Application of Neuropathiazol in preparation of products for treating pancreatic cancer

Abstract

The invention discloses an application of Neuropathiazol in preparation of a product for treating pancreatic cancer, and belongs to the technical field of tumor drugs. The compound is obtained by screening through experiments such as Cell Counting Kit-8, promoter luciferase, cell immunofluorescence (Ki-67, CC3, Map2 and Tuj1) dyeing and the like, evaluation is further performed by utilizing an immunodeficiency type mouse transplantation tumor model and a wild type C57 mouse orthotopic transplantation tumor model, and experimental results show that the compound not only can remarkably inhibit proliferation of pancreatic cancer cell strains Panc-1 and SW1990 cultured in vitro, but also can remarkably inhibit proliferation of pancreatic cancer cells, and has a good application prospect. The compound can promote tumor cells to be differentiated into neurons, can effectively inhibit the growth of transplanted tumors in a mouse body, and has a good application prospect when being developed into a novel pancreatic cancer treatment medicine due to the safe, efficient and ideal anti-cancer effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor drugs, and in particular relates to the application of Neuropathiazol in the preparation of products for treating pancreatic cancer. Background Art

[0002] Pancreatic cancer is a malignant tumor originating from the pancreatic ductal epithelium and acinar cells. It is extremely malignant, has an insidious onset, is difficult to diagnose early, progresses rapidly, and has a short survival time. It is one of the malignant tumors with the worst prognosis and is known as the "king of cancer". Its incidence and mortality rates are among the top ten malignant tumors in the world and in my country, and the incidence and mortality rates of pancreatic cancer are on the rise worldwide. According to data from the National Comprehensive Cancer Network of the United States in 2019, approximately 55,440 patients were diagnosed with pancreatic cancer in the United States in 2018, and approximately 43,330 patients died of pancreatic cancer. According to data from the National Cancer Center of China in 2019, pancreatic cancer ranks 10th in the incidence of malignant tumors in China and 6th in the mortality rate of malignant tumors. At present, the overall 5-year survival rate is only 8.5% through a combination of surgery combined with chemotherapy, immunotherapy and targeted therapy. Only a few cases that are discovered early and undergo timely surgery can achieve a cure. Although this treatment model can benefit some patients and improve their overall survival rate, its long-term effects such as tumor recurrence and other risks also increase accordingly. In addition, radiotherapy and chemotherapy also bring serious side effects such as hair loss, nausea and vomiting, and thrombocytopenia. It is urgent to explore the molecular mechanisms related to the occurrence, development, recurrence and metastasis of pancreatic cancer. Improving the therapeutic efficacy and reducing the toxicity of treatment are important directions for clinical trials and basic research on pancreatic cancer, in order to find new molecular targets for intervention and develop new therapeutic drugs.

[0003] Previous studies have found that mouse erythroleukemia cells can be induced to differentiate in vitro by dimethyl sulfoxide, which has pioneered the study of tumor cell differentiation. As a result, the concept of differentiation therapy was proposed. Tumor differentiation therapy is a treatment method that promotes the differentiation of tumor cells to mature stages or other normal cells through inducers, and changes their malignant biological behavior to make them lose the characteristics of tumor cells such as malignant proliferation and metastasis. The core concept of this treatment method is that through specific inducers, tumor cells can be returned to or closer to the state of normal cells, thereby losing their cancer cell characteristics. Induced differentiation therapy for pancreatic cancer forces tumor cells to transdifferentiate into mature neurons. Although it cannot cause tumor death like traditional cytotoxic therapy, it can significantly inhibit the growth and proliferation of tumor cells, minimize the impact on normal cells, and achieve a relatively high benefit-risk anti-cancer therapy, which brings a safer and more reliable treatment method to clinical pancreatic cancer patients.

[0004] Neuronal differentiation factor 1 (Neuronal differentiation factor 1, NeuroD1) is a transcription factor with an HLH (Helix-Loop-Helix) structure that can promote the differentiation of neurons, plays a vital role in the normal development of the nervous system, and is involved in the differentiation of neural crest cells. The inventor's previous article found that NeuroD1 can promote the transdifferentiation of medulloblastoma tumor cells into neurons (Cell Rep. 2020Jun 23; 31(12): 107782), and there are articles reporting that Neuro D1 can reverse the transdifferentiation of glial cells into neurons, but the functional study of Neuro D1 in pancreatic cancer has not been reported. Based on this, the applicant found in previous studies that NeuroD1 can also inhibit the proliferation of pancreatic cancer cells and promote the transdifferentiation of tumor cells into neurons, but the pharmacological mechanism of regulating NeuroD1 in pancreatic cancer is still unclear, so it is necessary to provide clues for the development of new means of inducing differentiation therapy for pancreatic cancer. Summary of the invention

[0005] The present invention provides an application of Neuropathiazol in the preparation of a product for treating pancreatic cancer, wherein the CAS Number of Neuropathiazol is 880090-88-0, the chemical name is Ethyl 4-[methyl-(2-phenyl-1,3-thiazol-4-yl)amino]benzoate, and the molecular formula is C 19 H 18 N2O2S, molecular weight is 338.42, structural formula see Figure 1 .

[0006] Preferably, the product is a medicine.

[0007] More preferably, Neuropathiazol is the only active ingredient in the medicament.

[0008] More preferably, the medicine further contains medically acceptable excipients.

[0009] The present invention also provides an application of a composition in preparing a product for treating pancreatic cancer, wherein the composition contains Neuropathiazol.

[0010] Preferably, the product is a medicine.

[0011] More preferably, the medicine further contains medically acceptable excipients.

[0012] More preferably, the dosage form of the drug is any one of tablets, capsules, pills, granules and powders.

[0013] More preferably, the tablet is any one of enteric-coated tablets, sugar-coated tablets, plain tablets, film-coated tablets, dispersible tablets, sustained-release tablets, and controlled-release tablets.

[0014] More preferably, the capsule is any one of a hard capsule, a soft capsule, an enteric-coated capsule, a sustained-release capsule, and a controlled-release capsule.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention found that the compound Neuropathiazol can not only significantly inhibit the proliferation of pancreatic cancer cell lines (Panc-1, SW1990) cultured in vitro, but also promote the differentiation of tumor cells into neurons, and can effectively inhibit the growth of transplanted tumors in mice. Due to its safe, efficient and ideal anti-cancer effect, it can be tried to be developed into a new therapeutic drug against pancreatic cancer, which will certainly have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the structural formula of the Neuropathiazol compound of the present invention.

[0018] Figure 2 The target compounds were obtained by screening the small molecule neuronal inducer compound library in Example 1, including A. screening process of small molecule neuronal inducer compounds (schematic diagram) and IC50 values ​​of compounds for treating two pancreatic cancer cells and fluorescence intensity of regulating Neuro D1 promoter transcriptional activity (table); B. IC50 values ​​of four compounds Neuropathiazol, gemcitabine, cisplatin and paclitaxel for treating Pan-1 pancreatic cancer cells; C. NeuroD1 promoter fluorescence values ​​of Pan-1 pancreatic cancer cells treated with four compounds; D. Neuro D1 mRNA expression levels of Pan-1 pancreatic cancer cells treated with four compounds; E. Neuro D1 protein expression levels of Pan-1 pancreatic cancer cells treated with four compounds.

[0019] Figure 3 The four compounds in Example 1 inhibit the proliferation of pancreatic cancer cells, including proliferation (Ki-67) and apoptosis (CC3) staining and statistical analysis of Pan-1 pancreatic cancer cells treated with AB. Neuropathiazol, gemcitabine, cisplatin and paclitaxel for 48 hours.

[0020] Figure 4 The compound in Example 1 induces pancreatic cancer cells to transdifferentiate into neurons. After treating tumor cells with Neuropathiazol for 0, 3, 5, and 7 days, the cells were collected for Map2 and Tuj1 immunofluorescence staining.

[0021] Figure 5 The compound in Example 1 inhibits the growth of subcutaneous transplanted tumors in mice, wherein A to C are the tumor growth picture size (A), growth change curve (B) and mouse weight change curve (C) of each treatment group; D to G are the tumor efficacy test results of each treatment group. DETAILED DESCRIPTION

[0022] Example 1

[0023] 1. Screening of target compounds using a small molecule neuron inducer compound library

[0024] (1) A preliminary screening was conducted using the small molecule neuron inducer compound library recommended by Shanghai Taoshu Biotechnology Co., Ltd. and literature research to obtain a screening list of candidate compounds.

[0025] (2) The candidate compounds were dissolved in DMSO to prepare 10 mM stock solutions, and the Panc-1 and SW1990 pancreatic cancer cell lines were seeded in 96-well plates at a density of 10,000 cells per well, respectively, and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 hours. The original culture medium was discarded, and the Panc-1 and SW1990 pancreatic cancer cell lines were treated in vitro with a drug concentration gradient of 0, 10 nM, 100 nM, 500 nM, 1 μM, 5 μM, 10 μM, 25 μM, 50 μM, and 100 μM. c-1, SW1990 tumor cells, each treatment group had 3 replicate wells. After the cells were cultured in the culture medium containing 100 μL of the drug in each well for 48 hours, 10 μL of CCK-8 reagent (CK04, Dojindo, Japan) was added to each well and incubated in a cell culture incubator for 2 hours. The absorbance was then read at a wavelength of 450 nm to evaluate the toxicity of the compound, and the chemotherapy drugs gemcitabine, cisplatin, and paclitaxel were used as positive controls for cytotoxic compounds.

[0026] (3) A tumor cell (Panc-1, SW1990 pancreatic cancer cell) screening system based on NeuroD1 promoter combined with luciferase was established. Tumor cells were treated in vitro at concentrations of 0, 0.5 μM, and 2 μM for 72 hours. The fluorescence intensity was detected at 560 nm using a luciferase detection kit (DL101-01, Vazyme, China). Low-toxic and high-efficiency neuronal inducers that could upregulate NeuroD1 transcriptional expression were screened based on the fluorescence intensity.

[0027] (4) After 72 hours of treatment of tumor cells in step (3), the protein and mRNA expression levels of NeuroD1 were detected by Western Blotting and qPCR, respectively, with anti-NeuroD1 (1:200; 12081-1-AP, Proteintech). The primer sequences are as follows:

[0028] Table 1

[0029]

[0030] 2. Compounds inhibit the proliferation of human pancreatic cancer cells

[0031] (1) After the target compound is screened, the effect of the compound on the proliferation and apoptosis of tumor cells is tested in vitro, as follows: Panc-1 and SW1990 pancreatic cancer cell lines are inoculated at a density of 100,000 cells per well in a 24-well plate on which a cell slide has been placed in advance. After the cells have attached to the plate for 6 hours, the drug culture medium is replaced with 0, 0.5 μM, and 2 μM. The tumor cells are treated with 1 μM gemcitabine, 2 μM cisplatin, and 5 μM paclitaxel for another 48 hours. The culture medium is then discarded, and 300 μL of 4% PFA is added to each well to fix the cells for 10 minutes, followed by washing three times with PBS.

[0032] (2) Add 300 μl of 10% NGS blocking solution prepared in PBST (PBS + 0.03% Triton X-100) to the washed cell slides and block at room temperature for 1 hour. After blocking, discard the blocking solution and add the primary antibody diluted in the blocking solution, incubate overnight at 4°C, wash three times with PBST the next day, shake for 10 minutes each time; add the secondary antibody diluted in the blocking solution, incubate at room temperature for 1 hour, discard the secondary antibody, and wash in the same way as above; add DAPI dye, incubate at room temperature for 5 minutes, then discard, and wash in the same way as above. After washing, take out the washed cell slides with tweezers and invert them on a slide with anti-fluorescence attenuation agent (Southern Biotechnology), place them in a dark place at room temperature, air dry for half an hour, and take immunofluorescence photos. The following antibodies were used: Ki67 (1:200; 27309-1-AP, Proteintech), CC3 (1:200; ab52101, Abcam), goat anti-mouse CoraLite594 (1:200; Proteintech, SA00013-3) and goat anti-rabbit CoraLite594 (1:200; Proteintech, SA00013-4).

[0033] 3. Compounds induce pancreatic cancer cells to transdifferentiate into neurons

[0034] (1) The ability of the compound to induce differentiation of pancreatic cancer cells was detected as follows: Panc-1 and SW1990 pancreatic cancer cell lines were inoculated at a density of 40,000 cells per well in a 24-well plate with a cell slide placed in advance. After 6 hours of cell adhesion, the drug culture medium was replaced with 0, 0.5 μM, and 2 μM, and the tumor cells were treated for 0 hour, 3 days, 5 days, and 7 days, respectively. The culture medium was then aspirated and discarded, and 300 μL of 4% PFA was added to each well to fix the cells for 10 minutes, and then washed three times with PBS.

[0035] (2) The washed cell slides were subjected to immunofluorescence staining of neuronal markers using the same method as above and the following antibodies: Map2 (1:1000; 17490-1-AP, Proteintech), Tuj1 (1:1000; 66375-1-Ig, Proteintech).

[0036] 4. Growth of Subcutaneous Tumor Xenografts in Compound-Transplanted Immunodeficient Mice

[0037] (1) Thirty 6-8 week old SCID female mice weighing approximately 20 g were raised. Six million Panc-1 human pancreatic cancer cells were inoculated into the left posterior abdomen of each mouse. After 3 weeks, the tumor grew to approximately 100 mm. 3 , randomly divided into 6 groups, 5 mice in each group. The first group was the control group, and 300μl PBS was injected intraperitoneally every other day; the second group was injected intraperitoneally with 300μl of Neuropathiazol at a concentration of 15mg / kg body weight every other day; the third group was injected intraperitoneally with 300μl of Neuropathiazol at a concentration of 50mg / kg body weight every other day; the fourth group was the control group, and 300μl of Gemcitabine at a concentration of 20mg / kg body weight was injected intraperitoneally every other day; the fifth group was the control group, and 300μl of Cisplatin at a concentration of 10mg / kg body weight was injected intraperitoneally every other day; the sixth group was the control group, and 300μl of Paclitaxel at a concentration of 15mg / kg body weight was injected intraperitoneally every other day. This method was used for continuous injection for 2 weeks, and the weight and tumor size of the mice were measured every 2 days, and the change in tumor volume was calculated according to the following formula: (W 2 ×L) / 2(W, width; L, length).

[0038] (2) After 2 weeks of administration, the tumor-bearing mice were killed, and the subcutaneous tumors were removed and fixed with 4% PFA. Immunohistochemical staining was performed to detect tumor cell proliferation (Ki-67 staining), apoptosis (CC3 staining), and tumor cell transdifferentiation (NeuN staining). The antibodies used were as follows: Ki67 (1:200; 27309-1-AP, Proteintech), CC3 (1:200; ab52101, Abcam), NeuN (1:200; ab104224, Abcam), goat anti-mouse CoraLite594 (1:200; Proteintech, SA00013-3) and goat anti-rabbit CoraLite594 (1:200; Proteintech, SA00013-4).

[0039] Statistics

[0040] All experiments in this study were repeated at least three times. Data were analyzed using GraphPad Prism 9 and Excel software and expressed as mean ± standard deviation (SEM). One-way ANOVA was used to analyze the differences among multiple groups, and Turkey was used for multiple comparisons. Kaplan-Meier was used to draw the curve for survival analysis, and Log-rank was used for statistical analysis (ns P>0.05, *P<0.05, **P<0.001, ***P<0.0001).

[0041] result

[0042] 1. Screening of target compounds using a small molecule neuron inducer compound library

[0043] Through the small molecule neuron inducer compound library recommended by Shanghai Taoshu Biological Company and combined with literature research, preliminary screening was carried out to obtain 16 candidate compounds. Panc-1 and SW1990 tumor cells were treated with a drug concentration gradient of 0, 10nM, 100nM, 500nM, 1μM, 5μM, 10μM, 25μM, 50μM, and 100μM in vitro. After 48 hours of culture, the toxicity of the compounds was evaluated by CCK-8 experiment, and the chemotherapy drugs gemcitabine, cisplatin, and paclitaxel were used as positive controls for cytotoxic compounds.

[0044] A tumor cell screening system based on NeuroD1 promoter combined with luciferase was established. Tumor cells were treated in vitro at concentrations of 0, 0.5 μM, and 2 μM for 72 hours. The fluorescence intensity was detected at 560 nm using a luciferase detection kit. The fluorescence intensity was correlated with the transcriptional expression of NeuroD1. Figure 2As shown in Tables A and 2B, compared with other small molecule neuronal inducer compounds and three chemotherapeutic drugs, the IC50 values ​​of Neuropathiazol in treating two pancreatic cancer cells, i.e., the lower the cytotoxicity, the stronger the regulation of NeuroD1 promoter activity ( Figure 2 C). Then, the mRNA of NeuroD1 was detected by Western Blotting and qPCR after the cells were treated with the compounds ( Figure 2 D) and protein ( Figure 2 E) Expression level. The experimental results show that Neuropathiazol can inhibit the proliferation of pancreatic cancer cell lines at a relatively low concentration and can better regulate the expression of transcription factor NeuroD1.

[0045] 2. Compounds inhibit the proliferation of human pancreatic cancer cells

[0046] After the target compound Neuropathiazol was screened, cells were treated with drug concentrations of 0, 0.5 μM, and 2 μM in vitro. At the same time, 1 μM gemcitabine, 2 μM cisplatin, and 5 μM paclitaxel were used as positive cytotoxic drugs to treat tumor cells for 48 hours. Ki67 and CC3 immunofluorescence staining experiments were used to detect the effects of the compounds on tumor cell proliferation and apoptosis. Figure 3 As shown in A-3B, Neuropathiazol can effectively inhibit tumor cell proliferation at a concentration of 0.5 μM, showing concentration dependence, and compared with the three cytotoxic chemotherapy drugs, Neuropathiazol has very low toxicity to tumor cells.

[0047] 3. Compounds induce pancreatic cancer cells to transdifferentiate into neurons

[0048] After observing that the target compound has low toxicity and high efficiency in inhibiting tumor cell activity, Panc-1 cells were treated with a drug concentration of 2 μM in vitro for 0 days, 3 days, 5 days, and 7 days. After the tumor cells were fixed with 4% PAF, immunofluorescence staining of neuronal markers Map2 and Tuj1 was used to detect the effect of the compound on inducing transdifferentiation of tumor cells. Figure 4 It was shown that Neuropathiazol can effectively promote the transdifferentiation of tumor cells into neurons, and as time goes by, the synaptic structure of neurons becomes more obvious.

[0049] 4. The compound inhibits the growth of subcutaneous tumors in immunodeficient mice

[0050] Thirty 6-8 week old SCID female mice weighing about 20 grams were raised. Six million Panc-1 human pancreatic cancer cells were inoculated into the left posterior abdomen of each mouse. After 3 weeks, when the tumor grew to about 100 mm 3, randomly divided into 6 groups, 5 mice in each group. The first group was the control group, which was intraperitoneally injected with 300μl PBS every other day; the second group was intraperitoneally injected with 300μl of the compound at a concentration of 15mg / kg body weight every other day; the third group was intraperitoneally injected with 300μl of the compound at a concentration of 50mg / kg body weight every other day; the fourth group was the control group, which was intraperitoneally injected with 300μl of Gemcitabine at a concentration of 20mg / kg body weight every other day; the fifth group was the control group, which was intraperitoneally injected with 300μl of Cisplatin at a concentration of 10mg / kg body weight every other day; the sixth group was the control group, which was intraperitoneally injected with 300μl of Paclitaxel at a concentration of 15mg / kg body weight every other day. This method was used for continuous injections for 2 weeks, and the weight and tumor size of the mice were measured every 2 days. After 2 weeks of administration, the tumor-bearing mice were killed, and the subcutaneous tumor masses were dissected and photographed ( Figure 5 A), and calculate the tumor volume change curve ( Figure 5 B) and the weight change curve of mice ( Figure 5 C), and then fixed with 4% PFA, and immunohistochemical staining was performed to detect tumor cell proliferation (Ki-67), apoptosis (CC3), and tumor cell transdifferentiation (NeuN). Figure 5 The results showed that the compound at two doses of 15 mg / kg body weight and 50 mg / kg body weight could significantly inhibit the growth of mouse tumors, with a significant dose-effect relationship, and could well promote the transdifferentiation of tumor cells into neurons, with very low toxic and side effects on the weight of mice.

[0051] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of Neuropathiazol in the preparation of products for the treatment of pancreatic cancer.

2. The use according to claim 1, characterized in that: The product described is a drug.

3. The use according to claim 2, characterized in that: Neuropathiazol is the only active ingredient in the drug.

4. The use according to claim 2 or 3, characterized in that: The medicine also contains medically acceptable auxiliary materials.

5. Use of a composition in preparing a product for treating pancreatic cancer, characterized in that: The composition contains Neuropathiazol.

6. The use according to claim 5, characterized in that: The product described is a drug.

7. The use according to claim 6, characterized in that: The medicine also contains medically acceptable auxiliary materials.

8. The use according to claim 6 or 7, characterized in that: The dosage form of the medicine is any one of tablets, capsules, pills, granules and powders.

9. The use according to claim 8, characterized in that: The tablet is any one of enteric-coated tablets, sugar-coated tablets, plain tablets, film-coated tablets, dispersible tablets, sustained-release tablets and controlled-release tablets.

10. The use according to claim 8, characterized in that: The capsule is any one of a hard capsule, a soft capsule, an enteric-coated capsule, a sustained-release capsule, and a controlled-release capsule.

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