CN115137729B - A small molecule drug for preventing and/or treating CRC and its application - Google Patents

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

A small molecule drug for preventing and/or treating CRC and its application

Technical field

The present invention relates to the field of medical technology, and specifically to a small molecule drug for preventing and/or treating CRC and its application.

Background technique

Colorectal cancer (CRC) is the second most common malignant tumor in my country. Patients with advanced CRC need to receive radical surgery and adjuvant treatment. Although surgery or radiotherapy and chemotherapy have achieved certain results in improving patient survival, many patients with intermediate and advanced stages often suffer from drug resistance due to tumor cell resistance and toxic and side effects of drugs. The opportunity for treatment is lost; in addition, more than 30% of patients will still develop recurrence and metastasis after treatment, and the 5-year survival rate of CRC patients with recurrence and metastasis is only about 13%.

In recent years, immunotherapy has made breakthrough progress in the field of CRC treatment. Immunotherapy achieves the purpose of inhibiting and killing tumor cells by activating and enhancing the host body's anti-tumor immune response. PD-1 antibodies are 50% effective in treating metastatic CRC patients harboring mismatch repair deficiency or microsatellite instability high (dMMR/MSI-H); however, the ratio of dMMR/MSI-H in CRC is only About 10% and about 90% of CRC patients with normal mismatch repair (mismatch repair proficient, pMMR) or microsatellite stable (microsatellite stability, MSS) are not sensitive to immunotherapy. The therapeutic effect of other immunotherapy drugs in CRC remains to be confirmed.

Chemotherapy combined with targeted therapy is the main method for the treatment of advanced CRC. Overactivated ERK pathway drives the occurrence of most tumors, including CRC. There are currently two types of targeted drugs for CRC: anti-vascular endothelial growth factor VEGF and anti-epidermal growth factor receptor EGFR treatments. For patients with metastatic KRAS wild-type CRC, chemotherapy combined with cetuximab (anti-EGFR) can be used to prolong patient survival. However, about 40% of patients with KRAS mutations still face the dilemma of no available drugs. Since the oncogenic targets of CRC and their exact functions and mechanisms have not been fully understood, some patients have primary and secondary resistance to existing drugs, and different targeted drugs still have varying degrees of toxic and side effects. CRC Existing clinical interventions have very limited effects. Therefore, there is an urgent need to find new drugs with less side effects and effective treatment for CRC.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a small molecule drug for preventing and/or treating CRC and its application.

In order to achieve the above objects, the technical solutions adopted by the present invention are as follows:

In a first aspect, the present invention provides a small molecule drug for preventing and/or treating CRC, and the small molecule drug is Kinetin or a derivative thereof.

The molecular weight of Kinetin (6-Furfuryladenine) is 215, and its structural formula is as follows:

Kinetin (CAS:525-79-1) is a cytokinin, a plant hormone that can promote cell division. It is often used in plant tissue culture to induce callus formation and can induce the occurrence of regenerated buds. The half inhibitory concentrations (IC50) of cell viability of HCT116 and SW480 cells treated with Kinetin for 72 hours were 38.18 μM and 56.43 μM respectively; at the same time, proliferation experiments showed that Kinetin could inhibit the proliferation of CRC cells in a dose-dependent manner. Kinetin in human-derived CRC xenografts (PDX) can significantly inhibit tumor growth in vivo and even promote tumor regression, and has no pathological toxicity to the liver or kidneys. The present invention uses Kinetin or its derivatives as a new small molecule drug to treat, stabilize, prevent and/or delay the occurrence or progression of CRC.

As a preferred embodiment of the small molecule drug of the present invention, the derivatives include salts of Kinetin, tautomers of Kinetin, and salts of tautomers of Kinetin.

In a second aspect, the present invention provides a pharmaceutical composition for preventing and/or treating CRC, which pharmaceutical composition includes: a) Kinetin or a derivative thereof and b) 5-FU.

In the present invention, the combined use of Kinetin and the chemotherapeutic drug 5-FU can significantly inhibit tumor growth and even promote tumor regression without pathological toxicity to the liver and kidneys. When used in combination, Kinetin can significantly inhibit the liver and kidney damage caused by the chemotherapeutic drug 5-FU. Kidney pathological changes.

As a preferred embodiment of the pharmaceutical composition of the present invention, the derivatives include salts of Kinetin, tautomers of Kinetin, and salts of tautomers of Kinetin.

In a third aspect, the present invention provides a preparation for the prevention and/or treatment of CRC, which preparation includes the small molecule drug or the pharmaceutical composition, and at least one pharmaceutically acceptable carrier.

As a preferred embodiment of the preparation of the present invention, the preparation is an oral preparation or an injection.

In the fourth aspect, the present invention uses 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 application of the present invention, they can be administered alone or in combination.

As a preferred embodiment of the application of the present invention, the combined treatment includes radiotherapy, chemotherapy and surgical treatment.

As a preferred embodiment of the application of the present invention, the tumor includes small intestinal tumor or CRC.

Compared with the prior art, the beneficial effects of the present invention are:

The present invention uses Kinetin or its derivatives as a new small molecule drug to treat, stabilize, prevent and/or delay the occurrence or progression of CRC. Kinetin can significantly inhibit the growth of CRC cells and MEK phosphorylation. In animal experiments, Kinetin alone or combined with the chemotherapy drug 5-FU can significantly inhibit the growth of CRC tumors and promote their regression. When used in combination, Kinetin can significantly inhibit the growth of CRC cells and promote their regression. Inhibits the pathological changes in the liver and kidneys caused by the chemotherapy drug 5-FU. The small molecule drug of the present invention provides new directions and methods for the prevention and treatment of tumors such as CRC.

Description of the drawings

Figure 1 shows the CCK8 method to detect the effect of Kinetin on the cell viability of CRC cell HCT116;

Figure 2 shows the CCK8 method to detect the effect of Kinetin on the cell viability of CRC cell SW480;

Figure 3 shows the effect of Kinetin on CRC cell proliferation;

Figure 4 shows the Western blotting method to detect the effect of Kinetin on MEK phosphorylation in CRC cells;

Figure 5 shows the effects of Kinetin and the combination of Kinetin and 5-FU on the PDX animal model;

Figure 6 shows the pathological sections of the kidneys and liver of the PDX animal model under a microscope.

Detailed ways

In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described here are only used to explain the present invention and are not intended to limit the present invention.

Unless otherwise specified, the test methods used in the examples are conventional methods; unless otherwise specified, the materials and reagents used can be obtained from commercial sources. The Kinetin drug used in the examples was purchased from Selleck Company (S2316).

Example 1: Cell experiments verify the effect of Kinetin on the proliferation ability of CRC cells

(1) Cell culture

CRC cell lines HCT116 and SW480 were preserved by the Institute of Gastroenterology, Sun Yat-sen University. HCT116 and SW480 cells were cultured in RPMI-1640 (Gibco, USA) medium containing 10% fetal calf serum (Invitrogen, USA) and placed in a medium containing 5 Culture in a 37°C constant-temperature cell culture incubator with % CO2.

(2) CCK8 method detects the effect of Kinetin on CRC cell activity:

CCK8 method was used to detect the cell viability of CRC cell lines HCT116 and SW480.

The specific steps for detecting cell activity using the CCK8 method are:

1) The CRC cells HCT116 and SW480 cultured to the logarithmic growth phase were inoculated into a 96-well plate. Each well contained 5 × 10 ³ cells in 100 μL culture medium. A blank group, a control group and a drug-treated group were set up respectively. The blank group There are no cells but culture medium, the control group contains cells without drugs, and the drug treatment group contains different concentrations of Kinetin drug from 0 to 100 μM.

2) The next day, add 100 μL of culture medium or culture medium containing different drug concentrations to each well. After the cells were cultured in the incubator for 72 hours, 10 μL of CCK8 solution was added to each well and incubated in the dark for 2 hours.

Use a full-wavelength multifunctional microplate reader to measure the absorbance value at a wavelength of 450 nm.

The calculation method of cell viability is:

(OD value of the drug treatment group - OD value of the blank group)/(OD value of the control group - OD value of the blank group) × 100%.

IC50 values were calculated using GraphPad Prism 8.0.1 software.

The results of cell viability detection by CCK8 method are shown in Figure 1 and Figure 2. The IC50 values of Kinetin for CRC cell lines HCT116 and SW480 are 38.18 μM and 56.43 μM respectively.

(3) Effect of Kinetin on CRC cell proliferation

After treating HCT116 cells with different concentrations of Kinetin (0 μM, 50 μM, 75 μM) for 24 hours, digest the cells, resuspend the cells in complete culture medium, dilute the cells to a density of 5 × ¹⁰ cells/ml, and inoculate 100 μl in each well in a 96-well plate. , then place the 96-wells in a cell culture incubator for 140 hours, and use the Incucyte Zoom instrument placed in the cell culture incubator for real-time observation. GraphPad prism 8.0.1 statistical analysis results.

In vitro cell proliferation experiments are shown in Figure 3. Kinetin inhibits CRC cell proliferation in a dose-dependent manner.

Example 2: Kinetin inhibits MEK phosphorylation in CRC cells

Western blotting was used to detect the inhibitory effect of Kinetin on MEK phosphorylation in CRC cells. The specific steps are as follows: After treating HCT116 cells with blank control or 50 μM Kinetin for 24 hours, aspirate the culture medium, add protein lysis solution, place on ice for lysis, transfer the lysis solution to a 1.5 mL EP tube, and crush it with an ultrasonic machine at 4°C for 10 min. Centrifuge at 12,000 rpm for 10 min, aspirate the supernatant protein sample, and perform SDS-PAGE gel electrophoresis, transfer to membrane, blocking, incubation with primary antibodies (pMEK, MEK, GAPDH) at 4°C overnight, incubation with secondary antibodies at room temperature for 1 hour, and chemiluminescence development.

As shown in Figure 4, Kinetin can inhibit MEK phosphorylation in CRC cells.

Example 3: Animal experiments verify the effect of Kinetin on the proliferation ability of CRC cells

Kinetin was administered alone or in combination with the chemotherapy drug 5-FU to verify its effect in PDX animal models. details as follows:

Freshly resected tumor tissues from CRC patients were inoculated into the right anterior axilla of BALB/c nude mice with rich blood supply to construct a PDX model.

After inoculation, when the tumor size reaches about ⁵⁰ mm, the nude mice are randomly divided into groups: a control group (normal saline), a 5-FU single-drug treatment group (10 mg/kg), and a Kinetin single-drug treatment group (2 mg/kg). kg) and the combined treatment group of 5-FU and Kinetin (10mg/kg 5-FU+2mg/kg Kinetin), the administration method was intraperitoneal injection of 5-FU every other day, and intraperitoneal injection of Kinetin every day, for 22 consecutive days.

Tumor size was measured every 3 days. The calculation formula of tumor volume is: V=a ² × b × 0.58 (a is the minimum diameter of the tumor, and b is the diameter perpendicular to a).

After treatment, the nude mice were euthanized and the tumors were dissected. All operations in animal experiments were performed in strict accordance with the guidelines for the protection of animal experiments.

The results of the PDX animal model are shown in Figure 5. Kinetin administered alone or in combination with the chemotherapy drug 5-FU can significantly inhibit tumor growth and promote its regression. Kinetin alone has a better inhibitory effect on tumor growth than the chemotherapy drug 5-FU alone; Kinetin combined with the chemotherapy drug 5-FU has a better inhibitory effect on tumor growth than Kinetin alone.

Example 4: Animal experiments to evaluate the nephrotoxicity of Kinetin in vivo

Take the PDX animal model in Example 3, separate the tumors, remove the kidneys and liver, fix them with 10% formaldehyde, embed them in paraffin, section them, and stain them with HE, and observe the morphological changes of the kidneys and livers under a microscope.

The pathological section results of kidney and liver are shown in Figure 6.

The pathological changes in the liver treated by the chemotherapy drug 5-FU alone were obvious, with local inflammatory infiltration; however, there were no pathological changes in the liver when Kinetin was administered alone; and the combination of Kinetin and the chemotherapy drug 5-FU could significantly inhibit the effects of the chemotherapy drug 5-FU. liver pathological changes.

When the chemotherapy drug 5-FU is administered alone, there are pathological changes in the kidneys, and the renal tubules are slightly dilated, which leads to a decrease in the renal filtration rate. However, when Kinetin is administered alone, there are no pathological changes in the kidneys, and the combination of Kinetin and the chemotherapy drug 5-FU can significantly inhibit chemotherapy. Kidney pathological changes caused by the drug 5-FU.

It can be seen that Kinetin alone or in combination with the chemotherapy drug 5-FU has no pathological toxicity to the liver and kidneys of nude mice.

In summary, the present invention screened out the drug Kinetin (CAS: 525-79-1). In vitro cell experiment results showed that the IC50 values of Kinetin on CRC cells HCT116 and SW480 were 38.18 μM and 56.43 μM respectively; at the same time, the proliferation experiment showed that Kinetin can Inhibits the proliferation of CRC cells in a dose-dependent manner. Further constructing a PDX model, it was found that Kinetin alone or in combination with the Kinetin chemotherapy drug 5-FU can significantly inhibit tumor growth and even promote tumor regression. It has no pathological toxicity to the liver and kidneys of nude mice. It can be considered that Kinetin has no effect on nude mice. Rats are not poisonous. Kinetin inhibits CRC by blocking or partially blocking the propagation of ERK signals by inhibiting MEK phosphorylation. 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 used to illustrate the technical solutions of the present invention and do not limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that The technical solution of the present invention may be modified or equivalently substituted without departing from the essence and scope of the technical solution of the present invention.

Claims (5)

1. Application of Kinetin or Kinetin salts in the preparation of drugs for the treatment of colorectal cancer. 2. The use of a pharmaceutical composition in the preparation of a drug for treating colorectal cancer, the pharmaceutical composition consisting of Kinetin or a salt of Kinetin, and 5-FU. 3. The use of a preparation in the preparation of a drug for treating colorectal cancer, the preparation consisting of Kinetin or a salt of Kinetin, and at least one pharmaceutically acceptable carrier. 4. The use of a preparation in the preparation of a drug for treating colorectal cancer, the preparation consisting of the pharmaceutical composition according to claim 2 and at least one pharmaceutically acceptable carrier. 5. The application according to any one of claims 3-4, characterized in that the preparation is an oral preparation or an injection.