CN115137729B - Small molecule medicine for preventing and/or treating CRC and application thereof - Google Patents
Small molecule medicine for preventing and/or treating CRC and application thereof Download PDFInfo
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- CN115137729B CN115137729B CN202210913120.5A CN202210913120A CN115137729B CN 115137729 B CN115137729 B CN 115137729B CN 202210913120 A CN202210913120 A CN 202210913120A CN 115137729 B CN115137729 B CN 115137729B
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- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A—HUMAN NECESSITIES
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
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- A—HUMAN NECESSITIES
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Abstract
The invention belongs to the technical field of medicines, and discloses a small molecule drug for preventing and/or treating colorectal cancer (CRC) and application thereof. The invention treats, stabilizes, prevents and/or delays CRC occurrence or progression by using Kinetin or its derivatives as a novel small molecule drug. Kinetin may block or partially block CRC cell ERK signaling pathway activation by inhibiting MEK phosphorylation. Kinetin can significantly inhibit the growth of CRC cells. The Kinetin can obviously inhibit the growth of CRC tumor and promote the regression of CRC tumor when being taken alone or combined with the chemotherapeutic drug 5-fluorouracil, and can obviously inhibit pathological changes of liver and kidney caused by the chemotherapeutic drug 5-fluorouracil (5-FU) when being combined. The small molecule compound of the invention provides a new direction and method for preventing and treating tumors such as CRC.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a small molecule medicine for preventing and/or treating CRC and application thereof.
Background
Colorectal cancer (Colorectal cancer, CRC) is the second highest malignant tumor in China. Patients with advanced CRC need radical surgery and adjuvant therapy, and although surgery or radiotherapy and chemotherapy achieve a certain effect in improving survival of patients, many patients in middle and late stages often lose the opportunity of treatment due to drug resistance of tumor cells and toxic and side effects of drugs; in addition, more than 30% of patients still experience recurrent metastasis after treatment, with only about 13% of CRC patients experiencing recurrent metastasis survival for 5 years.
In recent years, immunotherapy has made a breakthrough progress in the field of CRC treatment. The immune therapy achieves the aim of inhibiting and killing tumor cells by activating and enhancing the anti-tumor immune response of a host organism. PD-1 antibodies are useful in the treatment of metastatic CRC patients with mismatch repair defects or microsatellite highly unstable (dMMR/MSI-H) with up to 50% efficacy; however, the ratio of dMMR/MSI-H in CRC is only about 10%, about 90% of CRC patients with normal mismatch repair (mismatch repair proficient, pMMR) or microsatellite stabilized (microsatellite stability, MSS) are insensitive to immunotherapy. While the therapeutic effect of other immunotherapeutic drugs in CRC remains to be demonstrated.
Chemotherapy in combination with targeted therapy is the primary means of advanced CRC treatment, with the hyperactive ERK pathway driving most tumorigenesis including CRC. There are two classes of current targeted drugs for CRC: treatment of anti-vascular endothelial growth factor VEGF and anti-EGFR. For KRAS wild-type CRC patients with metastasis, chemotherapy in combination with cetuximab (anti-EGFR) can be used to extend patient survival, however about 40% of KRAS mutant patients still face the dilemma of no drug availability. Because the carcinogenic target of CRC and the exact action and mechanism thereof are not fully known, partial patients have primary and secondary drug resistance to the existing drugs, and different targeted drugs have different degrees of toxic and side effects, the existing clinical intervention effect of CRC is very limited. Therefore, there is an urgent need to find new drugs with little toxic and side effects, which can effectively treat CRC.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a small molecule drug for preventing and/or treating CRC and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the present invention provides a small molecule drug for the prevention and/or treatment of CRC, which is Kinetin or a derivative thereof.
The molecular weight of Kinetin (6-Furfureladene) is 215, and the structural formula is shown as follows:
kinetin (CAS: 525-79-1) is a cytokinin, which is a plant hormone that promotes cell division. Is commonly used for plant tissue culture, induces callus formation, and can induce the occurrence of regeneration buds. The half maximal inhibitory concentrations (IC 50) of cell viability of Kinetin-treated CRC cells HCT116 and SW480 for 72h were 38.18 μm and 56.43 μm, respectively; meanwhile, proliferation experiments show that the Kinetin can inhibit proliferation of CRC cells and has a dose-dependent effect. The Kinetin in the human CRC xenograft (PDX) can obviously inhibit the growth of tumors in vivo, even promote tumor regression, and has no pathological toxicity to the liver and kidney. The invention treats, stabilizes, prevents and/or delays CRC occurrence or progression by using Kinetin or its derivatives as a novel small molecule drug.
As a preferred embodiment of the small molecule drug according to the present invention, the derivatives include salts of the Kinetin, tautomers of Kinetin, salts of tautomers of Kinetin.
In a second aspect, the present invention provides a pharmaceutical composition for preventing and/or treating CRC, comprising: a) Kinetin or a derivative thereof and b) 5-FU.
According to the invention, the combination of the Kinetin and the chemotherapeutic medicine 5-FU can obviously inhibit the growth of tumors, even promote the regression of tumors, and has no pathological toxicity to the liver and the kidney, and the Kinetin can obviously inhibit the pathological changes of the liver and the kidney caused by the chemotherapeutic medicine 5-FU during the combination.
As a preferred embodiment of the pharmaceutical composition according to the invention, the derivatives comprise salts of said Kinetin, tautomers of Kinetin, salts of tautomers of Kinetin.
In a third aspect, the present invention provides a formulation for use in the prevention and/or treatment of CRC, said formulation comprising said small molecule drug or said pharmaceutical composition, and at least one pharmaceutically acceptable carrier.
As a preferred embodiment of the formulation according to the present invention, the formulation is an oral formulation or an injection.
In a fourth aspect, the invention applies the small molecule drug, the pharmaceutical composition or the preparation in preparing a medicament for preventing and/or treating tumors.
As a preferred embodiment of the use according to the invention, it is possible to administer the drug alone or in combination with a therapy.
As a preferred embodiment of the use according to the invention, the combination therapy comprises radiotherapy, chemotherapy and surgical treatment.
As a preferred embodiment of the use according to the invention, the tumor comprises a small intestine tumor or CRC.
Compared with the prior art, the invention has the beneficial effects that:
the invention treats, stabilizes, prevents and/or delays the occurrence or progress of CRC by taking Kinetin or a derivative thereof as a novel small molecule drug. The Kinetin can obviously inhibit the growth of CRC cells and the phosphorylation of MEK, and can obviously inhibit the growth of CRC tumors and promote the regression of CRC tumors when being singly or combined with the chemotherapeutic drug 5-FU in animal experiments, and can obviously inhibit the pathological changes of livers and kidneys caused by the chemotherapeutic drug 5-FU when being combined. The small molecule medicine of the invention provides a new direction and method for preventing and treating tumors such as CRC.
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FIG. 1 shows the effect of the CCK8 method on the cell viability of CRC cell HCT116 by Kinetin;
FIG. 2 shows the effect of the CCK8 method on the cell viability of CRC cell SW480 by Kinetin;
FIG. 3 is the effect of Kinetin on CRC cell proliferation;
FIG. 4 shows the effect of Western immunoblotting to detect Kinetin on the phosphorylation of CRC cell MEK;
FIG. 5 is the effect of Kinetin and a combination of Kinetin and 5-FU on a PDX animal model;
fig. 6 is a view of kidney and liver pathology sections of a PDX animal model under a microscope.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples. It will be appreciated by persons skilled in the art that the specific embodiments described herein are for purposes of illustration only and are not intended to be limiting.
The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available. The Kinetin drugs used in the examples were purchased from Selleck corporation (S2316).
Example 1: cell experiments prove that the effect of Kinetin on CRC cell proliferation capacity
(1) Cell culture
CRC cell lines HCT116, SW480 were deposited by the university of Zhongshan gastroenterology institute, and HCT116 and SW480 cells were cultured in RPMI-1640 (Gibco, USA) medium containing 10% fetal bovine serum (Invitrogen, USA) and placed in a 37℃incubator containing 5% CO 2.
(2) CCK8 assay to detect effect of Kinetin on CRC cell activity:
the cell activity of the CRC cell lines HCT116 and SW480 was examined by the CCK8 method.
The CCK8 method for detecting the cell activity comprises the following specific steps:
1) CRC cells HCT and SW480 cultured to logarithmic growth phase were inoculated in 96-well plates containing 5X 10 in 100. Mu.L of medium per well 3 And (3) respectively setting a blank group, a control group and a drug treatment group, wherein the blank group does not contain cells and only contains a culture medium, the control group contains cells and no drug, and the drug treatment group contains the Kinetin drugs with different concentrations of 0-100 mu M.
2) The next day, 100 μl of medium or medium containing different drug concentrations was added to each well. After the cells were continuously cultured in the incubator for 72 hours, 10. Mu.L of CCK8 solution was added to each well and incubated for 2 hours in the dark.
Absorbance values were measured at a wavelength of 450nm using a full wavelength multifunctional microplate reader.
The cell viability calculation method comprises the following steps:
(drug treatment OD value-blank OD value)/(control OD value-blank OD value) ×100%.
IC50 values were calculated using GraphPad Prism 8.0.1 software.
The results of the CCK8 assay for cell viability are shown in FIGS. 1 and 2, and the IC50 values of Kinetin for CRC cell lines HCT116 and SW480 are 38.18. Mu.M and 56.43. Mu.M, respectively.
(3) Effect of Kinetin on CRC cell proliferation
After 24h treatment of HCT116 cells with different concentrations of Kinetin (0. Mu.M, 50. Mu.M, 75. Mu.M), the cells were digested, resuspended in complete medium and diluted to a cell density of 5X 10 4 Mu.l of each well was inoculated in 96 wells per mlIn the plate, 96 wells were then placed in a cell incubator for 140h and observed in real time using an Incucyte Zoom instrument placed in the cell incubator. GraphPad prism 8.0.1 statistical analysis results.
In vitro cell proliferation experiments as shown in figure 3, kinetin inhibited CRC cell proliferation in a dose dependent manner.
Example 2: kinetin inhibits MEK phosphorylation of CRC cells
The inhibition of the phosphorylation of CRC cell MEK by Kinetin was examined by western blotting. The method comprises the following specific steps: after HCT116 cells were treated with blank or 50. Mu.M Kinetin for 24h, the medium was aspirated, the protein lysate was added, and the cells were lysed on ice, the lysate was transferred to a 1.5mL EP tube, broken for 10min at 4℃with a sonicator, centrifuged for 10min at 12000rpm, and the supernatant samples were aspirated and subjected to SDS-PAGE gel electrophoresis, transfer, blocking, incubation at 4℃for overnight with primary antibody (pMEK, MEK, GAPDH), incubation at room temperature for 1h with secondary antibody, and chemiluminescent development.
As shown in fig. 4, kinetin can inhibit MEK phosphorylation of CRC cells.
Example 3: animal experiments prove that the effect of Kinetin on CRC cell proliferation capacity
The effect of Kinetin alone or in combination with the chemotherapeutic drug 5-FU was verified in PDX animal models. The method comprises the following steps:
tumor tissues of freshly resected CRC patients were inoculated into the right anterior axilla of blood-enriched BALB/c nude mice, and PDX models were constructed.
After inoculation, the tumor growth size is up to 50mm 3 After left and right, nude mice are randomly grouped, and a control group (normal saline), a 5-FU single drug treatment group (10 mg/kg), a Kinetin single drug treatment group (2 mg/kg) and a 5-FU and Kinetin combined drug treatment group (10 mg/kg 5-FU+2mg/kg Kinetin) are respectively arranged, wherein the administration mode is that the 5-FU is intraperitoneally injected every other day, and the Kinetin is intraperitoneally injected every day for 22 days of continuous treatment.
Tumor size was measured every 3 days. The calculation formula of the tumor volume is V=a 2 Xb.times.0.58 (a is the tumor minimum diameter, b is the diameter perpendicular to a).
Nude mice were euthanized after treatment and tumors were dissected. All operations of the animal experiment are strictly carried out according to the animal experiment protection criteria.
The results of the PDX animal model are shown in fig. 5, and Kinetin alone or in combination with the chemotherapeutic drug 5-FU can significantly inhibit tumor growth and promote its regression. The inhibition effect of the Kinetin alone on the tumor growth is superior to that of the chemotherapeutic drug 5-FU alone; the inhibition effect of the combination of the Kinetin and the chemotherapeutic drug 5-FU on the tumor growth is superior to that of the treatment of the Kinetin by single administration.
Example 4: animal experiments to evaluate in vivo nephrotoxicity of Kinetin
The PDX animal model of example 3 was taken, after tumor isolation, kidneys and livers were extracted, fixed with 10% formaldehyde by volume fraction, paraffin-embedded, sectioned, HE stained, and observed under microscope for morphological changes of kidneys and livers.
The results of kidney and liver pathological sections are shown in figure 6,
the pathological change of the liver is obvious after the chemotherapy drug 5-FU is singly administered, and the local inflammatory infiltration exists; while the liver has no pathological change when Kinetin is singly administered; and the combination of the Kinetin and the chemotherapeutic drug 5-FU can obviously inhibit the pathological change of the liver caused by the chemotherapeutic drug 5-FU.
The pathological change of the kidney exists when the chemotherapeutic 5-FU is singly administered, and the tubular is slightly expanded, so that the kidney filtration rate is reduced; the kidney of the Kinetin is not pathologically changed when being singly administrated, and the Kinetin and the chemotherapeutic 5-FU are combined to obviously inhibit the pathologic change of the kidney caused by the chemotherapeutic 5-FU.
It is known that the administration of Kinetin alone or in combination with the chemotherapeutic drug 5-FU has no pathological toxicity to the liver and kidney of nude mice.
In conclusion, the invention screens out the medicine Kinetin (CAS: 525-79-1), and the in vitro cell experiment result shows that the IC50 values of the Kinetin on CRC cells HCT116 and SW480 are 38.18 mu M and 56.43 mu M respectively; meanwhile, proliferation experiments show that the Kinetin can inhibit proliferation of CRC cells and has a dose-dependent effect. Further constructing a PDX model, finding that the Kinetin can obviously inhibit tumor growth and even promote tumor regression by singly or in combination with the Kinetin chemotherapeutic drug 5-FU, and has no pathological toxicity to the liver and kidney of a nude mouse, and the Kinetin can be considered to have no toxicity to the nude mouse. Kinetin inhibits CRC by inhibiting MEK phosphorylation block or partially block the transmission of ERK signals. Therefore, kinetin can be used as a small molecule drug for the treatment of CRC.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (5)
- Use of Kinetin or a salt of Kinetin for the preparation of a medicament for the treatment of colorectal cancer.
- 2. Use of a pharmaceutical composition consisting of Kinetin or a salt of Kinetin, and 5-FU, for the preparation of a medicament for the treatment of colorectal cancer.
- 3. Use of a formulation consisting of Kinetin or a salt of Kinetin, and at least one pharmaceutically acceptable carrier for the preparation of a medicament for the treatment of colorectal cancer.
- 4. Use of a formulation consisting of the pharmaceutical composition of claim 2, and at least one pharmaceutically acceptable carrier for the manufacture of a medicament for the treatment of colorectal cancer.
- 5. The use according to any one of claims 3 to 4, wherein the formulation is an oral formulation or an injection.
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| CN202210913120.5A CN115137729B (en) | 2022-07-29 | 2022-07-29 | Small molecule medicine for preventing and/or treating CRC and application thereof |
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| CN202210913120.5A CN115137729B (en) | 2022-07-29 | 2022-07-29 | Small molecule medicine for preventing and/or treating CRC and application thereof |
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| CN115137729A CN115137729A (en) | 2022-10-04 |
| CN115137729B true CN115137729B (en) | 2023-09-26 |
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Non-Patent Citations (1)
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| 植物细胞分裂素ortho-Topolin Riboside对人白血病细胞株THP-1的抗癌活性及机制初探;邓营营等;《食品工业科技》;20200331(第06期);第299-304页 * |
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