A composition and its use in combination with irinotecan to prepare an antitumor drug

Abstract

This invention discloses a composition and its use in combination with irinotecan to prepare an antitumor drug. This invention discovers that the composition of vardenafil and liraristine has a synergistic enhancing effect on the anti-colon cancer activity of irinotecan, while vardenafil or liraristine alone does not have a synergistic enhancing effect on the anti-colon cancer activity of irinotecan. Therefore, the composition of vardenafil and liraristine can be combined with irinotecan to prepare an anti-colon cancer drug.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to pharmaceutical compositions, specifically to a composition and its use in combination with irinotecan to prepare an antitumor drug. Background Technology

[0002] Colorectal 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, an estimated 150,000 people in the United States were diagnosed with colorectal cancer, and approximately 50,000 died from it. Surveys have found that in China, colorectal cancer is one of the most common cancers, second only to lung cancer in incidence, and the incidence rate is gradually increasing. Currently, surgery combined with postoperative chemotherapy is the main treatment for colorectal cancer. However, the efficacy of current chemotherapy drugs needs improvement.

[0003] Irinotecan (CPT-11) is a semi-synthetic, water-soluble camptothecin derivative, a first-line drug for advanced colorectal cancer, and can also be used as adjuvant chemotherapy after surgery; vardenafil is a drug for treating erectile dysfunction in men; linagliptin is a drug for regulating blood sugar levels. Currently, no studies, either domestically or internationally, have shown that vardenafil and / or linagliptin synergistically enhance the anti-colorectal cancer activity of irinotecan. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a composition and its use in combination with irinotecan to prepare an antitumor drug, so as to improve the drug's anti-colon cancer activity.

[0005] The above-mentioned objective of this invention is achieved through the following technical solution:

[0006] A composition comprising vardenafil and liraristine.

[0007] The above composition, in combination with irinotecan, is used to prepare an antitumor drug.

[0008] Preferably, the tumor is human colon cancer.

[0009] Beneficial effects:

[0010] This invention reveals that the combination of vardenafil and liraristine synergistically enhances the anti-colon cancer activity of irinotecan, while neither vardenafil nor liraristine alone synergistically enhances the anti-colon cancer activity of irinotecan. Therefore, the combination of vardenafil and liraristine can be used in conjunction with irinotecan to prepare an anti-colon cancer drug. Detailed Implementation

[0011] The following describes the substantive content of the present invention in detail with reference to embodiments, but this does not limit the scope of protection of the present invention.

[0012] I. Experimental Materials and Reagents

[0013] Cells: Human colon cancer HCT-116 cells (Chinese Academy of Sciences Cell Bank).

[0014] Drugs: Irinotecan (CPT-11, CAS No.:97682-44-5), Vardenafil hydrochloride (Vard, CAS No.:330808-88-3), and Linagliptin (Linag, CAS No.:668270-12-0), all purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0015] Reagents: DMEM high glucose medium (Gibco, USA); FBS (Gibco, USA); penicillin-streptomycin (Hyclone, USA); 0.25% trypsin-EDTA solution (Boster Biological, China); PBS (Boster Biological, China); DMSO (Sigma-Aldrich, USA); MTT (Beyotime, China).

[0016] Consumables: 10cm cell culture dish (SORFA, China); 15 and 50mL centrifuge tubes (JET BIOFIL, China); cell cryopreservation tubes (Corning, USA); 96-well cell culture plate (SORFA, China).

[0017] Instruments and Equipment: 2.5, 10, 100, and 1000 μL pipettes (Eppendorf, Germany); Biosafety cabinet (Thermo, USA); CO2 cell incubator (Thermo, USA); XD202 inverted biological microscope (Nanjing Jiangnan Yongxin Optical Co., Ltd., China); HW-12 electric thermostatic water bath (Shanghai Yiheng Scientific Instrument Co., Ltd., China); IC1000 automated cell counter (Shanghai Ruiyu Biotechnology Co., Ltd., China); TDL-50B low-speed benchtop centrifuge (Shanghai Anting Scientific Instrument Factory, China); Vortex mixer (Haimen Qilinbei Instrument Manufacturing Co., Ltd., China); Microplate constant temperature shaker (Hangzhou Aosheng Instrument Co., Ltd., China); Multifunctional microplate reader (Tecan Infinite M200Pro, Switzerland); YCD-EL260 medical refrigerator / freezer (Zhongke Meiling Cryogenic Technology Co., Ltd., China).

[0018] II. Experimental Methods

[0019] 1. Inhibitory effect of Vard combined with CPT-11 on the proliferation of HCT-116 cells

[0020] HCT-116 cells in logarithmic growth phase were collected after digestion with 0.25% trypsin, and the cell suspension concentration was adjusted to 6 × 10⁻⁶. 7 The cells / L were seeded at 100 μL per well in a 96-well plate, which is approximately 6 × 10⁶ cells per well. 3 The experiment included a blank control group, a control group, a 15 μM Vard group, four concentrations of CPT-11 (2.5, 5, 10, and 20 μM) groups, and a combination of 15 μM Vard and four concentrations of CPT-11 (2.5, 5, 10, and 20 μM) in each group. Each group had three replicates to investigate the inhibitory effect of Vard combined with CPT-11 on HCT-116 cell proliferation. Cells were cultured overnight at 37°C in a 5% CO2 incubator. The blank and control groups were replaced with fresh complete culture medium, while the drug-treated groups were replaced with fresh complete culture medium containing different concentrations of the respective drugs, and cultured for another 48 h. After incubation, the cell growth inhibition rate was determined using the MTT assay. Specifically, 20 μL of MTT (5 mg / mL) solution was added to each well, and culture continued. After 4 h, the liquid in the wells was aspirated, and 100 μL of DMSO was added to each well. The wells were then shaken slowly for 10 min on a microplate shaker. The absorbance (optical density, OD) of each well was measured at a wavelength of 490 nm. The growth inhibition rate of cells in each group was calculated using the following formula: Cell growth inhibition rate = [1 – (OD)] 给药组 –OD 空白组 ) / (OD 对照组 –OD 空白组 )]×100%. The experiment was repeated three times.

[0021] 2. Inhibitory effect of Linag combined with CPT-11 on the proliferation of HCT-116 cells

[0022] HCT-116 cells in logarithmic growth phase were collected after digestion with 0.25% trypsin, and the cell suspension concentration was adjusted to 6 × 10⁻⁶. 7 The cells / L were seeded at 100 μL per well in a 96-well plate, which is approximately 6 × 10⁶ cells per well. 3The study included a blank control group, a control group, a 15 μM Linag group, four concentrations of CPT-11 (2.5, 5, 10, and 20 μM) groups, and a combination of 15 μM Linag and four concentrations of CPT-11 (2.5, 5, 10, and 20 μM) groups. Each group had three replicates to investigate the inhibitory effect of Linag combined with CPT-11 on the proliferation of HCT-116 cells. Cells were cultured overnight at 37°C in a 5% CO2 incubator. The blank and control groups were replaced with fresh complete culture medium, while the drug-treated groups were replaced with fresh complete culture medium containing different drug concentrations, and cultured for another 48 h. After incubation, the cell growth inhibition rate was determined using the MTT assay. Specifically, 20 μL of MTT (5 mg / mL) solution was added to each well, and culture continued. After 4 h, the liquid in the wells was aspirated, and 100 μL of LDMSO was added to each well. The wells were then shaken slowly for 10 min on a microplate shaker. The OD value of each well was measured at 490 nm. Calculate the growth inhibition rate of each group of cells using the following formula: Cell growth inhibition rate = [1 – (OD)] / ... 给药组 –OD 空白组 ) / (OD 对照组 –OD 空白组 )]×100%. The experiment was repeated three times.

[0023] 3. The inhibitory effect of Vard+Linag combination with CPT-11 on the proliferation of HCT-116 cells.

[0024] HCT-116 cells in logarithmic growth phase were collected after digestion with 0.25% trypsin, and the cell suspension concentration was adjusted to 6 × 10⁻⁶. 7 The cells / L were seeded at 100 μL per well in a 96-well plate, which is approximately 6 × 10⁶ cells per well. 3The study included a blank control group, a control group, a (15 μM Vard + 15 μM Linag) group, four concentrations of CPT-11 (2.5, 5, 10, and 20 μM) groups, and a (15 μM Vard + 15 μM Linag) combined with four concentrations of CPT-11 (2.5, 5, 10, and 20 μM) group, with three replicates per group. The inhibitory effect of (Vard + Linag) combined with CPT-11 on HCT-116 cell proliferation was investigated. Cells were cultured overnight at 37°C in a 5% CO2 incubator. The blank and control groups were replaced with fresh complete culture medium, while the drug-treated groups were replaced with fresh complete culture medium containing different drug concentrations, and cultured for another 48 h. After incubation, the cell growth inhibition rate was determined using the MTT assay, specifically by adding 20 μM MTT (5 mg / mL) solution to each well and continuing culture. After 4 hours, the liquid in the wells was aspirated, and 100 μL of DMSO was added to each well. The wells were then placed on a microplate shaker and shaken at low speed for 10 minutes. The OD value of each well was measured at a wavelength of 490 nm. The cell growth inhibition rate of each group was calculated using the following formula: Cell growth inhibition rate = [1 – (OD value)] 给药组 –OD 空白组 ) / (OD 对照组 –OD 空白组 )]×100%. The experiment was repeated three times.

[0025] Experimental data were processed using Graphpad Prism 7.0 software, and the results are expressed as mean ± SD. Two-tailed unpaired t-tests were used to compare statistical significance between two independent samples. * P<0.05, ** P<0.01, *** P<0.001.

[0026] The efficacy of each drug combined with CPT-11 was determined based on the average q-value of the inhibition rate; q = E a+b / (E a +E b -E a ×E b E a and E b These represent the inhibition rates of the two drugs used alone, E a+b The formula represents the inhibition rate of combined drug use; the numerator represents the "measured combined effect", and the denominator is the "expected combined effect"; q < 0.85 is the antagonistic effect, 0.85 ≤ q < 1.15 is the additive effect, and q ≥ 1.15 is the synergistic effect.

[0027] III. Experimental Results

[0028] 1. Inhibitory effect of Vard combined with CPT-11 on the proliferation of HCT-116 cells

[0029] The results (Table 1) showed that the combination of 15 μM Vard and CPT-11 did not enhance the inhibition of HCT-116 cell proliferation by various concentrations of CPT-11. Meanwhile, the q values ​​of 15 μM Vard combined with various concentrations of CPT-11 ranged from 0.85 to 1.15, exhibiting an additive effect, indicating that the inhibition of HCT-116 cell proliferation by the combination of Vard and CPT-11 is additive.

[0030] Table 1. Inhibitory effect of 15 μM Vard combined with CPT-11 on the proliferation of HCT-116 cells ( n=3)

[0031]

[0032] 2. Inhibitory effect of Linag combined with CPT-11 on the proliferation of HCT-116 cells

[0033] The results (Table 2) showed that the combination of 15 μM Linag and CPT-11 significantly enhanced the inhibitory effect of 20 μM CPT-11 on HCT-116 cell proliferation. The combination of 15 μM Linag and 2.5 μM CPT-11 showed an antagonistic effect (q value of 0.76); the combination of 15 μM Linag with 5, 10, and 20 μM CPT-11 showed an additive effect (q values ​​ranging from 0.85 to 1.15). This indicates that the combination of Linag and CPT-11 has an antagonistic or additive effect on the proliferation of HCT-116 cells.

[0034] Table 2. Inhibitory effect of 15 μM Linag combined with CPT-11 on the proliferation of HCT-116 cells ( n=3)

[0035]

[0036] Note: Compared with the corresponding CPT-11 group, * P≤0.05.

[0037] 3. The inhibitory effect of Vard+Linag combination with CPT-11 on the proliferation of HCT-116 cells.

[0038] The results (Table 3) showed that the combination of (15 μM Vard + 15 μM Linag) and CPT-11 significantly enhanced the inhibitory effect of various concentrations of CPT-11 on the proliferation of HCT-116 cells. Furthermore, the combination of (15 μM Vard + 15 μM Linag) with 2.5, 5, 10, and 20 μM CPT-11 yielded q values ​​of 1.39, 1.40, 1.30, and 1.25, respectively, demonstrating a synergistic effect. This indicates that the combination of (Vard + Linag) and CPT-11 has a synergistic inhibitory effect on the proliferation of HCT-116 cells.

[0039] Table 3. Inhibitory effect of (15 μM Vard + 15 μM Linag) combined with CPT-11 on HCT-116 cell proliferation. n=3)

[0040]

[0041] Note: Compared with the corresponding CPT-11 group, ** P≤0.01, *** P≤0.001.

[0042] In summary, Vard and Linag did not have a synergistic effect when used in combination with CPT-11, while the (Vard+Linag) combination had a synergistic effect when used in combination with CPT-11, and could synergistically enhance the inhibitory effect of CPT-11 on HCT-116 cell proliferation.

[0043] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. Use of a composition in the manufacture of a medicament for the treatment of human colon cancer in combination with irinotecan; said composition consisting of vardenafil and liratrigine in a ratio of 15:15:2.5, 15:15:5, 15:15:10 or 15:15:20 of the amount of substance of vardenafil, liratrigine and irinotecan, respectively.

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