CN114796257B - New use of pharmaceutical compound in treating solid tumor - Google Patents
New use of pharmaceutical compound in treating solid tumor Download PDFInfo
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- A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
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- A—HUMAN NECESSITIES
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Abstract
The invention relates to the technical field of pharmacy, in particular to a novel application of a pharmaceutical compound in treating solid tumors. In particular to the application of S- (5' -adenosine) -L-methionine chloride in preparing a medicine for treating solid tumors. The application of the S- (5' -adenosine) -L-methionine chloride in preparing the medicine for treating the solid tumor increases the optional range of the medicine for treating the solid tumor, has remarkable prevention and treatment effects on lung cancer, and has higher pertinence and higher inhibition rate on lung cancer compared with the traditional chemotherapy medicine.
Description
Technical Field
The invention relates to an application of a pharmaceutical compound in preparing a medicine for treating solid tumor lung cancer, in particular to a novel pharmaceutical application of S- (5' -adenosine) -L-methionine chloride dihydrochloride (SAM), and more particularly relates to an application of SAM in treating small cell lung cancer (Small Cell Lung Cancer), belonging to the field of biological medicine.
Background
Lung cancer is the malignant tumor with leading Chinese mortality, more than 70 thousands of new cases exist each year, and the survival rate of 5 years is lower than 17 percent. Lung cancer can be broadly classified into Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC) according to its pathological characteristics. SCLC is a type of lung cancer with neuroendocrine properties, accounting for 15% of all lung cancers. SCLC is not usually treated surgically because of its extremely strong metastatic capacity in the early stages. SCLC at first diagnosis is often sensitive to chemotherapy, and current chemotherapy regimens of etoposide + cisplatin (EP) were routinely practiced since the 1980 s. Most patients quickly relapse resistant. The effect of the latest immunotherapy is also very limited.
Disclosure of Invention
The invention aims at: aiming at the problems of high mortality rate of lung cancer and high recurrence rate of traditional harmful chemotherapeutics in the prior art, the novel application of the drug compound in treating lung cancer is provided.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
use of S- (5' -adenosine) -L-methionine chloride in the manufacture of a medicament for the treatment of solid tumours.
Further, the solid tumor is lung cancer, liver cancer, ovarian cancer, and bladder cancer. Preferably, the lung cancer is non-small cell lung cancer or small cell lung cancer. Especially for small cell lung cancer.
Further, the S- (5 '-adenosine) -L-methionine chloride is S- (5' -adenosine) -L-methionine chloride dihydrochloride.
Preferably, the S- (5' -adenosine) -L-methionine chloride dihydrochloride has the following structural formula:
s- (5' -adenosine) -L-methionine chloride dihydrochloride has a CAS number of 86867-01-8;
its molecular formula is C 15 H 23 ClN 6 O 5 S·2HCl。
In summary, due to the adoption of the technical scheme, the beneficial effects of the invention are as follows:
1. the inventor verifies and confirms the high-efficiency inhibition effect of S- (5 '-adenosine) -L-methionine chloride on lung cancer cells or lung cancer tissues through cell experiments and animal experiments, determines that the S- (5' -adenosine) -L-methionine chloride has remarkable inhibition effect on lung cancer cells, is controllable in cytotoxicity, and has brand-new drug development potential for treating lung cancer. Especially has the inhibiting effect on the metastasis of lung cancer cells and has obvious curative effect on preventing and treating lung cancer.
2. The application of the S- (5' -adenosine) -L-methionine chloride in preparing the medicine for treating the solid tumor increases the optional range of the medicine for treating the solid tumor, has remarkable prevention and treatment effects on lung cancer, and has higher pertinence and higher inhibition rate on lung cancer compared with the traditional chemotherapy medicine.
Drawings
FIG. 1A is a chemical structural formula of S- (5' -adenosine) -L-methionine chloride dihydrochloride.
FIG. 1B is a graph showing relative organoid numbers at 48h, 72h, 96h, 120h after treatment of mouse small cell lung cancer organoids with SAM drugs at different concentrations.
FIG. 1C shows organoid changes in small cell lung cancer after 0.02mM SAM, with fewer mutations after drug action.
FIG. 1D is a plot of the proportion of organoids with synapses after drug action.
FIG. 2 is a graph showing the results of in vivo verification that SAM has an inhibitory effect on the growth and metastasis of mouse small cell lung cancer. Wherein, (A) the fluorescence signal intensity of the lungs of mice in the administration group and the solvent group is detected by using a luciferase living body imaging system; (B) flow-analyzing the proportion of circulating tumor cells in two groups of mice; (C) a statistical graph of the proportion of circulating tumor cells in two groups of mice; (D) liver metastasis plots for mice in dosing and solvent groups; (E) a liver metastasis statistical map; (F) HE staining of liver sections.
FIG. 3A is a graph showing relative organoid numbers at 48h, 72h, 96h, 120h after treatment of a small cell lung cancer patient HX20200923 organoid with SAM drugs at different concentrations.
FIG. 3B is a graph showing relative organoid numbers at 48h, 72h, 96h, 120h after treatment of a small cell lung cancer patient HX20201103 organoid with SAM drugs at different concentrations.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings.
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. Reagents for use in the present invention are commercially available unless specified.
Brief description of the invention the experimental procedure is as follows: fresh small cell lung cancer tissue of mice is obtained, collagenase is adopted to be digested into single cells, the single cells are mixed with matrigel to construct a 3D culture environment, and the 3D culture environment is prepared in a conditioned medium containing growth factors such as FGF10, RSPO1, wnt3a and the like at 37 ℃ and 5 percent CO 2 Culturing small cell lung cancer organoids in an incubator. Organoid transplantation using in situ transplantation techniques, monitoring tumor formation by small animal luciferase in vivo imaging, and diagnosis of tumor formation by H&E staining and immunofluorescence staining to perform pathological analysis on tumor, and the successful construction of the model is in vivo and in vitro drug testThe assay provides a new approach. The model can be applied to drug screening, drug toxicity tests, immunotherapy tests and the like.
Specifically, the cell test and animal test protocols of the present invention were carried out according to the following three parts in total of examples 1 to 3.
Example 1
In vitro validation of S- (5' -adenosine) -L-methionine chloride dihydrochloride against mouse small cell lung carcinoma organoids
Growth has inhibiting effect
Mouse small cell lung carcinoma organoids were 3D cultured in vitro using matrigel (BD Matrixgel) and multiple cytokines. In 96-well plates, SAM drugs were treated with a plurality of concentration gradients of 0.0,0.1,0.5,2.5mM. The relative organoid number of each well was calculated using microscopic photographs counted organoid number, with solvent set as control. The number of organoids with synapses was counted and the ratio was calculated to evaluate the metastatic capacity of small cell lung cancer cells.
Specifically, the experimental method comprises the following steps:
(1) Fresh small cell lung cancer tissue of the mice is taken and sheared on ice.
(2) Collagenase (1 mg/mL collagenase I and 0.5mg/mL collagenase IV) resuspension the minced tissue pieces was placed in a 50mL centrifuge tube and blown with a 10mL pipette to promote lysis of the tissue pieces. The amount of the sheared tissue blocks is 1-2 g, and the amount of collagenase is 10mL.
(3) A50 mL centrifuge tube containing collagenase and tissue blocks is placed in a shaking table at 37 ℃ and at a speed of 220rpm for digestion for 10min, and then taken out and blown by a pipetting gun to fully disperse tissue cells.
(4) Placing in a shaking table for digestion for 10min, taking out, and blowing with a pipetting gun.
(5) Filtering the liquid containing the mouse lung cancer cells treated in the step (4) by using a 100 mu m cell screen.
(6) After filtration, the supernatant was removed by centrifugation at 1500rpm for 5min at room temperature.
(7) 5ml of DMEM/F12 (Gibco, GREF#C11330500 BT) was added and resuspended, and the supernatant was removed by centrifugation at 1500rpm for 5min at room temperature.
(8) After cell counting, approximately 30 μl of Martrigel was mixed per 10000 cells and dropped into the center of the 48-well plate well.
(9) Transfer to 37℃and 5% CO 2 The Martrigel is solidified for 10-20min.
(10) 150. Mu.L of cell culture medium was added to each well and cultured in a cell culture incubator.
(11) After 2-3 days of culture, the medium in the wells was removed, the cells were resuspended in centrifuge tubes with TrypLE and bathed in a 37 degree celsius water bath for 10min. Taking out and blowing once in the middle.
(12) Centrifugation was performed at 1500rpm for 5min at room temperature, and the supernatant was removed. After cell counting, approximately 10 μl of Martrigel was mixed per 3000 cells and dropped into the center of the 96-well plate well.
(13) After 24h, the cell culture medium was changed to that containing SAM drugs at a concentration gradient of 0.0,0.1,0.5,2.5mM.
(14) The organoid numbers were counted by microscopic photographing after 48h, 72h, 96h and 120h of drug action, respectively.
(15) The relative organoid number per well was calculated compared to the solvent control. The calculation method comprises the following steps: taking SAM well as an example, relative organoid number= (organoid number of SAM well)/(organoid number of solvent well before dosing).
As shown in fig. 1B, 1C and 1D, the experimental results of in vitro verification that SAM has an inhibitory effect on the growth of mouse small cell lung cancer organoids show that SAM concentration is 2.5mM, and lung cancer organoids are completely inhibited 48 hours after administration; at a SAM concentration of 0.1mM, lung cancer organoid survival was essentially zero after 96 hours of action.
FIG. 1B shows relative organoid numbers at 48h, 72h, 96h, 120h after treatment of mouse small cell lung cancer organoids with different concentrations of SAM drugs.
FIG. 1C shows organoid changes in small cell lung cancer after 0.02mM SAM, with fewer mutations after drug action.
Figure 1D shows the proportion of organoids with synapses after drug action.
Example 2
In vivo validation of growth and in vivo validation of S- (5' -adenosine) -L-methionine chloride dihydrochloride against small cell lung cancer in mice
Metastasis has inhibiting effect
The mouse small cell lung cancer cells were transplanted in situ into the mouse lung. After 10 days, mice were examined for tumor burden by a luciferase biopsy system and intragastric administration was initiated. The administration dose is 100mg/kg. The administration was continued for 14 days, and the tumor burden of the mice was detected by a luciferase biopsy system during the administration. Peripheral blood is collected through the eyebox of the mouse, and the proportion of circulating tumor cells is analyzed by flow. Mice were dissected after dosing was completed, and lung in situ tumor size and other organ metastases were counted.
Specifically, the cell experiment method comprises the following steps:
(1) The lung cancer tissue of the mice was digested with Trpl E and digested into single cells.
(2) About 1 x 10 5 Cells were resuspended with 25. Mu.L LPBS, and then mixed by pipetting with 25. Mu.L Matrigel.
(3) The cell suspension was transplanted in situ into the left lung of mice using an insulin needle.
(4) Tumors in mice were detected by a luciferase in vivo imaging system 10 days after implantation. The detection method comprises the following steps: mice were intraperitoneally administered 150mg/kg of D-potassium fluorescein (Biovision, cat #7903-10 PK) and imaged on an IVIS spectroscopic in vivo imaging system (Perkinelmer) after 10-15 minutes of injection.
(5) Mice were divided into dosing and solvent groups based on fluorescence signal, 3 mice per group, and two with similar initial fluorescence signal values were paired.
(6) The mice were administered by gavage once daily at a dose of 100mg/kg for 14 days.
(7) Tumors in mice were detected by luciferase biopsy system 7 and 14 days after initiation of dosing. Peripheral blood is collected through the eyebox of the mouse, and the proportion of circulating tumor cells is analyzed by flow.
(8) After the administration is completed, the mice are opened, and the size of lung in-situ tumor is counted, and the metastasis of organs such as liver, lymph node, kidney and the like is counted.
The experimental results are shown in fig. 2, and the experimental results show that the tumor load of the administration group is smaller than that of the control group, and the flow analysis shows that the circulating tumor cells are obviously reduced. Mice were dissected and the number of liver metastases was found to be significantly reduced in the dosed group compared to the control group.
Figure 2 shows that SAM has an inhibitory effect on growth and metastasis of mouse small cell lung cancer. In fig. 2, (a) fluorescence signal intensities of the lungs of mice in the dosing group and the solvent group were detected using a luciferase biopsy system; (B) flow-analyzing the proportion of circulating tumor cells in two groups of mice; (C) a statistical graph of the proportion of circulating tumor cells in two groups of mice; (D) liver metastasis plots for mice in dosing and solvent groups; (E) a liver metastasis statistical map; (F) HE staining of liver sections.
Example 3
In vitro validation of S- (5' -adenosine) -L-methionine chloride dihydrochloride on human small cell lung carcinoma organoids
Long has inhibiting effect
In vitro verification shows that SAM has inhibiting effect on growth of human small cell lung cancer organoid. Taking breast water of a small cell lung cancer patient, collecting tumor cells, and culturing in vitro to form organoids. SAM drug was added to the organoid medium at a concentration of 0.0,0.1,0.5,2.5mM. The relative organoid number of each well was calculated using microscopic photographs counted organoid number, with solvent set as control.
Specifically, the cell experiment method comprises the following steps:
(1) The breast water of the small cell lung cancer patient is filtered by a 100 mu m cell screen.
(2) After filtration, the supernatant was removed by centrifugation at 1500rpm for 5min at room temperature.
(3) The cell pellet was resuspended in red blood cell lysate, red blood cells were lysed on ice for 3min, centrifuged at 1500rpm for 3min, and the supernatant removed.
(4) 5ml DMEM/F12 was added to resuspend, centrifuged at 1500rpm for 5min at room temperature, the supernatant removed and the tumor cells collected.
(5) After cell counting, about 10 μl of Martrigel (BD, cat# 354230) was mixed per 3000 cells and dropped in the middle of 96-well plate wells.
(6) After 24h, the cell culture medium was changed to that containing SAM drugs at a concentration gradient of 0.0,0.1,0.5,2.5mM.
(7) The organoid numbers were counted by microscopic photographing after 48h, 72h, 96h and 120h of drug action, respectively.
(8) The relative organoid number per well was calculated compared to the solvent control. The calculation method comprises the following steps: taking SAM well as an example, relative organoid number= (organoid number of SAM well)/(organoid number of solvent well before dosing).
The experimental results are shown in fig. 3A and 3B, and the experimental results show that the growth of small cell lung cancer organoids of patients is significantly inhibited under the action of SAM of 0.1mM, 0.5mM and 2.5mM, and the patient has dose dependency. Wherein, FIG. 3A shows the relative organoid numbers at 48h, 72h, 96h, 120h after treatment of the HX20200923 organoids of small cell lung cancer patients with SAM drugs at different concentrations. FIG. 3B shows the relative organoid numbers at 48h, 72h, 96h, 120h after treatment of the HX20201103 organoids of small cell lung cancer patients with SAM drugs at different concentrations.
Claims (1)
- Use of s- (5' -adenosine) -L-methionine chloride dihydrochloride in the manufacture of a medicament for the treatment of small cell lung cancer;the structural formula of the S- (5' -adenosine) -L-methionine chloride dihydrochloride is as follows:。/>
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