CN111904957A - Application of oxicaine in preparing antitumor drug - Google Patents

Abstract

The invention discloses an application of oxicaine in preparing antineoplastic drug, wherein the oxicaine (oxenthazaine) has the chemical name: 2-bis (N-methyl-N-phenyl tert-butyl-carbamoylmethyl) aminoethanol having CAS number 126-27-2 and molecular formula C₂₈H41N₃O₃And the molecular weight is 467.64. The anti-tumor drug is a drug for treating esophageal cancer or gastric cancer. Experiments prove that when the oxicaine is used for esophageal squamous cell carcinoma cells (KYSE 150 cells and KYSE450 cells) or gastric cancer cells (HGC 27 cells and AGS cells), the oxicaine can play a role in inhibiting growth and transformation of the esophageal squamous cell carcinoma cells or gastric cancer cells, and the appropriate concentration of the oxicaine for inhibiting proliferation and transformation of the esophageal squamous cell carcinoma cells or gastric cancer cells is 1.0-10 mu M.

Description

Application of oxicaine in preparing antitumor drug

Technical Field

The application belongs to the field of biological medicine, and particularly relates to application of oxicaine in preparation of antitumor drugs.

Background

Worldwide, cancer is a leading cause of morbidity and mortality. According to WHO reports, in 2012, cancer is estimated to occur in 1400 million new cases worldwide, and will rise to 2200 million per year and death will rise to 1300 million per year in the next 20 years.

Cancer treatment is one of the difficult and difficult problems in the world at present, not only is complete cure difficult, but also the improvement of the life cycle of patients has a lot of difficulties. Statistics show that Chinese esophageal cancer and gastric cancer are the most common types of cancer and the most common causes of cancer death.

Esophageal cancer, among the most common tumors causing cancer death in china, is located in the sixth place, with very poor prognosis and a five-year survival rate of only 15-20%. Esophageal Squamous Cell Carcinoma (ESCC) and Esophageal Adenocarcinoma (EAC) are the two major histological types of esophageal cancer. ESCC is the most common subtype in developing countries, but esophageal cancer is predominantly EAC in the united states and other western countries. Esophageal cancer typically has progressive dysphagia, which is characterized by difficulty swallowing dry food, followed by semifluid food, and finally, water and saliva.

Gastric cancer (gastric cancer) is a malignant tumor originated from gastric mucosal epithelium, the incidence rate of the gastric cancer is the first in various malignant tumors in China, the incidence rate of the gastric cancer is obviously different regionally, and the incidence rate of the gastric cancer is obviously higher in northwest and east coastal areas of China than in south areas. Gastric cancer can occur at any age, and the symptoms of gastric cancer are not obvious in early stage, which is called invisible killer, and most of gastric cancers are diagnosed in the middle and late stage. Gastric cancer tends to be younger due to changes in dietary structure, increased working pressure, infection with helicobacter pylori, and the like. Gastric cancer generally has no typical clinical manifestations, and common symptoms are gastral cavity distending pain, emaciation, anorexia, hypodynamia, acid regurgitation, dark stool and the like.

In recent years, despite the great progress in the early diagnosis and early detection of esophageal and gastric cancers, and the clinical application of standard chemotherapy and radiotherapy for decades, the overall survival rate of most patients in the middle and late stages of the clinic is not improved significantly, suggesting that only early detection and treatment of tumors are emphasized, while tumor prevention is ignored, with little success.

Although treatment regimens for cancer are rapidly evolving, treatment is challenged by drug toxicity and postoperative recurrence due to drug resistance. New safer and more effective treatment options are therefore highly desirable. With the introduction of the concept of "Old Drugs for New use" (Liu Q. Editorial: Old Drugs Learn New locks: Advances and Applications for Drug delivery. Curr Top Med Chem 2016; 16: 3627. 3628.), more and more doctors and scientists believe that clinical non-antineoplastic Drugs which have been popularized and applied can be explored and developed into New anti-cancer Drugs if the basic and clinical verification is carried out. The method can save the development cost and time of the new medicine and also can ensure the physicochemical property, the pharmacokinetics and the safety of the medicine to a certain extent.

Oxicaine is a potent local anesthetic that acts to relieve dysphagia and relieve pain due to reflux, chronic gastritis and duodenal ulcers. Oxicaine inhibits gastric acid secretion by inhibiting gastrin secretion. In addition, the medicine has local anesthetic effect on gastric mucosa and unique chemical characteristics, however, the medicine has no report on the effect of inhibiting the proliferation and growth of esophageal cancer and gastric cancer, and no related patent application exists.

Disclosure of Invention

The invention aims to provide a new application of oxicaine in the technical field of medicines, namely an application of oxicaine in preparing anti-tumor medicines, in particular to a medicine for treating and preventing esophageal cancer or gastric cancer.

Based on the purpose, the invention adopts the following technical scheme:

application of oxicaine in preparing antitumor drugs. Oxicaine (Oxethazaine), chemical name: 2-bis (N-methyl-N-phenyl tert-butyl-carbamoylmethyl) aminoethanol having CAS number 126-27-2 and molecular formula C₂₈H41N₃O₃Molecular weight 467.64, structural formula:

。

the anti-tumor drug is a drug for treating esophageal cancer or gastric cancer. The anti-tumor medicine comprises oxicaine, pharmaceutically acceptable salts and esters of the oxicaine, or combinations of the oxicaine and the pharmaceutically acceptable salts and esters, or combinations of the oxicaine and other compounds and medicines.

The anti-tumor drug can be applied to preventing tumor occurrence, treating tumor and preventing tumor recurrence.

The anti-tumor medicament is prepared into medicinal formulations, wherein the formulations comprise sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquid, buccal agents, granules, medicinal granules, pills, pellets, suspensions, powder, vinum, preparations, drops, injection, powder injections, creams, sustained-release agents, targeting agents and the like, and the anti-tumor medicament is used as a medicament for preventing, treating and preventing recurrent cancers.

The administration mode of the antitumor drug comprises oral administration, injection, implantation, external application, spraying, inhalation or the combination of the oral administration, the injection, the implantation, the external application, the spraying and the inhalation.

Specifically, the application of the oxicaine in the medicines for treating and preventing the esophageal cancer or the gastric cancer is realized by using an esophageal cancer cell line KYSE150, KYSE450 and a gastric cancer cell line HGC27 and AGS through a cytotoxicity experiment, a cell proliferation experiment and a soft agar clone formation experiment, treating the clinical non-anticancer application medicine oxicaine in cells, and finding that the oxicaine has a toxic effect on esophageal squamous cell KYSE150 cells and KYSE450 and the gastric cancer cell line HGC27 and AGS cells at 12.5 mu M and has a certain inhibition effect on the proliferation of the cancer cells at 1 mu M, so the oxicaine has the function of inhibiting the proliferation and clone formation capabilities of the esophageal cancer and the gastric cancer cell lines at the concentration of 1-10 mu M.

Furthermore, the invention utilizes an esophageal cancer PDX (pdx) (Patientderiversedxenogrft) model, namely a subcutaneous transplanted tumor model which is established on an immunodeficient mouse by utilizing cancer tissues of a cancer patient and grows by depending on a microenvironment provided by the mouse, shows a remarkable anti-tumor growth effect and can play a role in preventing esophageal cancer relapse. According to the invention, the tumor treatment concentration of the oxicaine in SCID mice is 6-48 mg/kg, and the oxicaine has no obvious influence on the body weight of the mice, so that the oxicaine is the acceptable safe drug concentration.

The invention has the beneficial effects that: the invention discloses that the oxicaine can be used for treating and preventing esophageal cancer, gastric cancer and other tumors for the first time, inhibiting growth and proliferation of esophageal cancer and gastric cancer cells, inhibiting in-vitro clone formation, and has a remarkable effect on prevention of a humanized transplanted tumor model (PDX) of the esophageal cancer. The implementation of the invention widens the medical application of the oxicaine, provides a new idea for treating human esophageal cancer, gastric cancer and other tumors, and has important significance for treating, preventing and recrudescence of the tumors.

Drawings

FIG. 1 is a graph of the results of the effect of oxicaine on esophageal cancer cells; a is an experimental result graph of toxicity of the oxicaine on esophageal cancer cells; b is an analysis chart of the influence of the oxicaine on the proliferation activity of the esophageal cancer cells KYSE150 and KYSE 450;

FIG. 2 is a graph showing the effect of oxicaine on the ability of the esophageal cancer cells KYSE150 and KYSE450 to clone in vitro;

FIG. 3 is a graph showing the results of the effect of oxybivacaine on gastric cancer cells; a is an experimental result graph of the cytotoxicity of the oxicaine on the gastric cancer;

b is an analysis chart of the effect of the oxicaine on the proliferation activity of gastric cancer cells HGC27 and AGS;

FIG. 4 is a graph showing the effect of treatment of tumor growth of oxybivacaine in a mouse model with esophageal cancer accession number EG20, wherein A is the trend of the body weight of mice treated with oxybivacaine in a PDX mouse model of tumor tissue of patients with esophageal squamous cell carcinoma during administration; b is a graph of the change trend of the tumor volume of a mouse treated by the oxybivacaine in a PDX mouse model of the tumor tissue of the esophageal squamous cell carcinoma patient during administration; c is the weight statistical result of the tumor weighed by the PDX mouse model of the tumor tissue of the esophageal squamous cell carcinoma patient after the mouse is killed after the treatment of the oxybivacaine; d is a tumor picture of a PDX mouse model of tumor tissues of an esophageal squamous carcinoma patient, which is treated by the oxybivacaine, of a control group and an administration group after the mouse is killed;

in fig. 1 to 4, xp<0.05, **p<0.01, ***p<0.001。

Detailed Description

The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention. The experimental procedures in the examples, unless otherwise specified, were carried out by conventional techniques in the art and the experimental reagents were all purchased commercially.

Materials and methods

Material

Esophageal squamous carcinoma cells KYSE150 and KYSE450 and gastric carcinoma cells HGC27 and AGS from Zhengzhou university basic medical college pathophysiology research room

Cell lines: KYSE150 cell line, wherein the cell line adopts 1640 culture medium containing 10% FBS, and is constant in temperature of 37 ℃ and 5% CO₂Culturing under the condition for later use.

KYSE450 cell strain, wherein the cell strain adopts DMEM medium containing 10% FBS, is constant in temperature of 37 ℃ and contains 5% CO₂Culturing under the condition for later use.

HGC27 cell line, the cell line adopts 1640 culture medium containing 10% FBS, the temperature is constant at 37 ℃, and 5% CO₂Culturing under the condition for later use.

AGS cell line, wherein the cell line adopts F12K culture medium containing 10% FBS, and is constant in temperature of 37 ℃ and 5% CO₂Culturing under the condition for later use.

Tumor tissue

The present invention used 1 human esophageal cancer tissue specimen, numbered EG20 (from tumor hospital, patient, male, 46 years old, hospital number 2042083, T2N0M0 ii, department of south fluvial province, mid-differentiation).

Laboratory animal

SCID CB-17 mice in this example were purchased from Experimental animals technology, Inc. of Wei Tongliwa, Beijing. The license number is: SCXK (jing) 2012-0001. SPF grade, SCID mice are 6-8 weeks old, 16-18 g in weight, female. Mouse feeds were purchased from Aojieli feeds, Inc., Beijing, Ke. The license number is SCXK (Jing) 2014-0010.

Reagent

RPMI-1640 medium, DMEM medium 500ml one bottle (Biological Industries); F12K (Gibco); FBS (biological industries); Trypsin-EDTA pancreatin digestive juice (Shanghai Bin Yuntian Biotechnology Co., Ltd.); PBS (beijing solibao technologies ltd); a cell culture dish; pentobarbital sodium (national pharmaceutical group chemical agents limited): weighing sodium pentobarbital, dissolving in sterile distilled water, and filtering; double resistance: penicillin and streptomycin were purchased from north China pharmaceutical Co., Ltd, both at a concentration of 500U/ml; physiological saline 500 ml/bottle (Chenxin pharmaceutical Co., Ltd.); oxicaine: the in vitro experimental drug was purchased from TargetMol, and the in vivo experimental drug was purchased from TRC.

Apparatus and equipment

A medical heat preservation refrigerator for Haler; an electronic balance; a Thermo clean bench; an electronic balance; cell culture case, ophthalmology scissors, surgical forceps, aseptic culture dish, scalpel, medicine dissolving needle, syringe.

Example 1 in vitro assay of Oxicaine to inhibit proliferation of esophageal cancer cells

Cytotoxicity test

The experimental process is as follows: KYSE150 cells at 8X 10³Each well was inoculated in a 96-well plate (10% FBS/1640, 37 ℃ C., 5% CO)₂) KYSE450 cells at 1.2X 10⁴Each well was inoculated in a 96-well plate (10% FBS/DMEM, 37 ℃, 5% CO)₂) 14-16 hours later, the culture medium was replaced with fresh medium and the final concentration of the oxybivacaine in the culture medium was 0. mu.M, 3.125. mu.M, 6.25. mu.M, 12.5. mu.M, 25. mu.M, 50. mu.M, 75. mu.M, 100. mu.M, and after 24 and 48 hours of culture, the cells were removed from the incubator, the original medium was poured off, and washed twice with 1 XPBS. PBS was then discarded, 4% paraformaldehyde was added at 100. mu.L/well, fixed for 30 minutes, paraformaldehyde discarded, and washed twice with 1 XPBS. PBS was then discarded, stained with DAPI (DAPI stock: 1 XPBS = 1: 5000 dilution, Beijing Solibao technologies Co., Ltd.), 100. mu.L/well, incubated at 37 ℃ for 20 minutes, and the number of cells in each well was counted.

The results of the experiment are shown in FIG. 1A. As can be seen from FIG. 1A, when the concentration of the oxicaine was 12.5. mu.M, the cell survival rate was 75% (KYSE 150, 48 h) and 28% (KYSE 450, 48 h), and a certain cytotoxicity was exhibited, and safe drug concentrations were selected and determined to be 0. mu.M, 1. mu.M, 2.5. mu.M, 5. mu.M, and 10. mu.M, and further proliferation experiments were performed.

Cell proliferation assay

The experimental process is as follows: KYSE150 cells at 3X 10³Each well was inoculated in a 96-well plate (10% FBS/1640, 37 ℃ C., 5% CO)₂) KYSE450 cells at 5X 10³Each well was inoculated in a 96-well plate (10% FBS/DMEM, 37 ℃, 5% CO)₂) 14-16 hours later, the fresh medium was replaced and the final concentration of the oxicaine in the medium was 0. mu.M, 1. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M, added (DMSO prepared as a solution), after culturing for 0, 24, 48, 72 and 96 hours respectively, the cells were removed from the incubator, the original medium was decanted, and washed twice with 1 XPBS. PBS was then discarded, 4% paraformaldehyde was added at 100. mu.L/well, fixed for 30 minutes, paraformaldehyde discarded, and washed twice with 1 XPBS. PBS was then discarded, stained with DAPI (DAPI stock: 1 XPBS = 1: 5000 dilution, Beijing Solibao technologies Co., Ltd.), 100. mu.L/well, incubated at 37 ℃ for 20 minutes, and the number of cells in each well was counted.

The results of the experiment are shown in FIG. 1B. As can be seen in fig. 1B, oxybivacaine has an inhibitory effect on esophageal cancer cells and appears to be time and dose dependent.

As can be seen in fig. 1B, oxybivacaine has an inhibitory effect on esophageal cancer cells and appears to be time and dose dependent. The growth inhibitory effect on KYSE150 cells was significant in the cases of the concentration of oxicaine at 1.5. mu.M, 2. mu.M and 2.5. mu.M, and the growth inhibitory effect on KYSE450 cells was significant in the cases of the concentration of oxicaine at 1.5. mu.M, 2. mu.M and 2.5. mu.M.

Example 2 in vitro assay of the ability of Oxicaine to inhibit clonogenic potential of esophageal cancer cells

The experimental process is as follows: 3 mL of BME medium (containing 10% FBS and 0.5% agar) was plated per well on a 6-well plate, and esophageal cancer cells suspended therein (KYSE 150: 8X 10) were plated after coagulation³Root/pore or KYSE450：8×10³Pieces/well) of supernatant (1 mL of BME medium containing 10% FBS and 0.33% agar, final concentrations of drug in the medium being 0. mu.M, 1. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M, respectively), in an incubator (37 ℃, 5% CO)₂) After 7-14 days of culture or depending on the Cell condition, to be cloned, the number of clones was counted by IN Cell Analyzer 3000, and FIG. 2 shows the result obtained after 9 days of culture.

The results of the experiment are shown in FIG. 2. As can be seen from FIG. 2, the number of cell clones is reduced after the treatment of the oxicaine, the size of a single colony of the cell is obviously reduced, which shows that the oxicaine can inhibit the in vitro cloning formation capability of the esophageal cancer cell, the concentration of the oxicaine is 2.5. mu.M, 5. mu.M and 10. mu.M, the oxicaine has an inhibiting effect on the cloning formation of the KYSE150 cell or the KYSE450 cell, and the concentration of the oxicaine is 5. mu.M and 10. mu.M, the inhibiting effect on the cloning formation of the KYSE150 cell or the KYSE450 cell is obvious.

Example 3 in vitro experiment of Oxicaine to inhibit gastric cancer cell proliferation

Cytotoxicity test

The experimental process is as follows: HGC27 cells at 6X 10³Each well was inoculated in a 96-well plate (10% FBS/1640, 37 ℃ C., 5% CO)₂) AGS cells at 8X 10³Each well was inoculated in a 96-well plate (10% FBS/F12k, 37 ℃, 5% CO)₂) 14-16 hours later, the fresh medium was replaced and the final concentration of the oxicaine in the medium was 0. mu.M, 3.125. mu.M, 6.25. mu.M, 12.5. mu.M, 25. mu.M, 50. mu.M, 100. mu.M by adding the oxicaine (DMSO prepared as a solution), after 24 and 48 hours of culture, the cells were taken out of the incubator, the original medium was poured off, and washed twice with 1 XPBS. PBS was then discarded, 4% paraformaldehyde was added at 100. mu.L/well, fixed for 30 minutes, paraformaldehyde discarded, and washed twice with 1 XPBS. PBS was then discarded, stained with DAPI (DAPI stock: 1 XPBS = 1: 5000 dilution, Beijing Solibao technologies Co., Ltd.), 100. mu.L/well, incubated at 37 ℃ for 20 minutes, and the number of cells in each well was counted.

The results of the experiment are shown in FIG. 3A. As can be seen from FIG. 3A, when the concentration of oxicaine was 12.5. mu.M, the cell survival rate was 43% (HGC 27, 48 h) and 35% (AGS, 48 h), and a certain cytotoxicity was exhibited, and safe drug concentrations were selected and determined to be 0. mu.M, 1. mu.M, 2.5. mu.M, 5. mu.M, and 10. mu.M, and further proliferation experiments were performed.

Cell proliferation

The experimental process is as follows: HGC27 cells at 3X 10³Each well was inoculated in a 96-well plate (10% FBS/1640, 37 ℃ C., 5% CO)₂) AGS cells at 3X 10³Each well was inoculated in a 96-well plate (10% FBS/F12k, 37 ℃, 5% CO)₂) 14-16 hours later, the fresh medium was replaced and the final concentration of the oxicaine in the medium was 0. mu.M, 1. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M, added (DMSO prepared as a solution), after culturing for 0, 24, 48, 72 and 96 hours respectively, the cells were removed from the incubator, the original medium was decanted, and washed twice with 1 XPBS. PBS was then discarded, 4% paraformaldehyde was added at 100. mu.L/well, fixed for 30 minutes, paraformaldehyde discarded, and washed twice with 1 XPBS. PBS was then discarded, stained with DAPI (DAPI stock: 1 XPBS = 1: 5000 dilution, Beijing Solibao technologies Co., Ltd.), 100. mu.L/well, incubated at 37 ℃ for 20 minutes, and the number of cells in each well was counted.

The results of the experiment are shown in FIG. 3B. As can be seen from fig. 3B, the proliferative activity of gastric cancer cells was decreased as the concentration of oxicaine was increased, and the effect was more significant as the treatment time was prolonged. The inhibitory effect on HGC27 cell proliferation was significant in 72h and 96h cultures at 2.5 μ M, 5 μ M, and 10 μ M concentrations of oxicaine, p <0.05,. p <0.01,. p <0.001 at 72h and 96h cultures at 1 μ M, 2.5 μ M, 5 μ M, and 10 μ M concentrations of oxicaine.

Example 4 establishment of a model of human esophageal cancer immunodeficiency mouse melanoma

Fresh tumor tissue from the patient was removed, the tissue was trimmed to size, rinsed with PBS containing penicillin streptomycin (PBS: 50:1 diad) and placed on ice for inoculation before tissue inoculation. Selecting 6-8 week female SCID mouse, anesthetizing, pricking a small opening with a drug dissolving needle, spreading with forceps, and placing tumor tissueThe mice were subcutaneously returned to the sterile rearing room after they were anesthetized and recovered. After the wound on the neck and the back of the mouse healed after about 3 to 5 days, the tumor volume of the mouse is measured once every fixed time until the tumor volume reaches 1000 mm³At that time, mice were sacrificed and tumor tissue was removed. Subcultured to the subcutaneous stage of new SCID mice in the same manner (passage 2). When the transplanted tumor is stably transferred to 3 generations, the success establishment of the esophageal cancer transplanted tumor model is proved.

Example 5 Oxicaine inhibits tumor growth in human esophageal carcinoma xenograft mice

One or two weeks after inoculation, the mice begin to be grouped when the tumor nodules on the backs of the mice grow to about 200 cubic millimeters, namely the mice are uniformly distributed to each group according to the volume of the tumors, and each group is more than 10 mice. The 3 groups of mice were individually gavaged with physiological saline (control group), 6 mg/kg/d of oxicaine (low dose group), and 48 mg/kg/d of oxicaine (high dose group, high and low dose groups, where the oxicaine was dissolved in physiological saline to make the desired drug concentration). Weigh twice weekly and measure tumor volume. When the tumor volume of the control mice grows to about 1000 cubic millimeters, the experiment is terminated, tumor tissues are taken out, and the tumor weight is weighed and photographed.

The results of the experiment are shown in FIG. 4. As can be seen from FIG. 4, after the treatment with the benzocaine, the volume and the weight of the tumor tissues of the mice are both obviously reduced, and the weight of the mice is not obviously changed, which indicates that the benzocaine has no toxic or side effect. The method can find that the tumor growth treatment effect of the oxicaine on the esophageal cancer PDX mouse model is obvious, the tumor volume is obviously inhibited in both the high-dose group and the low-dose group, the effect of the high-dose group is superior to that of the low-dose group, and the weight of the mouse is not obviously affected. The oxybivacaine can obviously inhibit the growth of the tumor after being treated at the concentration of 6-48 mg/kg.

The above embodiments are only for illustrating the preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention within the knowledge of those skilled in the art should be considered as the protection scope of the present application.

Claims (9)

1. The application of the oxicaine in preparing the anti-tumor medicament is characterized in that the anti-tumor medicament is a medicament for treating esophageal cancer or gastric cancer.

2. The use according to claim 1, wherein the use of oxicaine in the manufacture of a medicament for inhibiting proliferation of cells of esophageal squamous carcinoma.

3. The use according to claim 2, wherein the amount and size of the oxybivacaine in the concentration of 1.0 μ M to 10 μ M inhibits the proliferation and colony formation of esophageal squamous cell carcinoma cells.

4. The use as claimed in claim 3 wherein the esophageal squamous cancer cells are KYSE150 cells and/or KYSE450 cells.

5. The use according to claim 1, wherein the use of oxicaine in the manufacture of a medicament for inhibiting gastric cancer cell proliferation.

6. The use according to claim 5, wherein the oxybivacaine is capable of inhibiting proliferation of gastric cancer cells at a concentration of 1.0 μ M to 10 μ M.

7. The use according to claim 6, wherein the gastric cancer cells are HGC27 cells and/or AGS cells.

8. The use according to claim 1, wherein the use of oxicaine in the manufacture of a medicament for inhibiting the growth of tumours in a humanized transplanted tumour model of oesophageal cancer.

9. The use of claim 8, wherein the oxybivacaine inhibits growth of the tumor model of the humanized graft tumor of esophageal cancer at a concentration of 6mg/kg to 48 mg/kg.

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