阅读说明：本技术 一种组合物及其联合伊立替康制备抗肿瘤药物的用途 (Composition and application of composition and irinotecan combined to preparation of antitumor drug ) 是由 许风国 许瑞洁 王笛 张培 张尊建 于 2021-01-25 设计创作，主要内容包括：本发明公开了一种组合物及其联合伊立替康制备抗肿瘤药物的用途。本发明发现,伐地那非与利拉利汀的组合物对以伊立替康的抗结肠癌活性具有协同增强作用,而伐地那非或利拉利汀单独对伊立替康的抗结肠癌活性无协同增强作用。因此,伐地那非和利拉利汀的组合物可以联合伊立替康制备抗结肠癌的药物。(The invention discloses a composition and application thereof in preparing an antitumor drug by combining irinotecan. The invention discovers that the composition of the vardenafil and the linagliptin has a synergistic enhancement effect on the anti-colon cancer activity of irinotecan, and the vardenafil or the linagliptin has no synergistic enhancement effect on the anti-colon cancer activity of irinotecan alone. Therefore, the combination of the vardenafil and the linagliptin can be combined with irinotecan to prepare the anti-colon cancer medicament.)

1. A composition characterized by: consists of vardenafil and linagliptin.

2. Use of a composition according to claim 1 in combination with irinotecan for the preparation of an anti-neoplastic drug.

3. Use according to claim 2, characterized in that: the tumor is human colon cancer.

Technical Field

The invention belongs to the field of medicines, relates to a pharmaceutical composition, and particularly relates to a composition and application thereof in preparation of an antitumor drug by combining irinotecan.

Background

Colon cancer is a disease caused by the interaction of genetic and environmental factors and is one of the most common malignant tumors worldwide. Statistics show that in 2020, nearly 15 million people in the united states are expected to be diagnosed with colon cancer and about 5 million people die as a result. According to the investigation, colon cancer is one of the most common tumors in China, the incidence rate is second to that of lung cancer, and the incidence rate is gradually increased. Currently, surgery combined with post-operative chemotherapy is the primary treatment modality for colon cancer. However, the therapeutic effect of current chemotherapeutic drugs is to be improved.

Irinotecan (Irinotecan, CPT-11) is a semi-synthetic water-soluble camptothecin derivative, is a first-line medicament for advanced colon cancer, and can also be used for postoperative adjuvant chemotherapy; vardenafil (Vardenafil) is a drug for the treatment of male erectile dysfunction; linagliptin (Linagliptin) is a drug that regulates blood glucose levels. At present, no research at home and abroad shows that the vardenafil and/or linagliptin have a synergistic enhancement effect on the colon cancer resisting activity of irinotecan.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a composition and application of the composition in preparation of an anti-tumor medicament by combining irinotecan so as to improve the anti-colon cancer activity of the medicament.

The above purpose of the invention is realized by the following technical scheme:

a composition comprising vardenafil and linagliptin.

The composition is combined with irinotecan to prepare the application of the antitumor drug.

Preferably, the tumor is human colon cancer.

Has the advantages that:

the invention discovers that the composition of the vardenafil and the linagliptin has a synergistic enhancement effect on the anti-colon cancer activity of irinotecan, and the vardenafil or the linagliptin has no synergistic enhancement effect on the anti-colon cancer activity of irinotecan alone. Therefore, the combination of the vardenafil and the linagliptin can be combined with irinotecan to prepare the anti-colon cancer medicament.

Detailed Description

The following examples are given to illustrate the essence of the present invention, but not to limit the scope of the present invention.

First, experimental material and reagent

Cell: human colon cancer HCT-116 cells (cell bank, Chinese academy of sciences).

Medicine preparation: irinotecan (Irinotecan, CPT-11, CAS No.:97682-44-5), Vardenafil hydrochloride (Vardenafil, Vard, CAS No.:330808-88-3), Linagliptin (Linagliptin, Linag, CAS No.:668270-12-0), all available from Shanghai Allantin Biotechnology, Inc.

Reagent: DMEM high glucose medium (Gibco, usa); FBS (Gibco, usa); penicillin-streptomycin (Hyclone, usa); 0.25% pancreatin-EDTA solution (bosd, china); PBS (bosch de, china); DMSO (Sigma-Aldrich, USA); MTT (bi yun tian, china).

Consumable material: 10cm cell culture dishes (SORFA, china); 15. 50mL centrifuge tubes (JET BIOFIL, China); cell cryopreservation tubes (Corning, usa); 96-well cell culture plates (SORFA, china).

The instrument equipment comprises: 2.5, 10, 100 and 1000. mu.L pipette gun (Eppendorf, Germany); biological safety cabinets (Thermo, usa); CO 2₂Cell culture chambers (Thermo, usa); XD202 inverted biomicroscope (south kyoto south china, perpetual new optics ltd); HW-12 model electric heating constant temperature water bath (Shanghai-Hengyue scientific instruments Co., Ltd., China); IC1000 automatic cell counter (Shanghai Rui Yu Biotech Co., Ltd., China); model TDL-50B low speed table centrifuge (shanghai an pavilion scientific instruments factory, china); vortex mixer (waybill instruments manufacturing ltd, china, hamam); microplate constant temperature oscillator (Hangzhou Osheng instruments Co., Ltd., China); multifunctional microplate reader (Tecan Infinite M200Pro, Switzerland); YCD-EL260 medical refrigerator-freezer (Mike's Mitsubishi, China).

Second, Experimental methods

1. Inhibition of HCT-116 cell proliferation by Vard in combination with CPT-11

Collecting HCT-116 cells in logarithmic growth phase, digesting with 0.25% pancreatin, collecting cells, and regulating cell suspension concentration to 6 × 10⁷cells/L, 100. mu.L per well, in 96-well plates, i.e., about 6X 10 cells per well³And (4) respectively. Experimental data A blank group, a control group, a 15. mu.M Vard group, four CPT-11(2.5, 5, 10 and 20. mu.M) concentration groups and 1The 5. mu.M Vard was administered in combination with CPT-11 at four concentrations (2.5, 5, 10 and 20. mu.M) in 3 duplicate wells per group, and the inhibitory effect of Vard in combination with CPT-11 on HCT-116 cell proliferation was examined. Cells at 37 ℃ and 5% CO₂After overnight culture in the incubator, the blank group and the control group were replaced with fresh complete medium, the administration group was replaced with fresh complete medium containing different concentrations of each drug, and culture was continued for 48 hours. After the incubation, the cell growth inhibition rate of each group was measured by MTT method, specifically by adding 20 μ LMTT (5mg/mL) solution to each well and continuing the culture. After 4h, the liquid in the wells was aspirated, 100. mu.L of DMSO was added to each well, and the mixture was placed on a microplate oscillator and shaken at low speed for 10 min. The absorbance (OD) of each well was measured at 490 nm. The growth inhibition rate of each group of cells was calculated according to the following formula: cell growth inhibition rate ═ 1- (OD)_{Administration set}–OD_{Blank group})/(OD_{Control group}–OD_{Blank group})]X 100%. The experiment was repeated three times.

2. Linag combined with CPT-11 inhibition of HCT-116 cell proliferation

Collecting HCT-116 cells in logarithmic growth phase, digesting with 0.25% pancreatin, collecting cells, and regulating cell suspension concentration to 6 × 10⁷cells/L, 100. mu.L per well, in 96-well plates, i.e., about 6X 10 cells per well³And (4) respectively. A blank group, a control group, a 15 mu M Linag group, four CPT-11(2.5, 5, 10 and 20 mu M) concentration groups and a 15 mu M Linag group were set as a combined administration group with four CPT-11(2.5, 5, 10 and 20 mu M) concentration groups, 3 duplicate wells were set for each group, and the inhibitory effect of Linag combined CPT-11 administration on HCT-116 cell proliferation was examined. Cells at 37 ℃ and 5% CO₂After the incubator is used for overnight culture, the blank group and the control group are replaced by fresh complete culture mediums, the administration group is replaced by fresh complete culture mediums containing different drugs with different concentrations, and the culture is continued for 48 h. After the incubation, the cell growth inhibition rate of each group was measured by the MTT method, in which 20. mu.L of MTT (5mg/mL) solution was added to each well, and the culture was continued. After 4h, the liquid in the wells was aspirated, 100. mu. LDMSO was added to each well, and the mixture was placed on a microplate oscillator and shaken at low speed for 10 min. The OD of each well was measured at 490 nm. The growth inhibition rate of each group of cells was calculated according to the following formula: inhibition of cell growth＝[1–(OD_{Administration set}–OD_{Blank group})/(OD_{Control group}–OD_{Blank group})]X 100%. The experiment was repeated three times.

3. Combination of Vard + Linag with CPT-11 for inhibition of HCT-116 cell proliferation

Collecting HCT-116 cells in logarithmic growth phase, digesting with 0.25% pancreatin, collecting cells, and regulating cell suspension concentration to 6 × 10⁷cells/L, 100. mu.L per well, in 96-well plates, i.e., about 6X 10 cells per well³And (4) respectively. A blank group, a control group, a (15. mu.M Vard + 15. mu.M Linag) group, four CPT-11(2.5, 5, 10 and 20. mu.M) concentration groups and a (15. mu.M Vard + 15. mu.M Linag) group were prepared, and the effect of (Vard + Linag) combined with CPT-11(2.5, 5, 10 and 20. mu.M) administration to HCT-116 cell proliferation was examined in 3 duplicate wells per group. Cells at 37 ℃ and 5% CO₂After the incubator is used for overnight culture, the blank group and the control group are replaced by fresh complete culture mediums, the administration group is replaced by fresh complete culture mediums containing different drugs with different concentrations, and the culture is continued for 48 h. After the incubation, the cell growth inhibition rate of each group was measured by MTT method, specifically by adding 20 μ LMTT (5mg/mL) solution to each well and continuing the culture. After 4h, the liquid in the wells was aspirated, 100. mu.L of DMSO was added to each well, and the mixture was placed on a microplate oscillator and shaken at low speed for 10 min. The OD of each well was measured at 490 nm. The growth inhibition rate of each group of cells was calculated according to the following formula: cell growth inhibition rate ═ 1- (OD)_{Administration set}–OD_{Blank group})/(OD_{Control group}–OD_{Blank group})]X 100%. The experiment was repeated three times.

The experimental data were processed using Graphpad Prism 7.0 software and the experimental results are expressed as mean ± SD. Two independent samples are compared by adopting double-sided unpaired t test to count the significant difference,^*P<0.05，^**P<0.01，^***P<0.001。

judging the effect of the combination of each drug and CPT-11 according to the positive average q value of the inhibition rate; q ═ E_a+b/(E_a+E_b-E_a×E_b)，E_aAnd E_bInhibition rates for two drugs alone, respectively, E_a+bThe inhibition rate of the combination drug; the numerator in the formula represents the 'actually measured merging effect', and the denominator is the 'expected merging effect'; q is less than 0.85 for antagonistic effect, q is more than or equal to 0.85 and less than 1.15 for additive effect, and q is more than or equal to 1.15 for synergistic effect.

Third, experimental results

1. Inhibition of HCT-116 cell proliferation by Vard in combination with CPT-11

The results show (Table 1) that 15. mu.M Vard in combination with CPT-11 did not enhance cell proliferation of HCT-116 inhibited by CPT-11 at various concentrations. Meanwhile, the q value of the combination of 15 mu M Vard and CPT-11 at each concentration is between 0.85 and 1.15, which shows an additive effect, and the combination of Vard and CPT-11 has an additive effect on the inhibition of HCT-116 cell proliferation.

TABLE 115 μ M Vard in combination with CPT-11 inhibition of HCT-116 cell proliferation ((n＝3)

2. Linag combined with CPT-11 inhibition of HCT-116 cell proliferation

The results show (Table 2) that 15. mu.M Linag in combination with CPT-11 significantly enhanced the inhibitory effect of 20. mu.M CPT-11 on HCT-116 cell proliferation only. 15 mu M Linag and 2.5 mu M CPT-11 are used together, the q value is 0.76, and antagonism is shown; linag 15 μ M was used in combination with CPT-11 5, 10 and 20 μ M, and q values were all between 0.85 and 1.15, showing an additive effect. Indicating that Linag in combination with CPT-11 antagonizes or additively inhibits HCT-116 cell proliferation.

TABLE 215 μ M Linag in combination with CPT-11 inhibition of HCT-116 cell proliferation ((n＝3)

Note: in comparison to the corresponding CPT-11 group,^*P≤0.05。

3. combination of Vard + Linag with CPT-11 for inhibition of HCT-116 cell proliferation

The results show (Table 3) that the combination of (15. mu.M Vard + 15. mu.M Linag) with CPT-11 significantly enhanced the inhibitory effect of CPT-11 at each concentration on HCT-116 cell proliferation. At the same time (15. mu.M Vard + 15. mu.M Linag) was used in combination with 2.5, 5, 10 and 20. mu.M CPT-11, with q values of 1.39, 1.40, 1.30 and 1.25, respectively, showing a synergistic effect. Indicating that the combination of (Vard + Linag) with CPT-11 is synergistic in inhibiting HCT-116 cell proliferation.

TABLE 3 (15. mu.M Vard + 15. mu.M Linag) in combination with CPT-11 inhibition of HCT-116 cell proliferation ((C))n＝3)

Note: in comparison to the corresponding CPT-11 group,^**P≤0.01，^***P≤0.001。

in conclusion, Vard and Linag have no synergistic effect in combination with CPT-11, respectively, whereas the (Vard + Linag) composition has a synergistic effect in combination with CPT-11, and is capable of synergistically enhancing the effect of CPT-11 in inhibiting HCT-116 cell proliferation.

The above-described embodiments are intended to be illustrative of the nature of the invention, but those skilled in the art will recognize that the scope of the invention is not limited to the specific embodiments.