Use of neuropathiazol in the preparation of a product for the treatment of pancreatic cancer

Abstract

The application discloses application of Neuropathiazol in preparation of a product for treating pancreatic cancer and belongs to the technical field of tumor drugs. The compound is screened through Cell Counting Kit-8, promoter luciferase, cell immunofluorescence (Ki-67, CC3, Map2, Tuj1) staining and other experiments, and is further evaluated by using an immunodeficient mouse transplanted tumor model and a wild-type C57 mouse orthotopic transplanted tumor model. The experimental results show that the compound can not only significantly inhibit the proliferation of in-vitro cultured pancreatic cancer cell strains Panc-1 and SW1990, but also promote the differentiation of tumor cells into neurons, and can effectively inhibit the growth of transplanted tumors in mice. Since the compound is safe, efficient and has an ideal anticancer effect, the compound will be developed into a new type of therapeutic drug for resisting pancreatic cancer, and will certainly have a good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of tumor drugs, and particularly relates to application of Neuropathiazol in preparation of a product for treating pancreatic cancer. BACKGROUND

[0002] Pancreatic cancer is a malignant tumor originating from pancreatic duct epithelial and acinar cells, which has extremely high malignancy, is difficult to diagnose in the early stage, progresses rapidly, and has a short survival time, and is one of the malignant tumors with the worst prognosis, is called the "king of cancer", and has high morbidity and mortality, ranking in the top ten of malignant tumors in the world and China. The data of the National Comprehensive Cancer Network of the United States in 2019 showed that about 55440 patients were diagnosed with pancreatic cancer in the United States in 2018, and about 43330 patients died of pancreatic cancer. The data of the China National Cancer Center in 2019 showed that pancreatic cancer ranked the 10th in the morbidity of malignant tumors in China, and the 6th in the mortality of malignant tumors. At present, through the comprehensive means of surgery combined with chemotherapy, immunotherapy and targeted therapy, the 5-year overall survival rate is only 8.5%. Only a small number of cases of early detection and timely surgery can achieve a cure effect, although this treatment mode can benefit some patients and improve the overall survival rate, but the long-term effects such as tumor recurrence also increase. In addition, radiotherapy and chemotherapy also bring serious side effects such as hair loss, nausea and vomiting, and reduced platelets. It is urgent to explore the molecular mechanism related to the occurrence, development, recurrence and metastasis of pancreatic cancer, and to improve the therapeutic effect and reduce the toxicity of treatment, which is an important direction for clinical trials and basic research on pancreatic cancer at present, in order to find new intervention molecular targets 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 opened up the research of tumor cell differentiation. Therefore, the concept of differentiation therapy has been proposed, which is a treatment method that promotes tumor cells to differentiate into mature stages or other normal cells through inducers, changes their malignant biological behavior, and makes them lose the characteristics of tumor cell malignant proliferation and metastasis. The core idea of this treatment is that through specific inducers, tumor cells can return to or be closer to the state of normal cells, thereby losing their cancer cell characteristics. Induced differentiation therapy of pancreatic cancer forces tumor cells to transdifferentiate into mature neurons, although it cannot cause tumor death like traditional cytotoxic therapy, but can significantly inhibit the growth and proliferation of tumor cells, and minimize the impact on normal cells, achieving a high-risk benefit cure anti-cancer therapy, and bringing a safer and more reliable treatment for clinical pancreatic cancer patients.

[0004] Neuronal differentiation factor 1 (NeuroD1) is a transcription factor with a HLH (Helix-Loop-Helix) structure, which can promote the differentiation of neurons, plays a crucial role in the normal development of the nervous system, and is involved in the differentiation of neural crest cells. The inventors' previous article found that NeuroD1 can promote the transdifferentiation of medulloblastoma tumor cells into neurons (Cell Rep. 2020 Jun 23; 31(12): 107782), and some articles have reported 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 the previous study 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 for inducing pancreatic cancer differentiation therapy. SUMMARY

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

[0006] Preferably, the product is a medicament.

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

[0008] More preferably, the medicament further contains a medically acceptable excipient.

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

[0010] Preferably, the product is a medicament.

[0011] More preferably, the medicament further contains a medically acceptable excipient.

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

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

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

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

[0016] The present application finds that the compound Neuropathiazol can not only significantly inhibit the proliferation of in-vitro cultured pancreatic cancer cell lines (Panc-1, S W1990), but also promote the differentiation of tumor cells into neurons, and effectively inhibit the growth of transplanted tumors in mice. Due to its safety, high efficiency and ideal anticancer effect, it can be tried to develop it as a new type of therapeutic drug for pancreatic cancer, which must have good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The structural formula of the Neuropathiazol compound in the present application.

[0018] Figure 2 The purpose compound obtained by screening the small molecule neuron inducer compound library in Example 1, wherein A. small molecule neuron inducer compound screening process (schematic diagram) and IC50 value of compound treatment of two kinds of pancreatic cancer cells and fluorescence intensity of regulation of Neuro D1 promoter transcription activity (table); B. IC50 value of Neuropathiazol, gemcitabine, cisplatin, paclitaxel four kinds of compounds treating Pan-1 pancreatic cancer cells; C. NeuroD1 promoter fluorescence value of four kinds of compounds treating Pan-1 pancreatic cancer cells; D. mRNA expression level of Neuro D1 of four kinds of compounds treating Pan-1 pancreatic cancer cells; E. Neuro D1 protein expression level of four kinds of compounds treating pancreatic cancer cells Pan-1.

[0019] Figure 3 The proliferation of pancreatic cancer cells treated by four kinds of compounds in Example 1, wherein A-B. Staining and statistical analysis of proliferation (Ki-67) and apoptosis (CC3) of Pan-1 pancreatic cancer cells treated by Neuropathiazol, gemcitabine, cisplatin and paclitaxel for 48 hours.

[0020] Figure 4 The transdifferentiation of pancreatic cancer cells into neurons induced by the compound in Example 1, after Neuropathiazol treated tumor cells for 0 days, 3 days, 5 days and 7 days, the cells were collected for Map2 and Tuj1 immunofluorescence staining.

[0021] Figure 5 To implement the compound in Example 1 to inhibit the growth of subcutaneous transplanted tumors in mice, wherein A-C. is the picture size of tumor growth (A), the growth curve (B) and the weight change curve (C) of each treatment group; D-G is the tumor efficacy test result of each treatment group. DETAILED DESCRIPTION

[0022] Example 1

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

[0024] (1) Through the small molecule neuron inducer compound library recommended by Shanghai Taoshu Biological Company, combined with literature research, a preliminary screening of candidate compounds was carried out to obtain a screening list of candidate compounds.

[0025] (2) Dissolve the candidate compounds in DMSO to prepare 10 mM stock solution, respectively. Panc-1, SW1990 pancreatic cancer cell lines were inoculated in 96-well plates at a density of 10,000 cells per well, and cultured in a 37℃, 5% CO2 cell incubator for 24 hours. The original culture medium was aspirated, and the Panc-1, SW1990 tumor cells were treated in vitro with drug concentration gradients of 0, 10 nM, 100 nM, 500 nM, 1 μM, 5 μM, 10 μM, 25 μM, 50 μM, and 100 μM. Each treatment group had 3 replicate wells. After 48 hours of continuous culture in each well with 100 μL of drug-containing medium, 10 μL of CCK-8 reagent (CK04, Dojindo, Japan) was added to each well, and incubated in the cell incubator for 2 hours. Then the absorbance was read at 450 nm wavelength to evaluate the toxicity of the compounds, and the chemotherapeutic 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 binding luciferase was established. The tumor cells were treated in vitro with 0, 0.5 μM, and 2 μM concentrations for 72 hours. The luciferase detection kit (DL101-01, Vazyme, China) was used to detect the fluorescence intensity at 560 nm. According to the fluorescence intensity, low-toxicity and high-efficiency neuron inducers that can up-regulate NeuroD1 transcription expression were screened.

[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. Anti-NeuroD1 (1:200; 12081-1-AP, Proteintech), and the primer sequences are as follows:

[0028] Table 1

[0029]

[0030] 2. The compound inhibits 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 detected in vitro, as follows: Panc-1, SW1990 pancreatic cancer cell strains are inoculated in 24-well plates with cell climbing sheets at a density of 100,000 cells per well. After 6 hours of cell adhesion, the medium is replaced with 0, 0.5 μM, and 2 μM of the drug, 1 μM of gemcitabine, 2 μM of cisplatin, and 5 μM of paclitaxel to continue treating the tumor cells for 48 hours. The culture medium is aspirated, and 300 μL of 4% PFA is added to fix the cells for 10 minutes, followed by three washes with PBS.

[0032] (2) After washing, 300 μl of 10% NGS blocking solution prepared in PBST (PBS + 0.03% Triton X-100) is added to the cell climbing sheet and incubated at room temperature for 1 hour. After blocking, the blocking solution is discarded, and the primary antibody diluted in blocking solution is added for overnight incubation at 4°C. The next day, the cells are washed three times with PBST for 10 minutes each time on a shaker. The secondary antibody diluted in blocking solution is added and incubated at room temperature for 1 hour. The secondary antibody is discarded, and the cells are washed as above. DAPI dye is added and incubated at room temperature for 5 minutes, then discarded and washed as above. After washing, the cell climbing sheet is removed with tweezers and inverted onto a glass slide with anti-fluorescence quencher (Southern Biotechnology), placed in a dark place at room temperature, and air-dried for half an hour. Immunofluorescence is photographed. The antibodies used are as follows: Ki67 (1:200; 27309-1-AP, Proteintech), CC3 (1:200; ab52101, Abeam), goat anti-mouse CoraLite594 (1:200; Proteintech, SA00013-3), and goat anti-rabbit CoraLite594 (1:200; Proteintech, SA00013-4).

[0033] 3. The compound induces the transdifferentiation of pancreatic cancer cells into neurons

[0034] (1) Detection of the ability of the compound to induce differentiation of pancreatic cancer cells, specifically as follows: 40,000 cells of Panc-1, SW1990 pancreatic cancer cell lines were inoculated in 24-well plates in advance with cell climbing sheets, and after 6 hours of cell adhesion, the medium was replaced with 0, 0.5 μM, and 2 μM of the drug, and the tumor cells were treated for 0 hours, 3 days, 5 days, and 7 days, respectively. Then the culture medium was aspirated, and 300 μL of 4% PFA was added to fix the cells for 10 minutes, followed by washing with PBS three times.

[0035] (2) The washed cell climbing sheet was subjected to neuronal marker immunofluorescence staining, and the method was the same as above, and the antibodies used were as follows: Map2 (1:1000; 17490-1-AP, Proteintech), Tuj1 (1:1000; 66375-1-Ig, Proteintech).

[0036] 4. Growth of subcutaneous transplanted tumors in immunodeficient mice transplanted with compounds

[0037] (1) 30 immunodeficient (SCID) female mice, 6-8 weeks old, weighing about 20 grams, were fed. 6 million Panc-1 human pancreatic cancer cells were inoculated into the left posterior abdomen of each mouse, and after 3 weeks, the tumor was grown to about 100 mm 3 , and randomly divided into 6 groups, 5 in each group. The first group was the control group, and 300 μl of PBS was injected intraperitoneally every other day; the second group was injected intraperitoneally with 300 μl of compound Neuropathiazol at a concentration of 15 mg / kg body weight every other day; the third group was injected intraperitoneally with 300 μl of compound Neuropathiazol at a concentration of 50 mg / kg body weight every other day; the fourth group was the control group, and 300 μl of Gemcitabine at a concentration of 20 mg / kg body weight was injected intraperitoneally every other day; the fifth group was the control group, and 300 μl of Cisplatin at a concentration of 10 mg / kg body weight was injected intraperitoneally every other day; the sixth group was the control group, and 300 μl of Paclitaxel at a concentration of 15 mg / kg body weight was injected intraperitoneally every other day. The method was continuously injected for 2 weeks, and the mouse weight and tumor size were measured every other day, and the tumor volume change 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 sacrificed, the subcutaneous tumor mass was dissected, fixed with 4% PFA, and immunohistochemical staining was performed to detect tumor cell proliferation (Ki-67 staining), apoptosis (CC3 staining), and the degree of tumor cell transdifferentiation (NeuN staining). The antibodies used are as follows: Ki67 (1:200; 27309-1-AP, Proteintech), CC3 (1:200; ab52101, Abeam), NeuN (1:200; ab104224, Abeam), goat anti-mouse CoraLite594 (1:200; Proteintech, SA00013-3) and goat anti-rabbit CoraLite594 (1:200; Proteintech, SA00013-4).

[0039] Data statistics

[0040] All experiments in this study were repeated at least three times. Data were analyzed using GraphPad Prism 9 and Excel software, and are expressed as the mean (Mean) ± standard error (SEM). Statistical analysis of differences between multiple groups was performed using one-way ANOVA, and multiple comparisons were made using Turkey. Survival analysis was performed using the Kaplan-Meier curve, and Log-rank was used for statistical analysis (ns P>0.05, *P<0.05, **P<0.001, ***P<0.0001).

[0041] Results

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

[0043] Through the small molecule neuron-inducing compound library recommended by Shanghai Taoshu Biological Company, combined with literature research, 16 candidate compounds were obtained. In vitro, Panc-1 and SW1990 tumor cells were treated with 0, 10 nM, 100 nM, 500 nM, 1 μM, 5 μM, 10 μM, 25 μM, 50 μM, and 100 μM drug concentration gradients, and cultured for 48 hours. CCK-8 experiment was used to evaluate the toxicity of the compounds, and the chemotherapeutic drugs gemcitabine, cisplatin, and paclitaxel were used as positive controls for cytotoxicity.

[0044] A tumor cell screening system based on NeuroD1 promoter binding luciferase was established. In vitro, tumor cells were treated with 0, 0.5 μM, and 2 μM concentrations for 72 hours. The luciferase detection kit was used to detect the fluorescence intensity at 560 nm, and the fluorescence intensity was correlated with NeuroD1 transcription expression. The results are as follows Figure 2A and 2B show that, compared with other small-molecule neuron-inducing compounds and three chemotherapeutic drugs, Neuropathiazol has lower IC50 values, i.e. lower cytotoxicity, and stronger regulation of NeuroD1 promoter activity in two pancreatic cancer cell lines Figure 2 C). Then, the mRNA Figure 2 D) and protein Figure 2 E) expression levels of NeuroD1 in cells treated with the compounds were detected by Western Blotting and qPCR, respectively. The results showed that Neuropathiazol could inhibit the proliferation of pancreatic cancer cell lines at a lower concentration and better regulate the expression of transcription factor NeuroD1.

[0045] 2. Compound inhibits the proliferation of human pancreatic cancer cells

[0046] After the target compound Neuropathiazol was screened, the cells were treated with 0, 0.5 μM, and 2 μM of the drug in vitro, and 1 μM of gemcitabine, 2 μM of cisplatin, and 5 μM of paclitaxel were used as positive cytotoxic drugs to treat tumor cells for 48 hours. The effects of the compound on tumor cell proliferation and apoptosis were detected by Ki67 and CC3 immunofluorescence staining experiments. As shown in Figure 3 A-3B, Neuropathiazol could well inhibit tumor cell proliferation at a concentration of 0.5 μM, showing a concentration-dependent manner, and the toxicity of Neuropathiazol to tumor cells was much lower than that of the three cytotoxic chemotherapeutic drugs.

[0047] 3. Compound induces transdifferentiation of pancreatic cancer cells into neurons

[0048] After observing the low-toxicity and high-efficiency inhibition of tumor cells by the target compound, Panc-1 cells were treated with 2 μM of the drug for 0 days, 3 days, 5 days, and 7 days in vitro. After 4% PAF fixation of the tumor cells, the effects of the compound on the induction of transdifferentiation of tumor cells were detected by immunofluorescence staining of neuron markers Map2 and Tuj1. As shown in Figure 4 Neuropathiazol could well promote the transdifferentiation of tumor cells into neurons, and the synaptic structure of neurons became more obvious with the increase of time.

[0049] 4. Compound inhibits the growth of subcutaneous transplanted tumors in immunodeficient mice

[0050] Thirty 6-8-week-old immunodeficient (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 three weeks, the tumor body grew to about 100 mm 3, randomly divided into 6 groups, 5 in each group. The first group is the control group, intraperitoneal injection of 300 μl PBS every other day; the second group intraperitoneal injection of 300 μl of the compound with a concentration of 15 mg / kg body weight every other day; the third group intraperitoneal injection of 300 μl of the compound with a concentration of 50 mg / kg body weight every other day; the fourth group is the control group, intraperitoneal injection of 300 μl of Gemcitabine with a concentration of 20 mg / kg body weight every other day; the fifth group is the control group, intraperitoneal injection of 300 μl of Cisplatin with a concentration of 10 mg / kg body weight every other day; the sixth group is the control group, intraperitoneal injection of 300 μl of Paclitaxel with a concentration of 15 mg / kg body weight every other day. According to the method, continuous injection for 2 weeks, every 2 days to measure the body weight and tumor size of mice. After 2 weeks of administration, the tumor-bearing mice were sacrificed, and the subcutaneous tumor mass was dissected, photographed Figure 5 A), and the tumor volume change curve Figure 5 B) and the body weight change curve of mice Figure 5 C) were calculated, then fixed with 4% PFA, and immunohistochemical staining was performed to detect tumor cell proliferation (Ki-67), apoptosis (CC3), and tumor cell transdifferentiation degree (NeuN), as shown in Figure 5 D-G. The results show that the compound can significantly inhibit the growth of mouse tumors at two doses of 15 mg / kg body weight and 50 mg / kg body weight, and the dose-effect relationship is obvious, and can well promote the transdifferentiation of tumor cells into neurons, and has very low toxic side effects on the body weight of mice.

[0051] The above-described embodiments are only descriptions of the preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the scope of protection determined by the claims of the present application.

Claims

1. Use of neuropathiazol for the preparation of a medicament for the treatment of pancreatic cancer.

2. Use according to claim 1, characterized in that, Neuropathiazol is the only active ingredient in the medicament.

3. Use according to claim 1 or 2, characterized in that, The medicament also contains a pharmaceutically acceptable excipient.

4. Use of a composition for the manufacture of a medicament for the treatment of pancreatic cancer, characterized in that, The composition contains neuropathiazol.

5. Use according to claim 4, characterized in that, The medicament also contains a pharmaceutically acceptable excipient.

6. Use according to claim 4 or 5, characterized in that, The dosage form of the medicament is one of a tablet, a capsule, a pill, a granule, and a powder.

7. Use according to claim 6, characterized in that, The tablet is one of an enteric-coated tablet, a sugar-coated tablet, a plain tablet, a film-coated tablet, a dispersible tablet, a sustained-release tablet, and a controlled-release tablet.

8. Use according to claim 6, characterized in that, The capsule is 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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