The results of the experiments showed that amiloride hydrochloride on Bxpc-3, PANC-1, Aspc-1 and CFPAC-1 cells increased the cytotoxic effect of erlotinib hydrochloride (as shown in FIG. 2).
Example 4 Effect of amiloride hydrochloride in combination with erlotinib hydrochloride on the Bxpc-3 cell cycle
Experimental materials:
propidium Iodide (PI), RNase enzyme was purchased from Sigma. The remaining materials were the same as in example 1.
The experimental method comprises the following steps:
1. well-differentiated cells were trypsinized, suspended in conventional medium and adjusted to a cell density of 2X 105Cells/ml. 2.5 mL/well was inoculated into 6-well plates and incubated overnight in an incubator.
After 2.48h, the drug-containing medium was replaced so that the final concentration of amiloride hydrochloride in the single drug group was 20 μ M and the final concentration of erlotinib hydrochloride was 3 μ M, and the drug combination group contained the two drugs at the same final concentrations. Blank control contained DMSO 0.7%.
3. The cells after administration were incubated at 37 ℃ with 5% CO2Culturing for 24h under the condition.
4. The cells were collected by digestion, washed once with ice PBS, added with a solution containing PI and RNase at 300. mu.l/well, incubated for 30min in the dark and then detected by flow cytometry.
The experimental results show that amiloride hydrochloride on Bxpc-3 cells increases the long-term inhibition effect of erlotinib hydrochloride on tumor cells (G1 phase retardation).
Example 5 effect of amiloride hydrochloride with erlotinib hydrochloride on EGFR and phosphorylation of its downstream signaling molecules experimental materials:
RIPA lysate and PMSF were purchased from Biyunstian Biotechnology institute (Jiangsu). The Protease inhibitor cocktail was purchased from Merck (germany). The remaining materials were the same as in example 1.
The experimental method comprises the following steps:
1. cell culture and drug delivery
Well-differentiated cells were trypsinized, suspended in conventional medium and adjusted to a cell density of 5X 103Cells/well were seeded in 6-well culture plates.
After 48h of culture, the drug-containing medium was added so that the final concentration of amiloride hydrochloride was 20. mu.M, the final concentration of erlotinib hydrochloride was 3. mu.M, and the blank control contained 0.3% DMSO.
The cells after administration were cultured at 37 ℃ under 5% CO2 for 24 hours.
2. Protein extraction:
adding PMSF (100 mM, 1: 100) and Protease Inhibitor Cocktail Set III (1: 200) into RIPA lysate before use;
pouring the culture medium from the cells cultured in the 6-well plate, washing the cells once by using PBS, and adding 150 mu l of lysate for ice bath lysis for 20 min;
transferring the cracked mixture into an EP tube, and centrifuging for 5min at 9000 g;
sucking the supernatant for quantification, simultaneously taking part of the supernatant, adding 1/4 volume of loading buffer solution (5 x), and decocting at 90-95 deg.C for 8 min;
the denatured proteins were stored in a freezer at-80 ℃.
Western Blot for detecting expression of P-EGFR, P-STAT3, P-Akt and P-Erk
Sample loading amount: the BCA protein quantification kit quantifies each group of extracted protein samples, and the corrected protein sample loading amount is 60 mu g;
the electrophoresis method comprises the following steps: concentrating the sample by constant voltage electrophoresis at 60V for 50 minutes; carrying out 120V electrophoresis for 1 hour for separating samples, and judging the electrophoresis degree according to a marker;
film transfer: performing wet rotation, 200mA, and performing transverse flow membrane conversion for 2 hours;
antibody hybridization: blocking with 5% BSA (blocking solution) for 1 hour at 37 ℃ on a shaker; diluting the primary antibody with a sealing solution according to the instruction, and hybridizing the transferred membrane in a hybridization bag at 4 ℃ overnight; washing the membrane with TBST four times (5 mL each time for 5min in a shaking table at 37 ℃); diluting the secondary antibody with the sealing solution, and hybridizing in a hybridization bag at 37 ℃ for 1 hour by a shaking table; carrying out chemiluminescence detection after washing the membrane for four times by TBST;
and (3) chemiluminescence detection: the detection of the target protein was carried out by an enhanced chemiluminescence method (ECL method).
The experimental results are as follows:
representative experimental results were selected as shown in fig. 3. The results show that amiloride hydrochloride on Bxpc-3 cells increases the inhibition of erlotinib hydrochloride on p-STAT3, p-Akt and p-Erk.
Claims (6)
1. A pharmaceutical composition for treating pancreatic cancer, comprising a therapeutically effective amount of amiloride or a derivative thereof having amiloride as the parent nucleus, wherein the pharmaceutical composition comprises: dimethylammocloride (DMA), 5-N-ethyl-N-isopropyl amiloride (5-ethylisopropropylamiloride, EIPA), Methylisobutylamide (MIBA) or 5-N, N-cyclohexane amiloride (5-N, N-hexamethyylene amide, HMA) and epidermal growth factor tyrosine kinase inhibitor (EGFR-TKI) Erlotinib (Erlotinib); wherein,
the effective treatment dose of the amiloride or the derivative taking the amiloride as the parent nucleus is 20 mu M, 50 mu M and 100 mu M respectively, and the effective treatment dose of the erlotinib is 3, 10 and 30 mu M respectively.
2. The anti-pancreatic cancer pharmaceutical composition according to claim 1, wherein amiloride or a derivative having amiloride as a parent nucleus is present in a base form or a salt form, the salt form being a hydrochloride form or a sulfate form.
3. The anti-pancreatic cancer pharmaceutical composition according to claim 1, characterized in that erlotinib is present in base form or in salt form, which is in the form of hydrochloride or in the form of sulfate.
4. The application of amiloride or the combination of amiloride-parent-nucleus derivative and erlotinib in preparing an anti-pancreatic cancer drug, wherein the therapeutic effective dose of the amiloride or the amiloride-parent-nucleus derivative is 20 mu M, 50 mu M and 100 mu M respectively, and the therapeutic effective dose of the erlotinib is 3, 10 and 30 mu M respectively;
the derivative taking amiloride as a parent nucleus is as follows: dimethylammocloride (DMA), 5-N-ethyl-N-isopropyl amiloride (EIPA), Methylisobutylamide (MIBA), or 5-N, N-cyclohexane amiloride (5-N, N-hexamethylene amiloride, HMA).
5. The use according to claim 4, wherein amiloride or a derivative thereof having amiloride as the parent nucleus is present in the base form or in the form of a salt, either as the hydrochloride salt or as the sulfate salt.
6. Use according to claim 4, wherein erlotinib is present in base form or in salt form, said salt being in the form of the hydrochloride or in the form of the sulfate.
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