CN114903901A - Medicine for treating intrahepatic bile duct cell cancer - Google Patents
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Abstract
The invention provides a medicine for treating tumors, in particular to a medicine for treating intrahepatic cholangiocellular carcinoma (intrahepatic cholangiocardiocarcinoma iCCA). The drug is a Cyclin Dependent Kinase (CDK) extensive inhibitor, Ronicolib. Research results show that the wide inhibitor Ronicolib of the Cyclin Dependent Kinase (CDK) has the effects of remarkably inhibiting the growth and migration of intrahepatic bile duct cancer cells and the growth of subcutaneous tumors of nude mice, and the Ronicolib is used as a pharmaceutical active ingredient and added with pharmaceutically acceptable additives or/and excipients or/and carriers to prepare the medicine for treating tumors.
Description
Technical Field
The invention belongs to the field of medicine and pharmacy, relates to a medicine for treating tumor, and particularly relates to a medicine for treating intrahepatic cholangiocellular carcinoma.
Background
Intrahepatic cholangiocellular carcinoma (intrahepatic cholangiococcoxicarnoma iCCA) is a malignant tumor originated from intrahepatic cholangiocarcinoma, is the second largest hepatic primary malignant tumor with the incidence rate only second to that of hepatocellular carcinoma, accounts for about 10% -15% of the hepatic primary malignant tumors, and has an increasing trend in recent years. Due to the characteristics of latent disease, low sensitivity of cytological and pathological diagnosis and the like, most patients are diagnosed as the disease progressive stage. At present, diagnosis of iCCA mainly depends on symptoms and signs of patients, imaging examination such as CT/MRI and the like, serological indexes such as CA-19-9 and the like, and a sensitive and specific diagnostic marker is lacked, most of patients are found to be in the progressive stage of diseases, the radical resection chance is lost, the prognosis of iCCA patients is extremely poor, and surgical resection of tumors is considered as a main treatment means, but only about 35 percent of early patients have the radical resection indication. The effect of the simple chemical drug therapy applied to intrahepatic cholangiocellular carcinoma is poor, and a drug therapy means for radically treating iCCA is still lacked at present. The peak onset of ICC is from 55 to 75 years of age, with less than 10% of cases occurring before the age of 45. The method is used for regularly screening the population with age greater than 45 years old and high risk factors, and is beneficial to early diagnosis and timely treatment of ICC. Unlike the high incidence of liver cancer in men, the incidence ratio of ICC in men and women is about 2: 3. The strong academician of yellow aspiration states that in the text of the development direction of hepatobiliary surgery, biliary duct cancer patients in China account for 55% of the world [ see: huang Zhi Qiang, the development of hepatobiliary surgery [ J ] surgical theory and practice, 2011,16(4): 329-. Considering the proportion of ICC in cholangiocarcinoma, the ICC patient population in China is quite large. The disease has high malignancy, poor prognosis and few long-term survivors. ICC has no obvious clinical symptoms at the early stage, and most patients lose the operation time when finding the ICC, so that higher requirements on early diagnosis and timely treatment of the ICC are provided. Surgical operation is the first choice of treatment for prolonging the life of ICC patients, and radical surgical resection is dependent on the location and size of cancer, and includes left and right hemihepatectomy, left and right hepatectomy, wedge hepatectomy, and hepatectomy. Radical surgical resection can significantly prolong the life of patients. No 5-year survivors were observed in patients with palliative surgical resection, conservative treatment and no treatment. ICC is lymphatic invasive and is more prone to lymph node metastasis. The recurrence rate after ICC surgery is high. For patients with large tumor volume or unsuitable position for operation, chemotherapy should be performed first, and then relevant operation treatment should be performed after the tumor is descended. There are many disputes about the factors affecting the treatment effect and recurrence of ICC, and many patients with ICC lose the operative time and have high recurrence rate after surgery, so that the clinician needs to select a proper auxiliary treatment mode to achieve the purpose of curing or reducing recurrence. The postoperative adjuvant chemotherapy should have better effect than simple operation, and chemical drug therapy is required when lymph node metastasis and/or blood vessel invasion exist. The pure chemical drug therapy is mainly applied to patients with extrahepatic metastasis and possible loss of other therapeutic measures. Because chemotherapy is now a possible way to prolong patient survival, current chemotherapy applied to ICC is not as effective. Therefore, the research and search of effective chemical drugs for treating ICC is currently an important issue.
Disclosure of Invention
The aim of the invention is to provide a medicament for treating tumors, in particular intrahepatic cholangiocellular carcinoma (intrahepatic cholangiocardiocarcinoma iCCA).
The technical scheme for realizing the invention is as follows:
the drug for treating intrahepatic cholangiocellular carcinoma (intrahepatic cholangiocardia iCCA) provided by the invention is a cyclin-dependent kinase (CDK) extensive inhibitor Ronicolib.
The molecular formula of the Cyclin Dependent Kinase (CDK) extensive inhibitor Ronicolib is C 18 H 21 F 3 N 4 O 3S Molecular weight of 430.44, and chemical formula as shown in the following formula:
life science agents suppliers such as shanghai pottery biochemical, mce (medchemexpress) can provide the inhibitors.
The invention discloses that a Cyclin Dependent Kinase (CDK) extensive inhibitor Ronicolib has the effects of obviously inhibiting the growth of tumor cells, obviously inhibiting the migration of the tumor cells and obviously inhibiting the growth of subcutaneous tumors of nude mice, and can be used for preparing a medicament for treating tumors.
In one embodiment of the invention, the hepatobiliary carcinoma cell line HCCC-9810 cells are paved into a 96-hole cell culture plate at 1000 cells/hole, 100 mu l of RPMI1640 culture medium (purchased from Hyclone) containing 10% Fetal Bovine Serum (FBS) is added, and 5% CO is contained at 37 DEG C 2 After culturing in the incubator for 16 hours, 100. mu.l of RPMI1640 medium containing 10% Fetal Bovine Serum (FBS) was replaced; the 96-well cell culture plate was divided into two zones, and one zone was added with 2.5X10 Ronicolib (MCE Co., Ltd., cat. No.: HY-13914) in 10mM of a mother solution in dimethyl sulfoxide (DMSO) solvent -4 μ l to a final concentration of 25nm as experimental group; in another zone, 2.5X10 solvent dimethyl sulfoxide (DMSO) without roniclib was added -4 μ l as non-medicated treatment group; the 96-well cell culture plate was incubated at 37 ℃ with 5% CO 2 After five days of culture in the incubator, cell proliferation was measured by the CCK8 method on the fifth day, which shows that roniclib can significantly inhibit tumor cell growth, as shown in table 1 in fig. 2 and example 1.
Another embodiment of the invention provides HCCC-9810 cells at 4X10 5 Individual cells/well were plated in 6-well plates at 37 ℃ with 5% CO 2 After the cells were attached to the wall for 16 hours in the incubator, 1.5ml of RPMI1640 medium containing 10% Fetal Bovine Serum (FBS) was added, and 10mM of Ronicolib 3.75X10 in a mother solution of dimethyl sulfoxide (DMSO) was added -3 After incubation of μ l to a final concentration of 25nm for 24 hours, the supernatant was discarded, cells were collected and counted, and 1 × 10 was added 5 HCCC-9810 cells were resuspended in 200. mu.l serum-free RPMI1640 medium, plated in 8 μm upper chamber of transwell cell transfer plate (Corning Corp., cat. No. 3422), 500. mu.l RPMI1640 medium containing 10% fetal bovine serum was added to the lower chamber, the same 6-well plate was divided into two zones, and 3.75X10 dimethyl sulfoxide (DMSO) solvent without Roniclil was added to the other zone -3 Mu.l of the cells were used as an untreated group, and after treatment under the same culture conditions and for the same time, the collected cells were plated in different upper chambers of the same transwell cell migration plate under the same cell number and conditions,after the same conditioned medium was added to the lower chamber, the plate was incubated at 37 ℃ with 5% CO 2 After 24 hours of incubation in the incubator, the cells migrated through the polycarbonate membrane were stained with 0.5% crystal violet and counting the number of cells by light microscopy showed that roniclib significantly inhibited tumor cell migration, see table 2 in fig. 3 and example 2.
Yet another embodiment of the present invention would be 4x10 6 HCCC-9810 cells are resuspended in 100 μ l PBS and injected into 5-week-old female nude mice subcutaneously, the nude mice are randomly divided into two groups of 8 mice each, and the tumor grows to 60mm 3 During the volume, the Ronicolib is dissolved into 600x mother liquor by DMSO, sulfobutyl-beta-cyclodextrin sodium is added to prepare 1.5mg/kg of dosage, a 1ml injector is adopted to cooperate with a No. 12 intragastric administration to perform intragastric administration on tumor-bearing nude mice for 200 mu l of volume, the intragastric administration is performed for 1 time every 1 day, 7 times of intragastric administration are taken as an experimental group, the other group is used as an non-administration treatment group by administering 200 mu l of DMSO mother liquor and sulfobutyl-beta-cyclodextrin sodium diluent through an intragastric administration needle with the same type for the same times, the nude mice are continuously fed after the intragastric administration is stopped for 8 days and then are killed, the tumor volume is measured every 5 days during the experimental period, and the tumors are stripped and weighed after the nude mice are killed, and the result shows that the Ronicolib can obviously inhibit the volume and the weight of the subcutaneous tumors of the nude mice, and the Ronicolib can be seen in a table 3 and a table 4 in a figure 4, a figure 5 and an embodiment 3.
The research result shows that the paniciclib which is a broad inhibitor of Cyclin Dependent Kinase (CDK) can obviously inhibit the growth and migration of intrahepatic bile duct cancer cells and the growth of subcutaneous tumors thereof. The pharmaceutical preparation for treating intrahepatic bile duct cell cancer, such as tablets, granules, liquid preparations and the like, can be prepared by taking the Ronicolib as a pharmaceutical active ingredient and adding pharmaceutically acceptable additives or/and excipients or/and carriers.
Drawings
FIG. 1: the chemical structural formula of Ronicolib.
FIG. 2: the CCK8 experiment detects intrahepatic bile duct cancer cell proliferation. The experiment is repeated three times, and the result shows that the Ronicolib obviously inhibits the growth of the tumor cell HCCC-9810 compared with the group without the drug. Ctrl, non-dosed group. Roniclib, roniclib experimental group. Absorbance, CCK8 test Absorbance values. P < 0.001.
FIG. 3: transwell cell migration assay detects intrahepatic bile duct cancer cell migration. The experiment is repeated three times, and the result shows that the Roniclib obviously inhibits the migration of the tumor cell HCCC-9810 compared with the cells of the group which are not added with drugs. Mock, non-medicated treatment group. Roniclib, roniclib experimental group. Number of grafted 9810cells, Number of HCCC-9810 cells migrated through the polycarbonate membrane. P < 0.001.
FIG. 4 is a schematic view of: and (3) carrying out intragastric administration treatment after subcutaneous tumor formation of the nude mice to detect the drug curative effect experiment. The major and broad diameters of the tumors were measured every 5 days during the animal experiments according to the formula: tumor volume was calculated as 0.52x length x width 2, indicating that roniclib significantly inhibited the size of subcutaneous tumors in nude mice. Ctrl, no drug-treated group. Roniclib, roniclib experimental group. Days, Days. Tumor volume (mm3), Tumor volume (cubic millimeters). P < 0.001.
FIG. 5 is a schematic view of: the tumor is stripped and weighed after the nude mice are sacrificed, and the result shows that the Ronicolib can obviously inhibit the weight of the subcutaneous tumor of the nude mice. Ctrl, non-dosed group. Roniclib, roniclib experimental group. Tumor weight (g), Tumor weight (g). P < 0.001.
Detailed Description
Example 1: ronicolib significantly inhibits tumor cell growth
Cells of intrahepatic bile duct cancer cell line HCCC-9810 were plated at 1000 cells/well in 96-well cell culture plates, 100. mu.l of RPMI1640 medium (purchased from Hyclone) containing 10% Fetal Bovine Serum (FBS) and containing 5% CO at 37 ℃ were added 2 After culturing in the incubator for 16 hours, 100. mu.l of RPMI1640 medium containing 10% Fetal Bovine Serum (FBS) was replaced; the 96-well cell culture plate was divided into two zones, and one zone was filled with 10mM of Ronicolib (MCE, cat. No.: HY-13914)2.5X10 in a mother solution of dimethyl sulfoxide (DMSO) as a solvent -4 μ l to a final concentration of 25nm as experimental group; in another zone, 2.5X10 solvent dimethyl sulfoxide (DMSO) without roniclib was added -4 μ l as non-medicated treatment group; the 96-well cell culture plate was incubated at 37 ℃ with 5% CO 2 Culturing in incubator for five days, and detecting cell proliferation condition by CCK8 method on the fifth dayIn this case, the results show that roniclib significantly inhibited tumor cell growth, see figure 2 and table 1.
Table 1 shows the results of three replicates of CCK8 testing cell proliferation for the non-medicated and Roniclib treated groups. Ctrl, no drug-treated group. Roniclib, roniclib experimental group. Absorbance, CCK8 test Absorbance values.
| Absorbance | Ctrl | Roniciclib |
| 1 | 1.459 | 0.034 |
| 2 | 1.434 | 0.037 |
| 3 | 1.531 | 0.000 |
Example 2: ronicolib significantly inhibits tumor cell migration
HCCC-9810 cells at 4X10 5 Individual cells/well were plated in 6-well plates with 5% CO at 37 ℃ 2 After the cells were attached to the surface for 16 hours in the incubator, 1.5ml of RPMI1640 medium containing 10% Fetal Bovine Serum (FBS) was added, and 10mM of Ronicolib 3.75X10 in a mother solution of Dimethylsulfoxide (DMSO) as a solvent was added -3 Mu.l to a final concentration of 25nm for 24 hours, discarding the supernatant, and collectingCells were pooled and counted, 1X10 5 HCCC-9810 cells were resuspended in 200. mu.l serum-free RPMI1640 medium, plated on a transwell cell transfer plate (Corning, Cat. 3422)8 μm upper chamber, 500. mu.l RPMI1640 medium containing 10% fetal bovine serum was added to the lower chamber, the same 6-well plate was divided into two zones, and 3.75X10 dimethyl sulfoxide (DMSO) solvent without Ronicolib was added to the other zone -3 Mu.l of the cells were collected as a non-drug-treated group, treated under the same culture conditions and time, plated in the same transwell cell migration plate in the same upper chamber and the same culture medium was added to the lower chamber, and the plate was incubated at 37 ℃ with 5% CO 2 After 24 hours in the incubator, the cells migrated through the polycarbonate membrane were stained with 0.5% crystal violet, and counting the number of cells with an optical microscope showed that roniclib significantly inhibited tumor cell migration (fig. 3, table 2).
Table 2 migration number of cells in Transwell experiment of the non-drug treatment group and the Roniclib treatment group, the experiment was repeated three times, and six observation fields were selected. Mock, non-medicated treatment group. Roniclib, roniclib experimental group.
Example 3: ronicolib significantly inhibits the growth of subcutaneous tumors in nude mice
Will 4x10 6 HCCC-9810 cells are resuspended in 100 mul PBS and injected into 5 weeks old female nude mice subcutaneously, the nude mice are randomly divided into two groups, each group comprises 8, when the tumor grows to 60mm 3 When the tumor size is measured, Ronicolib is dissolved into 600x mother liquor by DMSO, sulfobutyl betadex sodium is added to prepare 1.5mg/kg dose, a 1ml injector is adopted to match with a 12-grade gavage to perform gavage for a tumor-bearing nude mouse with 200 mu l of volume, the gavage is performed for 1 time every 1 day, the gavage is performed for 7 times in total as an experimental group, the other group uses a gavage needle with the same type to perform gavage for 200 mu l of volume of DMSO mother liquor and sulfobutyl betadex sodium diluent for the same times as a non-administration treatment group, the nude mouse is continuously raised after stopping the gavage for 8 days and then is killed, the tumor volume is measured every 5 days during the experimental period,tumors were dissected and weighed after sacrifice, and the results showed that roniclib significantly inhibited the size and weight of subcutaneous tumors in nude mice (fig. 4, fig. 5, table 3, table 4).
Table 3: volume size data of subcutaneous tumors in nude mice in control and roniclib treated groups. Ctrl, non-dosed group. Roniclib, roniclib experimental group. Days, Days. Volume (mm3), tumor Volume (cubic millimeters).
Table 4: weight size data of detached tumors in control and roniclib treated groups after sacrifice of nude mice. Ctrl, no drug-treated group. Roniclib, roniclib experimental group. Weight (g), tumor weight (g).
| Weight(g) | Ctrl | Roniciclib |
| 1 | 0.40 | 0.20 |
| 2 | 0.42 | 0.18 |
| 3 | 0.58 | 0.05 |
| 4 | 0.42 | 0.05 |
| 5 | 0.36 | 0.00 |
| 6 | 0.20 | 0.00 |
| 7 | 0.25 | 0.00 |
| 8 | 0.20 | 0.00 |
Claims (6)
1. Use of a Cyclin Dependent Kinase (CDK) pan-inhibitor roniiclib for the preparation of a medicament for the treatment of tumours.
2. The use according to claim 1 wherein the Cyclin Dependent Kinase (CDK) pan-inhibitor roniiclib has the formula:
3. a medicament for the treatment of tumors, comprising as an active ingredient a Cyclin Dependent Kinase (CDK) pan-inhibitor roniiclib.
4. A pharmaceutical preparation for treating tumors is characterized in that the pharmaceutical preparation is a medicament prepared by taking a Cyclin Dependent Kinase (CDK) extensive inhibitor Ronicolib as an active ingredient and adding pharmaceutically acceptable additives or/and excipients or/and carriers.
5. The pharmaceutical formulation of claim 4, wherein the pharmaceutical formulation is a tablet, a granule or a liquid formulation.
6. The tumor of claim 1, 3 or 4, which is intrahepatic cholangiocellular carcinoma.
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| CN202110183444.3A CN114903901A (en) | 2021-02-08 | 2021-02-08 | Medicine for treating intrahepatic bile duct cell cancer |
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Non-Patent Citations (2)
| Title |
|---|
| GERHARD SIEMEISTER等: "BAY 1000394, a Novel Cyclin-Dependent Kinase Inhibitor, with Potent Antitumor Activity in Mono- and in Combination Treatment upon Oral Application" * |
| MINAKO YAMAMURA等: "The cyclin-dependent kinase pathway involving CDK1 is a potential therapeutic target for cholangiocarcinoma" * |
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