Use of prex-in1 in the preparation of a tumor radiotherapy sensitizer or in the preparation of a medicament for treating colorectal cancer

Abstract

The application relates to an application of PREX-in1 in preparation of a tumor radiotherapy synergist. The PREX-in1 compound has the structural formula shown in the following formula (I): The application of the PREX-in1 in preparation of the tumor radiotherapy synergist can effectively inhibit in-vitro proliferation of colorectal cancer cells, can be used as a tumor radiotherapy synergist to enhance an immune response induced by radiotherapy, induce tumor cell death, and enhance the treatment effect of tumor radiotherapy. Meanwhile, the PREX-in1 has good safety, and is expected to be applied to treatment of colorectal cancer as an anti-tumor small molecule compound.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and pharmaceuticals, and more particularly to the application of PREX-in1 in the preparation of tumor radiotherapy enhancers or in the preparation of drugs for the treatment of colorectal cancer. Background Technology

[0002] Colorectal cancer (CRC) is a malignant tumor with an increasing incidence and mortality rate. Simultaneously, the number of deaths caused by colon cancer is also rising annually, accounting for 10% of all cancer deaths, seriously threatening the health and lives of the Chinese people. Due to the insidious onset, atypical early symptoms, and lack of effective early diagnostic methods, approximately 22% of colorectal cancer patients are already diagnosed with advanced distant metastases at initial diagnosis. With continuous improvements in clinical treatment protocols, the 5-year overall survival rate for colorectal cancer patients has reached 64.6%, but the median overall survival for patients with advanced metastatic colorectal cancer (mCRC) is only about 2 years.

[0003] Currently, the main clinical treatment for metastatic colorectal cancer is radiotherapy and chemotherapy. However, resistance to radiotherapy and chemotherapy by colorectal cancer cells remains a major obstacle to the treatment of colorectal cancer patients. Tumor recurrence, accompanied by tumor invasion and metastasis, seriously affects the prognosis of patients. Therefore, the main goal of clinical research is to implement appropriate strategies to overcome the drug resistance that patients develop during radiotherapy and chemotherapy. Exploring the molecular mechanisms of radiotherapy and chemotherapy resistance and identifying and defining new early diagnostic or therapeutic targets will help improve the cure rate of colorectal cancer patients.

[0004] Radiation therapy (RT) is one of the most effective local treatments for colorectal cancer, especially rectal cancer. It utilizes high-energy particles generated by high-energy ionizing rays to directly ionize tumor cell DNA molecules or indirectly interact with water and other organic matter in the tumor microenvironment, producing a series of reactive oxygen species (ROS). These highly reactive OOS, especially hydroxyl radicals, can strongly bind to single or double strands of tumor cell DNA, causing electron transfer and oxidation, ultimately inducing tumor cell apoptosis. However, during long-term development and evolution, tumors establish a strong immunosuppressive microenvironment. Repeated, multi-course preoperative radiotherapy leads to a severe inflammatory response in the tumor microenvironment, further inducing an immunosuppressive network that suppresses the anti-tumor T-cell response induced by conventional treatment, thus leading to treatment resistance. Therefore, finding new targeted small-molecule compounds to enhance the radiotherapy-induced immune response can effectively improve the radiosensitivity of tumors and improve patient prognosis. While there are many targeted therapies targeting traditional drug targets, very few are used for tumor radiosensitization, and their effectiveness is very limited.

[0005] Current radiosensitizing agents work in several ways: First, using sensitizers based on metallic elements (e.g., Au, Ta, rare earth elements, W, Pt, and i). These sensitizers can concentrate local radiation doses, enhance the photoelectric effect, and thus amplify the DNA damage effect of radiotherapy. Second, sensitizers that improve the hypoxic microenvironment of tumor tissue during radiotherapy. These sensitizers increase tumor O2 levels or tumor oxygenation through catalase (Cat), manganese dioxide (MnO2), and water-decomposing materials, thereby enhancing radiotherapy-induced DNA damage and achieving radiosensitization. Third, using DNA alkylating agents, platinum-based chemotherapy, purine-free / pyrimidine-free endonuclease inhibitors, and histone deacetylase inhibitors (HDACIs) to inhibit DNA repair in tumor cells after radiotherapy. Although these strategies can all achieve radiosensitization to some extent, most of them only target one aspect of radiotherapy tolerance factors (i.e., by enhancing DNA damage or inhibiting DNA repair), and it is difficult to address all aspects simultaneously. Therefore, their effectiveness in improving radiotherapy outcomes is not significant. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a PREX-in1 that can take into account both the advantages of promoting tumor radiation-induced immune response and radiosensitization in the preparation of tumor radiotherapy enhancers.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides the application of PREX-in1 in the preparation of tumor radiotherapy enhancers, wherein the structural formula of the PREX-in1 compound is shown in formula (I) below: The PREX-in1 (C 24 H 17 C l PREX-in1 (N4O3) is a small molecule inhibitor of PREX guanine-nucleotide exchange factor. It can block the Rac-GEF activity of full-length PREX1 and PREX2 and their isolated catalytic domains in vitro at low micromolar concentrations. This can enhance the therapeutic effect of radiotherapy by promoting tumor-induced immune responses. Furthermore, PREX-in1 can effectively inhibit the proliferation of colorectal cancer cells and can be used as an anti-tumor small molecule drug for the treatment of colorectal cancer.

[0008] In a preferred embodiment of the application described in this invention, the tumor is colorectal cancer.

[0009] As a preferred embodiment of the application described in this invention, the method of the application is as follows: administering a tumor radiotherapy enhancer during or before radiotherapy of the tumor; the PREX-in1 or its derivative is the active ingredient in the tumor radiotherapy enhancer.

[0010] This invention utilizes PREX-in1 in combination with radiotherapy, which enhances the efficacy of radiotherapy for tumors in vivo. This includes strengthening the immune system's ability to attack tumors and inducing tumor cell death. Compared to existing radiotherapy without sensitization, the combined application of PREX-in1 and radiotherapy for colorectal cancer resulted in a 50-60% reduction in tumor volume across different colorectal cancer cell lines, while maintaining tumor weight and volume at low levels. PREX-in1 and radiotherapy exhibit a good synergistic effect.

[0011] Furthermore, the PREX-in1 can be administered throughout the entire radiotherapy course to enhance the efficacy of tumor radiotherapy, and the dosing interval can be synchronized with the radiotherapy treatment.

[0012] In a preferred embodiment of the application described in this invention, the method of application is as follows: PREX-in1 is administered 2-5 hours before the radiotherapy process for the tumor. More preferably, PREX-in1 is administered 3 hours before the radiotherapy process for the tumor.

[0013] In a preferred embodiment of the application described in this invention, the tumor radiotherapy enhancer is administered to the tumor area via oral or injectable administration. Preferably, oral administration is performed via gavage or enema.

[0014] In a preferred embodiment of the application described in this invention, the dosage of the tumor radiotherapy enhancer is 0.5-1.0 mg / kg. Preferably, the dosage of the tumor radiotherapy enhancer is 0.8 mg / kg.

[0015] In a preferred embodiment of the application described in this invention, the concentration of PREX-in1 or its derivative in the tumor radiotherapy enhancer is 10 mM. PREX-in1 or its derivative is the active ingredient in the tumor radiotherapy enhancer.

[0016] As a preferred embodiment of the application described in this invention, PREX-in1 promotes radiotherapy-induced anti-colorectal cancer immune responses, including increasing CD3+ and CD8+ T cell infiltration after radiotherapy.

[0017] As a preferred embodiment of the application described in this invention, the radiotherapy includes treatment using α, β, and γ rays generated by radioactive isotopes; and / or X-rays emitted by X-ray therapy devices and accelerators; and / or treatment using various electron beams, proton beams, neutron beams, negative π meson beams, and other heavy ion beams.

[0018] Secondly, the present invention also provides the use of PREX-in1 or its derivatives in the preparation of drugs for treating colorectal cancer.

[0019] In a preferred embodiment of the application described in this invention, the concentration of PREX-in1 or its derivative in the drug for treating colorectal cancer is 50 μM-100 μM.

[0020] Preferably, the concentration of PREX-in1 or its derivative in the drug for treating colorectal cancer is 60 μM-80 μM. More preferably, the concentration of PREX-in1 or its derivative in the drug for treating colorectal cancer is 65 μM-75 μM.

[0021] PREX-in1 exhibits dose-dependent killing of colorectal cancer cell lines, and its inhibitory effect on cancer cells has a threshold effect. When used as a radiotherapy enhancer, the effective concentration of PREX-in1 is not less than 50 μM. At doses between 50 μM and 100 μM, PREX-in1 demonstrates significant cell inhibition, with an inhibition rate of approximately 60%-70%. This invention selects a more optimal PREX-in1 dose; when used as a radiotherapy enhancer, the effective concentration is 60 μM-80 μM. This results in the lowest cell survival rate and strongest inhibition of colorectal cancer cells, with a cell survival rate of only 20%.

[0022] As a preferred embodiment of the application described in this invention, the half-maximal inhibitory concentration (IC50) of PREX-in1 or its derivatives in the drug for treating colorectal cancer is... 50 The half-maximal concentration (50 μM-60 μM) is the optimal concentration. At this half-maximal concentration, the drug dose required to induce apoptosis is minimal, and the inhibitory effect on tumor cells is significant.

[0023] As a preferred embodiment of the application described in this invention, PREX-in1 inhibits the proliferation and spread of colorectal cancer cells, including reducing tumor volume and tumor weight.

[0024] As a preferred embodiment of the application described in this invention, the PREX-in1 derivative is a pharmaceutically acceptable derivative of PREX-in1, including one of its pharmaceutical salts, pharmaceutical esters, pharmaceutical ethers, pharmaceutical amides, or glycosides.

[0025] As a preferred embodiment of the application described in this invention, the PREX-in1 derivative is a PREX-in1 derivative that can be hydrolyzed in vivo to obtain a PREX-in1 derivative with the same pharmaceutical activity as PREX-in1.

[0026] Thirdly, the present invention provides a pharmaceutical formulation for enhancing the radiosensitivity of colorectal cancer, comprising PREX-in1 or a pharmaceutically acceptable derivative thereof.

[0027] As a preferred embodiment of the pharmaceutical formulation of the present invention, the pharmaceutically acceptable derivatives of PREX-in1 include pharmaceutical salts, pharmaceutical esters, pharmaceutical ethers, pharmaceutical amides, or glycosides of PREX-in1.

[0028] As a preferred embodiment of the pharmaceutical preparation of the present invention, the pharmaceutical preparation is a solid preparation, a semi-solid preparation, or a liquid preparation; the solid preparation includes tablets, capsules, granules, and / or pills; the semi-solid preparation includes gels, suppositories, and / or ointments; the liquid preparation includes emulsions, mixtures, suspensions, and / or solutions.

[0029] Fourthly, the present invention provides a pharmaceutical composition for treating colorectal cancer, comprising a pharmaceutical composition consisting of PREX-in1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier; wherein the concentration of PREX-in1 in the pharmaceutical composition is not less than 50 μM.

[0030] As a preferred embodiment of the pharmaceutical composition of the present invention, the pharmaceutically acceptable carrier includes solvents, solubilizers, cosolvents, emulsifiers, flavoring agents, odorants, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, pH regulators, stabilizers, surfactants and / or preservatives.

[0031] In a preferred embodiment of the pharmaceutical composition of the present invention, the pharmaceutical composition is a topical preparation. More preferably, the topical preparation is a suppository, a gavage, or an enema.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention provides the application of PREX-in1 in the preparation of tumor radiotherapy enhancers. In vivo experiments have shown that PREX-in1 can promote tumor radiation-induced immune responses and enhance the efficacy of tumor radiotherapy. Compared with existing tumor radiotherapy, this invention may have better therapeutic effects and can enhance the immune system's ability to attack tumors.

[0033] 2. This invention provides a pharmaceutical formulation for enhancing the radiosensitivity of colorectal cancer, comprising PREX-in1 or a pharmaceutically acceptable derivative thereof, which has been demonstrated through in vivo and in vitro experiments to have low toxicity and good tolerability within a certain dose range. Compared with existing tumor radiosensitizers, this invention may have better safety and reliability, and can reduce the side effects and risks of treatment.

[0034] 3. This invention provides a pharmaceutical composition for treating colorectal cancer, comprising an effective amount of PREX-in1. Compared to existing treatment methods, PREX-in1 can effectively inhibit the in vitro proliferation of colorectal cancer cells and induce tumor cell death, exhibiting better anti-tumor effects and more effectively inhibiting the growth and spread of colorectal cancer cells. Attached Figure Description

[0035] Figure 1 Here is the molecular structure diagram of PREX-in1; Figure 2 This is a graph showing the inhibitory effect of different PREX-in1 doses on colorectal cancer cells in Example 2. Figure 3 This is a diagram showing the changes in the appearance of subcutaneous tumors in C57 mice after PREX-in1 treatment in Example 3. Figure 4 This is a graph showing the changes in tumor volume and body weight after combined treatment with PREX-in1 and radiotherapy in Example 3; Figure 5 This is a flow cytometry graph of the combined PREX-in1 and radiotherapy treatment in Example 3; Figure 6 This is an immunohistochemical image of the combined PREX-in1 and radiotherapy treatment in Example 3. Detailed Implementation

[0036] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0037] Example 1: Inhibition of PREX-in1 and PREX2 by PREX-in1 PREX-in1 (C 24 H 17 C l N4O3) is 6-amino-4-[5-(4-chlorophenyl)-2-furanyl]-3-(4-methoxyphenyl)-2,4-dihydropyran[2,3-c]pyrazol-5-carbamate. According to molecular theory, PREX-in1 can be divided into four different regions, including: (a) chlorophenyl; (b) furan ring; (c) 6-amino-2,4-dihydropyro[2,3-c]pyrazol-5-acrylonitrile; (d) methoxyphenyl, such as Figure 1 As shown.

[0038] Structure-activity relationship (SAR) analysis was performed on PREX-in1 to identify the functional groups with biological functions. SAR tests showed that regions a, b, and c of PREX-in1 are all important for the inhibition of the Rac-GEF catalytic domain of PREX. Removing chlorine atoms from region (a) or making any significant changes to regions (b) would affect the inhibition effect, and removal from region (c) is generally also detrimental.

[0039] PREx1 and PREx2 are multi-domain Rho guanine nucleotide exchange factors (Rho-GEF). Their N-terminus catalyzes the Dbl homology domain (DH domain) and is tandemly linked with a membrane-targeting Pleckstrin homology domain (PH domain). They also include an interaction domain of the DEP domain (Dishevelled, Egl-10, and Pleckstrin) and two PDZ domains (PSD95, Dlg1, and Zo-1), as well as a carboxyl terminus with weak homology to inositol 4-phosphatase (IP4P), which lacks phosphatase activity.

[0040] PREX-in1, a small molecule inhibitor of PREx guanine-nucleotide exchange factor, can block the Rac-GEF activity of full-length PREx1 and PREx2 and their separated catalytic domains in vitro at low micromolar concentrations, without affecting the activity of other Rac-GEFs. Specifically, it targets the catalytic DH domains of PREx1 and PREx2.

[0041] With 10 µM PREX-in1, the Rac-GEF activity of the full-length PREx1 stimulated by PIP3 decreased by 56%, and the Rac-GEF activity of the isolated DH and DHPH domains was inhibited by 98% and 100%, respectively. Furthermore, 10 µM PREX-in1 inhibited the Rac-GEF activity of the full-length PREx2 stimulated by PIP3 (82%) and the Rac-GEF activity of the isolated PREx2 DH domain (92%).

[0042] Example 2: Inhibition of colorectal cancer cell lines by PREX-in1 1. Colorectal cancer cell culture The colorectal cancer cell line was derived from an existing cell line in our laboratory. The culture medium used for cell culture was purchased from Gibco, and fetal bovine serum was purchased from ExCell Bio. The cell line was cultured in RPMI-1640 medium containing 10% fetal bovine serum in an incubator at 37°C and 5% CO2. Cells in good growth condition were used for subsequent experiments.

[0043] Once the cell lines reached 80% confluence, they were digested with 0.05% trypsin, resuspended in RPMI-1640 complete medium, counted, and seeded into 96-well plates with 5000 cells per well, and cultured in 100 μl of medium.

[0044] 2. Inhibition of colorectal cancer cell lines by PREX-in1 PREX-in1 was dissolved in DMSO to prepare a 10 mM stock solution. After the cultured colorectal cancer cells adhered to the culture plate as a monolayer, PREX-in1 was added to achieve final concentrations of 1, 5, 10, 20, 50, 80, and 100 μM. Blank wells and wells containing 100% cell culture medium were set up as control groups. Each drug concentration was tested in triplicate for parallel experiments. The culture plates were returned to a 37°C, 5% CO2 incubator. After 24 hours of culture, 10 μL of CCK-8 was added to every 100 μL of culture medium. Incubation continued for 2 hours, and the absorbance at 450 nm was measured using a microplate reader.

[0045] Based on the OD value of each well, the inhibition rate of PREX-in1 on cells was calculated using the following formula. The inhibitory effect of PREX-in1 on colorectal cancer cells at different doses is shown in the figure. Figure 2 As shown.

[0046] Inhibition rate % = [(Absorbance of control wells - Absorbance of experimental wells) / (Absorbance of control wells - Absorbance of blank wells)] × 100%; Cell viability % = 1 - Inhibition rate.

[0047] from Figure 2It can be seen that PREX-in1's killing effect on colorectal cancer cell lines is dose-dependent, and its inhibitory effect on cancer cells exhibits a threshold effect. For both the SW480 and HCT116 cell lines, when PREX-in1 doses were between 1 μM and 50 μM, there was no inhibitory effect on tumor cells, with cell viability remaining at 80%-100%. However, when PREX-in1 doses were between 50 μM and 100 μM, it showed a significant inhibitory effect, with an inhibition rate of approximately 60%-70%. The IC50 of PREX-in1 against colorectal cancer cells... 50 At a concentration of 40-60 μM, it exhibits strong inhibition, with a cell survival rate of only 20%. At the same time, it requires the lowest drug dose to induce apoptosis and has a significant inhibitory effect on tumor cells.

[0048] Example 3: Experiment on the effect of PREX-in1 on tumor radiotherapy 1. Experimental design of mouse tumor formation and radiotherapy treatment groups Twenty-one female C57 mice aged 4-5 weeks were selected, and each mouse was subcutaneously injected with 1×10⁻⁶ oz. on both sides of its back. 6 After approximately 7 days, subcutaneous tumors were observed to form in 221 C57 mice with MC38 colorectal cancer cells. Once tumors had formed in all 221 C57 mice, the mice were randomly divided into three groups: a control group, a radiotherapy group, and a PREX-in1-treated radiotherapy group, with 7 C57 mice in each group.

[0049] In addition, 18 female C57 mice aged 4-5 weeks were selected, and each C57 mouse was subcutaneously injected with 1×10⁻⁶ oz. on both sides of its back. 6 After approximately 7 days, subcutaneous tumors were observed to form in C57 mice with radiotherapy-resistant colorectal cancer cells CMT93-RT. Once tumors had formed in all 18 C57 mice, the mice were randomly divided into three groups: a control group, a radiotherapy group, and a PREX-in1-treated radiotherapy group, with 6 C57 mice in each group.

[0050] 2. Experiment on the effect of PREX-in1 on tumor radiotherapy The treatment for the PREX-in1-treated radiotherapy group consisted of: PREX-in1 administered by gavage at 0.8 mg / kg 3 hours before each radiotherapy session, followed by 8 Gy of radiotherapy, for a total of three sessions, with each session two days apart. The PREX-in1 was dissolved in DMSO before gavage. The control group received 0.8 mg / kg of DMSO by gavage per session.

[0051] Figure 3The study investigated the changes in the appearance of subcutaneous tumors in C57 mice after PREX-in1 treatment. In CMT93-RT cells or MC38 cells, the tumors in the control group were the largest, while in the radiotherapy group, the tumor size decreased with increasing radiotherapy sessions. However, in the radiotherapy + PREX-in1 treatment group, the tumor size was significantly reduced. Administration of the drug 3 hours before radiotherapy showed that the living conditions of the C57 mice were unaffected, with normal eating and activity levels, indicating that PREX-in1 does not affect the activity of other non-cancerous cells.

[0052] Because the tumors in C57 mice develop at different times, drug administration or radiation therapy is initiated when all C57 mice in the same batch of experiments develop subcutaneous tumors. Treatment continues until any C57 mouse develops a subcutaneous tumor that reaches 1500 mm in size. 3 The endpoint is [not specified]. The size of the tumor is measured every other day using calipers, specifically the length (L) and width (W), and then calculated using the formula V (mm). 3 ) = LW 2 The volume of the tumor was calculated using a ratio of 2, and the weight of the subcutaneous tumor was measured using an electronic balance when the mouse was euthanized. Figure 4 This figure shows the changes in tumor volume and body weight after combined treatment with PREX-in1 and radiotherapy.

[0053] Calculations of tumor growth curves for each group revealed that the tumor growth rate in the radiotherapy group (IR) was lower than that in the control group. In particular, the tumor growth in the radiotherapy group (IR+PREX-in1) was significantly suppressed after PREX-in1 treatment: after MC38 cell therapy, the tumor volume in the radiosensitization group was less than 500 mm². 3 The tumor weight was below 0.5g, a reduction of approximately 75% compared to the control group and approximately 60% compared to the radiotherapy-only group. After IR-CMT93 cell therapy, the tumor volume in the radiosensitization group was below 50mm. 3 The tumor weight was 0.07g, which was about 65% lower than the control group and about 53% lower than the radiotherapy group. This shows that PREX-in1 can inhibit the growth of subcutaneous tumors in C57 mice and enhance the efficacy of radiotherapy in tumor treatment.

[0054] At the end of the experiment, tumors from 6 mice in each group were subjected to flow cytometry and immunohistochemistry to analyze the infiltration of immune cells within the tumors. Figure 5 The flow cytometry analysis of the combined PREX-in1 and radiotherapy treatment in Example 3 of this invention shows that, regardless of whether it is the MC38 or IR-CMT93 group, after radiosensitization (IR+PREX-in1) treatment, CD8 + Increased cell positivity, along with CD8 + Cells in CD45 + The percentage of cells was significantly higher compared to the control group and the radiotherapy-only group (IR). Figure 6 This image shows an immunohistochemical assay following combined PREX-in1 and radiotherapy. The number of positive immune cells was counted under a 20x microscope. Three fields of view were counted for each mouse section, and the average value was taken. (Note: The last sentence appears to be incomplete and possibly refers to a different image.) Figure 5 and Figure 6 Flow cytometry and immunohistochemical results showed that PREX-in1 combined with radiotherapy resulted in increased intratumoral CD3 levels. + Increased CD8+ T cell infiltration indicates that PREX-in1 can promote radiotherapy-induced antitumor immune responses.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Application of PREX-in1 combined with radiotherapy in the preparation of a drug for treating colorectal cancer; the dosage of PREX-in1 is 0.8 mg / kg; The structure of PREX-in1 is shown in the following formula: 。 2. The application as described in claim 1, characterized in that, The method of application is as follows: PREX-in1 is administered during or before radiotherapy for colorectal cancer.

3. The application according to claim 2, characterized in that, The method of application is as follows: PREX-in1 is administered 2-5 hours before radiotherapy for colorectal cancer.

4. The use of PREX-in1 in the preparation of a drug for treating colorectal cancer; wherein the concentration of PREX-in1 in the drug for treating colorectal cancer is 50 μM-100 μM; The structure of PREX-in1 is shown in the following formula: 。

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