Pyrvinium and its derivatives for the treatment of precancerous diseases

Abstract

Pyrvinium and its derivatives for reprogramming precancerous mucosa into a noncancerous state are described herein. Furthermore, pyrvinium and its derivatives for reducing the risk of cancer development in organisms containing metaplastic or dysplastic cells are described herein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 494,671, filed on April 6, 2023, which is incorporated herein by reference in its entirety.

[0002] Research sponsored by the federal government This invention was made with government support under grant numbers IBX000930 granted by the Department of Veterans Affairs, CA190172 granted by the Department of Defense, and R01 DK101332, R01 CA272687, and R37 CA244970 granted by the National Institutes of Health. The government has certain rights to this invention. [Background technology]

[0003] Gastric cancer (GC) is a significant global health concern, ranking as the fifth most common cancer and the third most common cause of cancer-related death. The risk of non-cardiac GC is primarily associated with Helicobacter pylori (H. pylori) infection, which causes a gradual progression from atrophic gastritis and intestinal deformity (IM) to dysplasia. Population-based screening programs have led to earlier detection and reduced mortality, and H. pylori eradication has been shown to reduce the risk of GC in countries with high GC incidence and to reduce the risk of metachronous GC in patients with early GC. However, long-term observational studies have shown that the incidence of metachronous cancer remains high even after H. pylori eradication. Furthermore, evidence suggests that patients with IM or dysplasia may not benefit from H. pylori treatment in terms of GC risk. The concept of a “point of no return” suggests that GC can develop after H. pylori eradication if histological changes have already progressed. Patients with gastric cystitis often present with varying degrees of precancerous changes in the gastric mucosa, such as mesenchymal dysplasia and multifocal dysplasia. However, there are no available treatment options for asymptomatic patients with advanced precancerous lesions. Therefore, there is an urgent need to identify chemotherapeutic agents that can effectively target these lesions.

[0004] There is a need for methods to use pyrvinium to reprogram certain metaplastic and dysplastic cells to reverse their metaplastic / dysplastic phenotype, as well as methods to induce dysplastic cell death by either (a) blocking MAPK signaling and / or (b) blocking STAT3 signaling, or both. [Overview of the Initiative]

[0005] One embodiment described herein is a method for reprogramming a precancerous mucosa into a noncancerous state, the method comprising identifying a precancerous mucosa containing metaplastic or dysplastic cells, and contacting the metaplastic or dysplastic cells with a compound of formula (I) or a salt thereof, [Chemical formula] In the formula, R 1 is C 1ー6 alkyl, R 2a and R 2b are each independently C 1ー6 alkyl, R 3 is C 6ー12 aryl, and R 3 is optionally substituted with 1 to 5 R X groups, R X is, in each occurrence, independently C 1ー6 alkyl, C 1ー4 haloalkyl, halogen, cyano, -N(R Xa )2, -OR Xb , -SR Xb , -C(O)R Xb , -CO2R Xb , -C(O)N(R Xa )2, -SO2R Xa , -L 2 -Y 2 , -O-L X -Y X , -S-L X -Y X , or -N(R Xa )-L X -Y X group, L X is, in each occurrence, independently C 1ー6 alkylene, C 2ー6 alkenylene, or C 2ー6 alkynylene, Y X is, in each occurrence, independently hydrogen, cyano, halogen, haloalkyl, -OH, -N(R Xa )2, -OR Xb , -SR Xb , -C(O)R Xb , -CO2R Xb , -C(O)N(R Xa )2, or -SO2R Xa group, R Xa In each appearance, independently, hydrogen and C 1ー4 Alkyl, or -C(O)C 1ー4 It is alkyl, R Xb In each appearance, independently, hydrogen and C 1ー4 Alkyl, C 1ー2 Haloalkyl, or -C(O)C 1ー4 It is alkyl.

[0006] In one embodiment, metaplastic or dysplastic cells are brought into contact with a salt of the compound of formula (I).

[0007] In another embodiment, a salt of the compound of formula (I) is of formula (IA), [ka] It is salt.

[0008] In another embodiment, R 1 It is methyl.

[0009] In another embodiment, R 2a and R 2b These are methyl compounds.

[0010] In another embodiment, R 3 It is an unsubstituted phenyl compound.

[0011] In another embodiment, the compound of formula (I) is pyrvinium.

[0012] In another embodiment, metaplastic cells or dysplastic cells are located within a living organism.

[0013] In another embodiment, the precancerous mucosa contains dysplastic cells.

[0014] In another embodiment, the method includes killing dysplastic cells.

[0015] In another embodiment, dysplastic cells are positive for CD133 expression, CD166 expression, and / or Trop2 expression.

[0016] In another embodiment, the dysplastic cells are dysplastic gastric cells, dysplastic esophageal cells, dysplastic pancreatic cells, dysplastic colonic cells, or dysplastic ovarian cells.

[0017] In another aspect, dysplastic gastric cells are part of organoids.

[0018] In another embodiment, the precancerous mucosa contains metaplastic cells.

[0019] In another embodiment, metaplastic gastric cells are positive for CD133, CD166, and / or Trop2 expression.

[0020] In another embodiment, metaplastic cells are metaplastic gastric cells, metaplastic esophageal cells, metaplastic pancreatic cells, metaplastic colonic cells, or metaplastic ovarian cells.

[0021] In another aspect, metaplastic gastric cells are part of organoids.

[0022] Another embodiment described herein is a method for reducing the risk of cancer development in a living organism containing metaplastic or dysplastic cells, the method being This includes administering a compound of formula (I) or a salt thereof to a living organism. [ka] During the ceremony, R 1 C 1ー6 It is alkyl, R 2a and R 2b Each of them is independent of C 1ー6 It is alkyl, R 3 C 6ー12 It is aryl, R 3 This is an optional selection of 1 to 5 R's. X Replaced by, R X In each occurrence, independently, C 1ー6 Alkyl, C 1ー4Haloalkyl, halogen, cyano, -N(R Xa )2, -OR Xb , -SR Xb , -C(O)R Xb , -CO2R Xb , -C(O)N(R Xa )2, -SO2R Xa , -L 2 -Y 2 , -O-L X -Y X , -S-L X -Y X , or -N(R Xa )-L X -Y X wherein, L X is, in each occurrence, independently, C 1ー6 alkylene, C 2ー6 alkenylene, or C 2ー6 alkynylene, Y X is, in each occurrence, independently, hydrogen, cyano, halogen, haloalkyl, -OH, -N(R Xa )2, -OR Xb , -SR Xb , -C(O)R Xb , -CO2R Xb , -C(O)N(R Xa )2, or -SO2R Xa wherein, R Xa is, in each occurrence, independently, hydrogen, C 1ー4 alkyl, or -C(O)C 1ー4 alkyl, R Xb is, in each occurrence, independently, hydrogen, C 1ー4 alkyl, C 1ー2 haloalkyl, or -C(O)C 1ー4 alkyl.

[0023] In another aspect, a salt of a compound of formula (I) is administered to a living body.

[0024] In another aspect, a salt of a compound of formula (I) is of formula (I-A), [Chemical formula] It is salt.

[0025] In another embodiment, the compound of formula (I) is pyrvinium.

[0026] In another embodiment, the method inhibits the STAT3 and MEK / ERK signaling pathways in metaplastic or dysplastic cells.

[0027] In another aspect, the living organism is a non-human animal.

[0028] In another embodiment, the organism is a human being.

[0029] In another aspect, humans are at risk of developing cancer.

[0030] In another embodiment, the method further includes administering a second drug to a living organism.

[0031] In another embodiment, the second agent includes a kinase inhibitor.

[0032] In another embodiment, the kinase inhibitor includes a MEK inhibitor.

[0033] In another embodiment, the MEK inhibitor includes trametinib, binimetinib, cobimetinib, and / or selumetinib.

[0034] Another embodiment described herein is the use of pyrvinium or a salt thereof in the manufacture of a pharmaceutical product for reprogramming precancerous mucosa to a noncancerous state or for reducing the risk of developing cancer. [Brief explanation of the drawing]

[0035] [Figure 1A]This figure shows that pyrvinium induces cell death in mouse dysplastic organoids. Phase contrast, hematoxylin & eosin (H&E) imaging, and co-immunostaining of CD44v9, Aqp5, and Trop2 in Meta3 and Meta4 organoids are shown. [Figure 1B] This figure shows that pyrvinium induces cell death in mouse dysplastic organoids. Figures 1B–1C show H&E and phase-contrast images of Meta3 (Figure 1B) and three different Meta4 organoids (Figure 1C) treated for 3 days with DMSO vehicle, 1 μM trametinib (Tra), 100 nM pyrvinium (Pyr), and combinations thereof. [Figure 1C] This figure shows that pyrvinium induces cell death in mouse dysplastic organoids. Figures 1B–1C show H&E and phase-contrast images of Meta3 (Figure 1B) and three different Meta4 organoids (Figure 1C) treated for 3 days with DMSO vehicle, 1 μM trametinib (Tra), 100 nM pyrvinium (Pyr), and combinations thereof. [Figure 1D] This figure shows that pyrvinium induces cell death in mouse dysplastic organoids. Figures 1D-1E show the quantitative analysis of organoid diameter before and after treatment. [Figure 1E] This figure shows that pyrvinium induces cell death in mouse dysplastic organoids. Figures 1D-1E show the quantitative analysis of organoid diameter before and after treatment. [Figure 1F] This figure shows that pyrvinium induces cell death in mouse dysplastic organoids. Phase-contrast images and live / dead (calcein AM / EthD-1) cell staining are shown with the percentage of live and dead cells in treated Meta4 organoids. [Figure 1G]This figure shows that pyrvinium induces cell death in mouse dysplastic organoids. Figures 1G-1H show phase contrast images (Figure 1G) and diameter quantification (Figure 1H) of Meta4 organoids before and after treatment with specific casein kinase 1α activators, 0, 0.2, 0.5, and 1 μM of SSTC3. Mean ± standard deviation. One-way ANOVA with Tukey's multiple comparisons. **P<0.01, ****P<0.0001. Scale bar = 500 μM. [Figure 1H] This figure shows that pyrvinium induces cell death in mouse dysplastic organoids. Figures 1G-1H show phase contrast images (Figure 1G) and diameter quantification (Figure 1H) of Meta4 organoids before and after treatment with specific casein kinase 1α activators, 0, 0.2, 0.5, and 1 μM of SSTC3. Mean ± standard deviation. One-way ANOVA with Tukey's multiple comparisons. **P<0.01, ****P<0.0001. Scale bar = 500 μM. [Figure 2A] This figure shows that a phosphoantibody array identifies pyrvinium-mediated downregulation of MAPK and STAT signaling pathways. The heatmap shows pathway enrichment analysis of differentially phosphorylated proteins after 3 days of pyrvinium (Pyr) treatment in Meta3 and Meta4 organoids, using the Molecular Signatures Database (MSigDB) Hallmark gene set collection. The shading scale represents standardized log2 (FDR) values. [Figure 2B] This figure shows that a phosphoantibody array identifies pyrvinium-mediated downregulation of MAPK and STAT signaling pathways. The log2-scale bar graphs of the signaling pathway changes in treated Meta3 and Meta4 organoids are shown. [Figure 2C] This figure shows that a phosphoantibody array identifies the downregulation of MAPK and STAT signaling pathways by pyrvinium. The heatmap shows upregulated or downregulated phosphoproteins in Meta4 organoids compared to treated Meta3. [Figure 2D] This figure shows how a phosphoantibody array identifies pyrvinium-mediated downregulation of MAPK and STAT signaling pathways. The graphs show changes in phosphorylation of STAT3Y705 and ERKT202 / T204 between Meta3 and Meta4 organoids (upper panel), or between DMSO and Pyr treatment of Meta4 organoids (lower panel). Each square represents the signal ratio of the antibody to the non-phosphorylated form of the site. [Figure 2E] This paper outlines how STAT3 and DPP in the RAS / MAPK pathway are affected by Pyr treatment in precancerous organoids. [Figure 3A] This figure shows that pyrvinium suppresses mouse dysplastic organoids via a dual blockade of the ERK and STAT3 signaling pathways. Figures 3A and 3B show Western blot analysis of phospho-STAT3 (p-STAT3) and phospho-ERK (p-ERK) in three Meta4 organoids after 3 days of treatment with trametinib (Tra), pyrvinium (Pyr), and combinations thereof (Figure 3A), as well as the ratios of p-STAT3 / total STAT3 and p-ERK / total ERK (Figure 3B). [Figure 3B] This figure shows that pyrvinium suppresses mouse dysplastic organoids via a dual blockade of the ERK and STAT3 signaling pathways. Figures 3A and 3B show Western blot analysis of phospho-STAT3 (p-STAT3) and phospho-ERK (p-ERK) in three Meta4 organoids after 3 days of treatment with trametinib (Tra), pyrvinium (Pyr), and combinations thereof (Figure 3A), as well as the ratios of p-STAT3 / total STAT3 and p-ERK / total ERK (Figure 3B). [Figure 3C]This figure shows that pyrvinium suppresses mouse dysplastic organoids through a dual blockade of the ERK and STAT3 signaling pathways. Figures 3C-3D show Western blot analysis of p-STAT3 and p-ERK in Meta3 and Meta4 organoids (Figure 3C), as well as the ratios of p-STAT3 / total STAT3 and p-ERK / total ERK (Figure 3D). [Figure 3D] This figure shows that pyrvinium suppresses mouse dysplastic organoids through a dual blockade of the ERK and STAT3 signaling pathways. Figures 3C-3D show Western blot analysis of p-STAT3 and p-ERK in Meta3 and Meta4 organoids (Figure 3C), as well as the ratios of p-STAT3 / total STAT3 and p-ERK / total ERK (Figure 3D). [Figure 3E] This figure shows that pyrvinium suppresses mouse dysplastic organoids via a double blockade of the ERK and STAT3 signaling pathways. Immunostaining of p-STAT3 in Meta3 and Meta4 organoids is shown. [Figure 3F] This figure shows that pyrvinium suppresses mouse dysplastic organoids via a dual blockade of the ERK and STAT3 signaling pathways. Figures 3F-3G show phase-contrast images (Figure 3F) of Meta4 organoids treated for 3 days with DMSO, Pyr (100 nM), STAT3-IN-1 (1 μM), Static (2 μM), and cryptotancinone (CPT) (5 μM), as well as quantification of organoid diameter before and after treatment (Figure 3G). [Figure 3G] This figure shows that pyrvinium suppresses mouse dysplastic organoids via a dual blockade of the ERK and STAT3 signaling pathways. Figures 3F-3G show phase-contrast images (Figure 3F) of Meta4 organoids treated for 3 days with DMSO, Pyr (100 nM), STAT3-IN-1 (1 μM), Static (2 μM), and cryptotancinone (CPT) (5 μM), as well as quantification of organoid diameter before and after treatment (Figure 3G). [Figure 3H]This figure shows that pyrvinium suppresses mouse dysplastic organoids via a dual blockade of the ERK and STAT3 signaling pathways. Figures 3H-3I show phase-contrast images (Figure 3H) of Meta4 organoids treated with DMSO, trametinib (Tra), STAT3-IN-1, a combination of Tra and STAT3-IN-1, and Pyr for 3 days, as well as quantification of organoid diameter before and after treatment (Figure 3I). [Figure 3I] This figure shows that pyrvinium suppresses mouse dysplastic organoids via a dual blockade of the ERK and STAT3 signaling pathways. Figures 3H-3I show phase-contrast images (Figure 3H) of Meta4 organoids treated with DMSO, trametinib (Tra), STAT3-IN-1, a combination of Tra and STAT3-IN-1, and Pyr for 3 days, as well as quantification of organoid diameter before and after treatment (Figure 3I). [Figure 3J] This figure shows that pyrvinium suppresses mouse dysplastic organoids via a dual blockade of the ERK and STAT3 signaling pathways. Figures 3J–3K show Western blots of p-STAT3 and p-ERK after treatment with Tra, Pyr, and SSTC3 (Figure 3J), as well as the ratios of p-STAT3 / t-STAT3 and p-ERK / t-ERK (Figure 3K). [Figure 3K] This figure shows that pyrvinium suppresses mouse dysplastic organoids via a dual blockade of the ERK and STAT3 signaling pathways. Figures 3J–3K show Western blots of p-STAT3 and p-ERK after treatment with Tra, Pyr, and SSTC3 (Figure 3J), as well as the ratios of p-STAT3 / t-STAT3 and p-ERK / t-ERK (Figure 3K). [Figure 3L]This figure shows that pyrvinium suppresses mouse dysplastic organoids via a double blockade of the ERK and STAT3 signaling pathways. A heatmap of quantitative real-time PCR analysis of STAT3 target genes in Meta4 organoids treated with DMSO or Pyr for 1 day is shown. Relative quantification of each gene expression level was normalized relative to GAPDH gene expression. The shading scale represents mRNA expression values. [Figure 3M] This figure shows that pyrvinium suppresses mouse dysplastic organoids via a double blockade of the ERK and STAT3 signaling pathways. Representative mRNAs of Pim-1 and Cyclin-B1 after treatment are shown. Mean ± standard deviation. Unpaired t-test or one-way ANOVA with Tukey's multiple comparisons. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. ns, not significant. Scale bar = 500 μM. [Figure 4A] This figure shows that pyrvinium targets stem cell populations in mouse dysplastic organoids. Figures 4A–4B show superimposed uniform manifold approximation and projection (UMAP) plots showing annotated clusters from DMSO-treated and pyrvinium (Pyr)-treated Meta4 cells (Figure 4A) and subpopulation-matched cluster overlays (Figure 4B). [Figure 4B] This figure shows that pyrvinium targets stem cell populations in mouse dysplastic organoids. Figures 4A–4B show superimposed uniform manifold approximation and projection (UMAP) plots showing annotated clusters from DMSO-treated and pyrvinium (Pyr)-treated Meta4 cells (Figure 4A) and subpopulation-matched cluster overlays (Figure 4B). [Figure 4C] This figure shows that pyrvinium targets stem cell populations in mouse dysplastic organoids. It also shows the PANTHER gene ontology classification results based on the top 300 differentially expressed genes in DMSO-treated and Pyr-treated Meta4 cells. [Figure 4D]This figure shows that pyrvinium targets the stem cell population in mouse dysplastic organoids. Figures 4D-4E show rasters of dysplastic stem cell (DSC) differentiated or damaged cells in an overlaid UMAP plot (Figure 4D), and their proportions in DMSO-treated and Pyr-treated Meta4 samples (Figure 4E). [Figure 4E] This figure shows that pyrvinium targets the stem cell population in mouse dysplastic organoids. Figures 4D-4E show rasters of dysplastic stem cell (DSC) differentiated or damaged cells in an overlaid UMAP plot (Figure 4D), and their proportions in DMSO-treated and Pyr-treated Meta4 samples (Figure 4E). [Figure 4F] This figure shows that pyrvinium targets stem cell populations in mouse dysplastic organoids. It also shows quantitative real-time PCR analysis illustrating the relative expression of DSC markers CD133 and CD166 in DMSO-treated Meta4 organoids versus Pyr-treated organoids. [Figure 4G] This figure shows that pyrvinium targets stem cell populations in mouse dysplastic organoids. It also shows fluorescence-activated cell sorting analysis of CD133 and CD166-positive cells in DMSO-treated and Pyr-treated Meta4 organoids. [Figure 4H] This figure shows that pyrvinium targets stem cell populations in mouse dysplastic organoids. Analysis of 7-AAD and annexin V expression in DMSO-treated and Pyr-treated Meta4 organoids is shown. [Figure 4I] This figure shows that pyrvinium targets stem cell populations in mouse dysplastic organoids. It also shows volcano plots of downregulated or upregulated transcripts in Pyr-treated Meta4 cells versus DMSO-treated Meta4 cells. [Figure 4J]This figure shows that pyrvinium targets stem cell populations in mouse dysplastic organoids. Figures 4J-4K show STAT3 target upregulatory (Fosb, Fos, and Jun) genes (Figure 4J) and downregulatory (Dusp4, Ddit3, and Areg) genes (Figure 4K) in superimposed UMAP plots. [Figure 4K] This figure shows that pyrvinium targets stem cell populations in mouse dysplastic organoids. Figures 4J-4K show STAT3 target upregulatory (Fosb, Fos, and Jun) genes (Figure 4J) and downregulatory (Dusp4, Ddit3, and Areg) genes (Figure 4K) in superimposed UMAP plots. [Figure 5A] This figure shows that pyrvinium treatment inhibits the progression to metaplasia in vivo. A schematic diagram of the experimental scheme for pyrvinium (Pyr) treatment in Mist1-Kras mice 3 months after tamoxifen injection is shown. [Figure 5B] This figure shows that pyrvinium treatment inhibits metaplastic progression to dysplasia in vivo. Schematic H&E (top) and immunostaining of UEA1 (center) and Ki-67 (bottom) in wild-type mice treated with Pyr for 2 weeks and Mist1-Kras mice treated with DMSO or Pyr for 1 or 2 weeks are shown. [Figure 5C] This figure shows that pyruvium treatment inhibits metaplastic progression to dysplasia in vivo. A schematic representation of Ki-67-positive cells per 20× field is shown. [Figure 5D] This figure shows that pyrvinium treatment inhibits metaplasia progression to dysplasia in vivo. A panoramic view of H / K-ATPase immunostaining in the gastric body of wild-type mice treated with Pyr for 2 weeks and Mist1-Kras mice treated with DMSO, Pyr for 1 or 2 weeks is shown. [Figure 5E] This figure shows that pyruvium treatment inhibits metaplasia progression to dysplasia in vivo. The figure quantifies the parietal cell-containing gastric units (GU) per 100 GU. [Figure 5F]This figure shows that pyrvinium treatment inhibits metaplasia progression to dysplasia in vivo. Representative H&E images of the gastric body from Mist1-Kras mice treated with DMSO or Pyr for 2 weeks are shown. [Figure 5G] This figure shows that pyruvium treatment inhibits metaplastic progression to dysplasia in vivo. The percentage of dysplastic glands in the remaining hyperplastic glands is shown. [Figure 5H] This figure shows that pyrvinium treatment inhibits metaplasia progression to dysplasia in vivo. Immunostaining of phospho-STAT3 (p-STAT3) and Pim-1 in the gastric body of Mist1-Kras mice treated with DMSO or Pyr for 2 weeks is shown. [Figure 5I] This figure shows that pyrvinium treatment inhibits metaplasia progression to dysplasia in vivo. Figures 5I-5J show the results of co-immunostaining of CD4 and CD8 (Figure 5I) and CD19 (Figure 5J) in the gastric body of Mist1-Kras mice treated with DMSO or Pyr for 2 weeks. [Figure 5J] This figure shows that pyrvinium treatment inhibits metaplasia progression to dysplasia in vivo. Figures 5I-5J show the results of co-immunostaining of CD4 and CD8 (Figure 5I) and CD19 (Figure 5J) in the gastric body of Mist1-Kras mice treated with DMSO or Pyr for 2 weeks. [Figure 5K] This figure shows that pyrvinium treatment inhibits metaplasia progression to dysplasia in vivo. Figures 5K-5L show co-immunostaining of CD3 and NK1.1 (Figure 5K) and immunostaining of CD68 or CD163 (Figure 5L) in the gastric body of Mist1-Kras mice treated with DMSO or Pyr for 2 weeks. [Figure 5L] This figure shows that pyrvinium treatment inhibits metaplasia progression to dysplasia in vivo. Figures 5K-5L show co-immunostaining of CD3 and NK1.1 (Figure 5K) and immunostaining of CD68 or CD163 (Figure 5L) in the gastric body of Mist1-Kras mice treated with DMSO or Pyr for 2 weeks. [Figure 5M]This figure shows that pyrvinium treatment inhibits metaplastic progression to dysplasia in vivo. It quantifies various immune cells per 20 × field in Mist1-Kras mice treated with DMSO or Pyr. Mean ± standard deviation. Unpaired t-test or one-way ANOVA with Tukey's multiple comparisons. ns, not significant. *P<0.05. **P<0.01. ***P<0.001. Scale bar = 100 μM. [Figure 6A] This figure shows that pyrvinium targets human precancerous organoids (hPCOs) with dysplastic characteristics. Figures 6A and 6B show representative phase-contrast images (Figure 6A) of three pyrvinium (Pyr)-sensitive hPCO series treated with DMSO, 1 μM trametinib (Tra), 100 nM pyrvinium, and combinations thereof over 6 days, as well as quantification of organoid diameter before and after treatment (Figure 6B). [Figure 6B] This figure shows that pyrvinium targets human precancerous organoids (hPCOs) with dysplastic characteristics. Figures 6A and 6B show representative phase-contrast images (Figure 6A) of three pyrvinium (Pyr)-sensitive hPCO series treated with DMSO, 1 μM trametinib (Tra), 100 nM pyrvinium, and combinations thereof over 6 days, as well as quantification of organoid diameter before and after treatment (Figure 6B). [Figure 6C] This figure shows that pyrvinium targets human precancerous organoids (hPCOs) with dysplastic characteristics. Figures 6C-6D show 6-day phase-contrast images (Figure 6C) of three Pyr-sensitive hPCO series treated with DMSO, 1 μM trametinib (Tra), 100 nM pyrvinium, and combinations thereof, as well as quantification of organoid diameter before and after treatment (Figure 6D). [Figure 6D]This figure shows that pyrvinium targets human precancerous organoids (hPCOs) with dysplastic characteristics. Figures 6C-6D show 6-day phase-contrast images (Figure 6C) of three Pyr-sensitive hPCO series treated with DMSO, 1 μM trametinib (Tra), 100 nM pyrvinium, and combinations thereof, as well as quantification of organoid diameter before and after treatment (Figure 6D). [Figure 6E] This figure shows that pyrvinium targets human precancerous organoids (hPCOs) with dysplastic characteristics. It also includes heatmaps showing the Pyr response, morphology, and expression of TROP2, AQP5, and CD44v9 in 20 hPCO series. [Figure 6F] This figure shows that pyrvinium targets human precancerous organoids (hPCOs) with dysplastic characteristics. A correlation graph of the Pyr response with morphology and TROP2 expression in the hPCO series is shown. [Figure 6G] This figure shows that pyrvinium targets human precancerous organoids (hPCOs) with dysplastic characteristics. A bar graph is shown illustrating the morphology or TROP2 expression score between Pyr-sensitive and Pyr-resistant hPCO lineages. [Figure 6H] This figure shows that pyrvinium targets human precancerous organoids (hPCOs) with dysplastic characteristics. Representative H&E images and co-immunostaining of CD44v9, AQP5, and TROP2 in three Pyr-sensitive and Pyr-resistant hPCO series are shown. Mean ± standard deviation. Unpaired t-test or one-way ANOVA with Tukey's multiple comparisons. **P<0.01, ***P<0.001, ****P<0.0001. Scale bar = 500 μM. [Figure 7A]This figure shows that pyrvinium induces cell death in dysplastic human precancerous (hPCO) organoids by blocking the ERK and STAT3 signaling pathways. Figures 7A and 7B show Western blot analysis of phospho-STAT3 (p-STAT3) and phospho-ERK (p-ERK) in three pyrvinium (Pyr)-sensitive hPCO series after Pyr treatment (Figure 7A), as well as the ratios of p-STAT3 / total STAT3 (t-STAT3) and p-ERK / total ERK (t-ERK) (Figure 7B). [Figure 7B] This figure shows that pyrvinium induces cell death in dysplastic human precancerous (hPCO) organoids by blocking the ERK and STAT3 signaling pathways. Figures 7A and 7B show Western blot analysis of phospho-STAT3 (p-STAT3) and phospho-ERK (p-ERK) in three pyrvinium (Pyr)-sensitive hPCO series after Pyr treatment (Figure 7A), as well as the ratios of p-STAT3 / total STAT3 (t-STAT3) and p-ERK / total ERK (t-ERK) (Figure 7B). [Figure 7C] This figure shows that pyrvinium induces cell death in dysplastic human precancerous (hPCO) organoids by blocking the ERK and STAT3 signaling pathways. Figures 7C–7D show Western blot analysis of p-STAT3 and p-ERK in three Pyr-resistant hPCO lineages after Pyr treatment (Figure 7C), as well as the ratios of p-STAT3 / t-STAT3 and p-ERK / t-ERK (Figure 7D). [Figure 7D] This figure shows that pyrvinium induces cell death in dysplastic human precancerous (hPCO) organoids by blocking the ERK and STAT3 signaling pathways. Figures 7C–7D show Western blot analysis of p-STAT3 and p-ERK in three Pyr-resistant hPCO lineages after Pyr treatment (Figure 7C), as well as the ratios of p-STAT3 / t-STAT3 and p-ERK / t-ERK (Figure 7D). [Figure 7E]This figure shows that pyrvinium induces cell death in dysplastic human precancerous (hPCO) organoids by blocking the ERK and STAT3 signaling pathways. Figures 7E–7F show Western blots of basal p-STAT3 and p-ERK levels in Pyr-sensitive and Pyr-resistant hPCO lineages (Figure 7E), as well as the ratios of p-STAT3 / t-STAT3 and p-ERK / t-ERK (Figure 7F). [Figure 7F] This figure shows that pyrvinium induces cell death in dysplastic human precancerous (hPCO) organoids by blocking the ERK and STAT3 signaling pathways. Figures 7E–7F show Western blots of basal p-STAT3 and p-ERK levels in Pyr-sensitive and Pyr-resistant hPCO lineages (Figure 7E), as well as the ratios of p-STAT3 / t-STAT3 and p-ERK / t-ERK (Figure 7F). [Figure 7G] This figure shows that pyrvinium induces cell death in dysplastic human precancerous (hPCO) organoids by blocking the ERK and STAT3 signaling pathways. Figures 7G–7H show Western blots of p-STAT3 and p-ERK in hPCO-3 after treatment with trametinib (Tra), Pyr, and combinations thereof (Figure 7G), as well as the ratios of p-STAT3 / t-STAT3 and p-ERK / t-ERK (Figure 7H). [Figure 7H] This figure shows that pyrvinium induces cell death in dysplastic human precancerous (hPCO) organoids by blocking the ERK and STAT3 signaling pathways. Figures 7G–7H show Western blots of p-STAT3 and p-ERK in hPCO-3 after treatment with trametinib (Tra), Pyr, and combinations thereof (Figure 7G), as well as the ratios of p-STAT3 / t-STAT3 and p-ERK / t-ERK (Figure 7H). [Figure 7I]This figure shows that pyrvinium induces cell death in dysplastic human precancerous (hPCO) organoids by blocking the ERK and STAT3 signaling pathways. Representative immunohistochemical staining images of p-STAT3 in Pyr-sensitive or Pyr-resistant hPCO lineages are shown. [Figure 7J] This figure shows that pyrvinium induces cell death in dysplastic human precancerous (hPCO) organoids by blocking the ERK and STAT3 signaling pathways. H&E and p-STAT3 immunostaining images of gastric tissue from Pyr-sensitive or Pyr-resistant hPCO lineages are shown. [Figure 7K] This figure shows that pyrvinium induces cell death in dysplastic human precancerous (hPCO) organoids by blocking the ERK and STAT3 signaling pathways. Figures 7K–7L show phase-contrast images (Figure 7K) of Pyr-sensitive hPCO-2 treated with DMSO, Pyr, STAT3-IN-1, Static, and cryptotancinone (CPT) for 6 days, as well as quantification of organoid diameter before and after treatment (Figure 7L). Mean ± standard deviation. Unpaired t-test. *P<0.05, ****P<0.0001. [Figure 7L] This figure shows that pyrvinium induces cell death in dysplastic human precancerous (hPCO) organoids by blocking the ERK and STAT3 signaling pathways. Figures 7K–7L show phase-contrast images (Figure 7K) of Pyr-sensitive hPCO-2 treated with DMSO, Pyr, STAT3-IN-1, Static, and cryptotancinone (CPT) for 6 days, as well as quantification of organoid diameter before and after treatment (Figure 7L). Mean ± standard deviation. Unpaired t-test. *P<0.05, ****P<0.0001. [Figure 8]This is an annotated photograph showing a linear gastric mucosal band (indicated by the dotted line above) obtained from a surgical specimen of a gastric cancer patient. This section was adjacent to the gastric cancerous area (indicated by the semicircular dotted line between the clips). To prevent contamination from cancer cells, the area near the cancerous lesion was marked with wire (indicated by the short vertical arrow). The mucosa enclosed within the rectangular dotted box was used to generate gastric organoids. [Figure 9] This figure shows the expression of Wnt / β-catein-related genes in dysplastic organoids after treatment with MEK inhibitors, pyrvinium, and CK1α inhibitors. Real-time PCR analysis of Wnt target genes, including Axin2, Cdca4, Ephb3, Lrg5, Rnf43, Sp5, and Znrf3, in Meta4 organoids after 6 or 24 hours of treatment with DMSO vehicle, trametinib (Tra, 1 μM), pyrvinium (Pyr, 100 nM), and SSTC3 (1 μM), respectively. Mean ± SD. One-way ANOVA using Tukey's multiple comparisons. [Figure 10A] This figure shows the effects of STAT3 inhibitors on Meta3 organoids. Phase-contrast images are shown of Meta3 organoids treated for 3 days with a DMSO vehicle, 100 nM pyrvinium (Pyr), or one of three STAT3 inhibitors, including STAT3-IN-1 (1 μM), Sttatic (2 μM), and cryptotancion (CPT, 5 μM). [Figure 10B] This figure shows the effect of STAT3 inhibitors on Meta3 organoids. It quantifies the organoid diameter before and after treatment. Mean ± SO₂. Bidirectional ANOVA using Tukey's test for pairwise comparison. Scale bar = 500 μm. [Figure 11] This bar graph shows the expression of STAT3 target genes in dysplastic organoids after treatment with pyrvinium (Pyr). Real-time PCR analysis of 15 STAT3 target genes in Meta4 organoids after 48 hours of pyrvinium (100 nM) treatment. Three copies were made. Two-sided Mann-Whitney test. [Figure 12]These images show normal tissue in multiple organs, including the stomach, esophagus, liver, kidneys, heart, lungs, and spleen, that were unaffected by in vivo pyrvinium (Pyr) treatment. Hematoxylin and eosin images were obtained from wild-type mice treated with either dimethyl sulfoxide (DMSO) or Pyr (4 mg / L in drinking water) for two weeks. Scale bar = 100 μm. [Figure 13A] This figure shows the changes in regulatory T cells in Mist1-Kras mice after pyrvinium treatment. It also shows immunohistochemical staining of CD3 and CD25 in the gastric body of Mist1-Kras mice treated with DMSO or Pyr for two weeks. [Figure 13B] This figure shows the changes in regulatory T cells in Mist1-Kras mice after pyrvinium treatment. It shows the quantification of various immune cells per 20× field and the ratio of regulatory T cells (Treg) to total T cells in wild-type and Mist1-Kras mice treated with DMSO or Pyr. Mean ± SD. Two-sided Mann-Whitney test. Scale bar = 100 μm. [Figure 14] This figure shows the gene expression analysis of Mist1-Kras mice 3 months after tamoxifen injection, administered either DMSO or pyrvinium (Pyr) for 2 weeks. Quantitative real-time PCR analysis was performed for bone marrow-derived suppressor cell markers including Slfn4, Nos2, and Arg1 and ltgam1. Relative quantification of each gene expression level was normalized to Gapdh gene expression. Mean ± standard deviation. Two-sided Mann-Whitney test. [Figure 15A] This figure shows the effects of pyrvinium (Pyr) treatment on 14 different human precancerous organoid (hPCO) series established from human gastric samples from gastric cancer patients. Co-immunostaining for H&E, CD44v9, Aqp5, and Trop2, as well as phase-contrast images of two Pyr-sensitive (Figure 15A), six Pyr-moderate (Figure 15B), and six Pyr-resistant (Figure 15C) hPCO series treated for 6 days with DMSO, 1 μM trametinib (Tra), 100 nM Pyr, and combinations thereof. Scale bar = 100 μm. [Figure 15B] This figure shows the effects of pyrvinium (Pyr) treatment on 14 different human precancerous organoid (hPCO) series established from human gastric samples from gastric cancer patients. Co-immunostaining for H&E, CD44v9, Aqp5, and Trop2, as well as phase-contrast images of two Pyr-sensitive (Figure 15A), six Pyr-moderate (Figure 15B), and six Pyr-resistant (Figure 15C) hPCO series treated for 6 days with DMSO, 1 μM trametinib (Tra), 100 nM Pyr, and combinations thereof. Scale bar = 100 μm. [Figure 15C] This figure shows the effects of pyrvinium (Pyr) treatment on 14 different human precancerous organoid (hPCO) series established from human gastric samples from gastric cancer patients. Co-immunostaining for H&E, CD44v9, Aqp5, and Trop2, as well as phase-contrast images of two Pyr-sensitive (Figure 15A), six Pyr-moderate (Figure 15B), and six Pyr-resistant (Figure 15C) hPCO series treated for 6 days with DMSO, 1 μM trametinib (Tra), 100 nM Pyr, and combinations thereof. Scale bar = 100 μm. [Figure 16] Images showing pyrvinium (Pyr)-induced cell death in the human precancerous organoid (hPCO) series. Live / dead cell analysis using double staining of calcein AM and ethidium homodimer-1 (EthD-1) after treatment with DMSO, trametinib (Tra, 1 μM), pyrvinium (Pyr, 100 nM), and combinations of Tra and Pyr in the Pyr-sensitive hPCO-34 series, as well as the percentage of live and dead cells after treatment. Scale bar = 500 μm. [Figure 17A] This figure shows the effects of casein kinase 1α (CK1α) activator treatment in the human precancerous organoid (hPCO) series. Phase-contrast images of pyrvinium-sensitive hPCO-34 treated for 6 days with a DMSO vehicle or with SSTC3, a specific CK1α activator, at various concentrations (0.1, 0.2, 0.5, and 1.0 μM) are shown. [Figure 17B]This figure shows the effect of casein kinase 1α (CK1α) activator treatment on human precancerous organoids (hPCOs). It quantifies organoid diameter before and after treatment. Mean ± SD. Bidirectional ANOVA (using Tukey's test for pairwise comparisons). Scale bar = 500 μm. [Modes for carrying out the invention]

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. For example, any nomenclature and techniques used in relation to biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry as described herein are well known and commonly used in the art. In case of any conflict, the disclosure including the definitions shall prevail. Similar or equivalent methods and materials may be used in carrying out or testing the embodiments and aspects described herein, but exemplary methods and materials are described below.

[0037] As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their general meanings as understood by a biochemist of the ordinary art in this field. Standard single-letter nucleotides (A, C, G, T, U) and standard single-letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0038] As used herein, terms such as “include,” “including,” “contain,” “containing,” and “having” mean “comprising.” This disclosure also intends to include, “essentially consist of,” and other embodiments “consisting of,” the embodiments or elements presented herein, whether expressly or otherwise. As used herein, “comprising” is a “non-restrictive” term that does not exclude additional unlisted elements or method steps. As used herein, “essentially consisting of” limits the scope of the claims to “a particular material or step of the claimed invention and which does not substantially affect the essential and novel features.” As used herein, “consisting of” excludes any element, step, or component not specified in the claims.

[0039] Where used herein, the terms “a,” “an,” and “the,” and similar terms as used in the context of this disclosure (particularly in the context of the claims), should be construed to cover both singular and plural forms unless otherwise indicated herein or unless clearly inconsistent with the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.

[0040] As used herein, the term "or" can be conjunctive or disjunctive.

[0041] As used herein, the terms "and / or" refer to both connective and disjunctive conjunctions.

[0042] As used herein, the term “substantially” means to a greater or more significant degree than “completely.”

[0043] As used herein, when applied to one or more values ​​of interest, the terms “about” or “approximately” mean a value similar to the stated reference value, or a value within the tolerance range for a particular value determined by those skilled in the art, which in part depends on how that value is measured or determined, for example, the limits of the measuring system. In one embodiment, the term “about” refers to any value, including both integer and fractional components, that are within ±10% variation of the value modified by the term “about.” Alternatively, “about” can mean three or more standard deviations, according to convention in the art. Alternatively, for example with respect to biological systems or processes, the term “about” can mean within one order of magnitude of the value, within five times in some embodiments, and within two times in some embodiments. As used herein, the symbol “~” means “about” or “approximately.”

[0044] All ranges disclosed herein include both endpoints as distinct values, as well as all integers and fractions specified within the range. For example, the range 0.1 to 2.0 includes 0.1, 0.2, 0.3, 0.4...2.0. Where an endpoint is qualified with the term "approximately", the specified range is extended by a variation of up to ±10% of any value within the range containing the endpoint or within three or more standard deviations, or as described in the definition of "approximately".

[0045] As used herein, the terms “active ingredient” or “active pharmaceutical ingredient” refer to a pharmaceutical, active ingredient, compound, or substance, composition, or mixture thereof that provides a pharmacological, often beneficial, effect.

[0046] As used herein, the terms “control” and “reference” are used synonymously. A “reference” or “control” level may be a predetermined value or range used as a baseline or benchmark for evaluating measurement results. “Control” also refers to a control experiment or control cells.

[0047] As used herein, the term “dose” refers to any form of active ingredient preparation or composition containing cells that contains an amount sufficient to initiate or produce a therapeutic effect with at least one dose. “Preparation” and “composition” are used synonymously herein.

[0048] As used herein, the term “prevention” means preventing or reducing the progression of a disability to a statistically significant degree or to a degree detectable by a person skilled in the art.

[0049] As used herein, the terms “effective dose” or “therapeutic dose” mean a substantially non-toxic but sufficient amount of an action, drug, composition, or cell(s) administered to a subject that prevents, treats, or improves, to some extent, one or more of the symptoms of a disease or condition that the subject is experiencing or is susceptible to. The result may be a reduction or mitigation of the signs, symptoms, or causes of the disease, or any other desirable change in the biological system. The effective dose may be based on individual factors for each subject, including but not limited to the subject’s age, size, type or severity of the disease, stage of the disease, route of administration, type or degree of adjunctive therapy used, ongoing disease process, and type of treatment desired.

[0050] As used herein, the term “subject” refers to an animal. Typically, the subject is a mammal. The subject may also refer to primates (e.g., humans, male or female, infant, adolescent, or adult), non-human primates, rats, mice, rabbits, pigs, cattle, sheep, goats, horses, dogs, cats, fish, birds, etc. In one embodiment, the subject is a primate. In one embodiment, the subject is a human.

[0051] As used herein, an object “in need of treatment” is defined as an object that derives a biological, medical, or quality of life benefit from such treatment. An object in need of treatment does not necessarily have symptoms, especially in the case of preventive or prophylactic treatment.

[0052] As used herein, the terms “inhibit,” “inhibit,” or “to inhibit” refer to a reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0053] As used herein, the terms “salt” or “salts” refer to acid-added or base-added salts of the compounds of the present invention. “Salt” includes, in particular, “pharmaceutically acceptable salts.” The term “pharmaceutically acceptable salt” refers to a salt that retains the biological efficacy and properties of the compounds of the present invention and is not typically biologically or otherwise unsuitable.

[0054] As used herein, “treatment” or “to treat” means preventing, stopping, inhibiting, suppressing, reversing, mitigating, improving, or inhibiting the progression of a biological process, including a disorder or disease, or completely eliminating the disease. Treatment may be carried out in an emergency or long-term manner. The term “treatment” also means reducing the severity of a disease or symptoms associated with such a disease before the suffering caused by the disease occurs. To “suppress” or “improve” a disease, disorder, or its symptoms involves administering the cells, compositions, or compounds described herein to the target after the clinical manifestation of such disease, disorder, or its symptoms. To “prevent” or “prevent” a disease, disorder, or its symptoms involves administering the cells, compositions, or compounds described herein to the target before the onset of the disease, disorder, or its symptoms. To “suppress” a disease or disorder involves administering the cells, compositions, or compounds described herein to the target after the induction of the disease or disorder, but before its clinical manifestation or the appearance of its symptoms.

[0055] As used herein, the terms “salt” or “salts” refer to acid-addition or base-addition salts of the compounds of this disclosure. “Salt” includes, in particular, “pharmaceutically acceptable salts.” “pharmaceutically acceptable salts” refers to salts that retain the biological efficacy and properties of the compounds of this disclosure and are typically not biologically or otherwise unsuitable. In many cases, the compounds described herein may form acid and / or base salts due to the presence of amino and / or carboxyl groups, or similar groups.

[0056] As used herein, “treatment” or “to treat” means preventing, stopping, inhibiting, suppressing, reversing, mitigating, improving, or inhibiting the progression of a biological process, including a disorder or disease, or completely eliminating the disease. Treatment may be carried out in an emergency or long-term manner. The term “treatment” also means reducing the severity of a disease or symptoms associated with such a disease before the suffering caused by the disease occurs. To “suppress” or “improve” a disease, disorder, or its symptoms involves administering the cells, compositions, or compounds described herein to the target after the clinical manifestation of such disease, disorder, or its symptoms. To “prevent” or “prevent” a disease, disorder, or its symptoms involves administering the cells, compositions, or compounds described herein to the target before the onset of the disease, disorder, or its symptoms. To “suppress” a disease or disorder involves administering the cells, compositions, or compounds described herein to the target after the induction of the disease or disorder, but before its clinical manifestation or the appearance of its symptoms.

[0057] The definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this disclosure, chemical elements are defined as follows: Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 thThe ed. and inside cover are specified, and specific functional groups are generally defined as described therein. Furthermore, the general principles of organic chemistry, as well as specific functional parts and reactivity, are as follows: Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999, Smith and March March's Advanced Organic Chemistry, 5 th Edition, John Wiley&Sons, Inc., New York, 2001, Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989, Carruthers, Some Modern Methods of Organic Synthesis, 3 rd This information is contained in Edition, Cambridge University Press, Cambridge, 1987, and the entire contents of each section are incorporated herein by reference.

[0058] As used herein, the term "alkoxy" refers to an -O-alkyl group. Typical examples of alkoxys include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

[0059] As used herein, the term "alkyl" means a straight-chain or branched saturated hydrocarbon chain. 1ー6 The term "alkyl" refers to a straight-chain or branched-chain hydrocarbon containing 1 to 6 carbon atoms. 1ー4The term "alkyl" refers to a linear or branched hydrocarbon containing 1 to 4 carbon atoms. Representative examples of alkyls include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0060] When used herein, the term "alkoxyalkyl" refers to an alkoxy group as defined herein that has been added to the parent molecule via an alkyl group as defined herein.

[0061] The term "amide," as used herein, means -C(O)NR- or -NRC(O)-, where R may be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclic, alkenyl, or heteroalkyl.

[0062] As used herein, the term "amino" is -NR x R y This means that in the formula, R x and R y The aminoalkyl group can be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclic, alkenyl, or heteroalkyl. If aminoalkyl or amino is any other part to which two other parts are added together, amino is -NR x - is acceptable, and in the formula, R x This can be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclic, alkenyl, or heteroalkyl.

[0063] The term "aryl," as used herein, refers to phenyl, or phenyl attached to the parent molecule and fused to a cycloalkane group (e.g., aryl may be indan-4-yl), phenyl fused to a six-membered arene group (i.e., aryl is naphthyl), or phenyl fused to a non-aromatic heterocycle (e.g., aryl may be benzo[d][1,3]dioxol-5-yl). The term "phenyl" is used when referring to a substituent, and the term "six-membered arene" is used when referring to a fused ring. A six-membered arene is monocyclic (e.g., benzene or benzo). Aryl can be monocyclic (phenyl) or bicyclic (e.g., a 9- to 12-membered fused bicyclic system).

[0064] As used herein, the terms "halogen" or "halo" mean Cl, Br, I, or F.

[0065] As used herein, the term "haloalkyl" means an alkyl group as defined herein, in which one, two, three, four, five, six, seven, or eight hydrogen atoms are replaced by halogens.

[0066] When used herein, the term "haloalkoxy" means that at least one haloalkyl group, as defined herein, is attached to the parent molecule via an oxygen atom.

[0067] As used herein, the term "halocycloalkyl" means a cycloalkyl group as defined herein, in which one or more hydrogen atoms are replaced by halogens.

[0068] The term "heteroalkyl," as used herein, means an alkyl group as defined herein, in which one or more carbon atoms are replaced by heteroatoms selected from S, O, P, and N. Typical examples of heteroalkyls include, but are not limited to, alkyl ethers, secondary and tertiary alkylamines, amides, and alkyl sulfides.

[0069] The terms "hydroxyl" or "hydroxy" as used herein mean the -OH group.

[0070] In certain cases, terms such as "alkyl" and "alkylene" may be preceded by a designation indicating the number of atoms present in the base (for example, "C"). 1ー4 "Alkyl" or "C 1ー4 "Alkylene"). These designations are used in a manner that is generally understood by those skilled in the art. For example, the expression "C" with a subscript indicates the number of carbon atoms present in the following group. Thus, "C3 alkyl" is an alkyl group having three carbon atoms (i.e., n-propyl, isopropyl). 1ー4 When a range is given, such as "C", the members of the subsequent group may have any number of carbon atoms within the listed range. For example, "C 1ー4 "Alkyl" refers to an alkyl group having 1 to 4 carbon atoms in any configuration (i.e., linear or branched).

[0071] The term "substituted" refers to a group that can be further substituted with one or more non-hydrogen substituents. Substituents include, but are not limited to, halogens, =O (oxo), =S (thioxo), cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, hydroxy, alkoxy, alkoxyalkyl, alkylene, amino, alkylamino, aminoalkyl, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylhonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, and acyl.

[0072] In certain cases, terms such as "alkyl" and "alkylene" may be preceded by a designation indicating the number of atoms present in the base (for example, "C"). 1ー4 "Alkyl", "C 1ー4"Alkylene"). These designations are used in a manner that is generally understood by those skilled in the art. For example, the expression "C" with a subscript indicates the number of carbon atoms present in the following group. Thus, "C3 alkyl" is an alkyl group having three carbon atoms (i.e., n-propyl, isopropyl). 1ー4 When a range is given, such as "C", the members of the subsequent group may have any number of carbon atoms within the listed range. For example, "C 1ー4 "Alkyl" refers to an alkyl group having 1 to 4 carbon atoms in any configuration (i.e., linear or branched).

[0073] The term "substituted" refers to a group that can be further substituted with one or more non-hydrogen substituents.

[0074] A method for reprogramming a precancerous mucosa into a noncancerous state is described herein, the method comprising identifying a precancerous mucosa containing metaplastic or dysplastic cells, and contacting the metaplastic or dysplastic cells with a compound of formula (I) or a salt thereof, [ka] During the ceremony, R 1 C 1ー6 It is alkyl, R 2a and R 2b Each of them is independent of C 1ー6 It is alkyl, R 3 C 6ー12 It is aryl, R 3 This is an optional selection of 1 to 5 R's. X Replaced by, R X In each occurrence, independently, C 1ー6 Alkyl, C 1ー4 Haloalkyl, halogen, cyano, -N(R) Xa )2, -OR Xb , -SR Xb , -C(O)R Xb , -CO2R Xb ,-C(O)N(RXa )2, -SO2R Xa , -L 2 -Y 2 ,-OL X -Y X -SL X -Y X , or -N(R Xa )-L X -Y X And, L X In each occurrence, independently, C 1ー6 Alkilen, C 2ー6 Alkenylene, or C 2ー6 It is alkynylene, Y X In each appearance, independently, hydrogen, cyano, halogen, haloalkyl, -OH, and -N(R) are present. Xa )2, -OR Xb , -SR Xb , -C(O)R Xb , -CO2R Xb ,-C(O)N(R Xa )2, or -SO2R Xa And, R Xa In each appearance, independently, hydrogen and C 1ー4 Alkyl, or -C(O)C 1ー4 It is alkyl, R Xb In each appearance, independently, hydrogen and C 1ー4 Alkyl, C 1ー2 Haloalkyl, or -C(O)C 1ー4 It is alkyl.

[0075] Metaplastic cells or dysplastic cells may be located within a living organism, such as a non-human animal or a human. As used herein, the term “living organism” means any biological system that functions as an individual living organism. Dysplastic cells may be positive for CD133, CD166, and Trop2 expression. Dysplastic cells may be dysplastic gastric cells, dysplastic esophageal cells, dysplastic pancreatic cells, dysplastic colonic cells, or dysplastic ovarian cells. Dysplastic gastric cells may be part of an organoid. Metaplastic gastric cells may be positive for CD133, CD166, and Trop2 expression. Metaplastic cells may be metaplastic gastric cells, metaplastic esophageal cells, metaplastic pancreatic cells, metaplastic colonic cells, or metaplastic ovarian cells. Metaplastic gastric cells may be part of an organoid.

[0076] In another embodiment, a method for reducing the risk of cancer development in a living organism is described herein, wherein the living organism contains metaplastic or dysplastic cells, and the method comprises administering to the living organism a compound of formula (I) or a salt thereof. The method may further comprise administering to the living organism a second agent. The second agent may be a kinase inhibitor, e.g., a MEK inhibitor, e.g., trametinib, binimetinib, cobimetinib, and / or selumetinib.

[0077] These and other aspects of this disclosure are described in detail below.

[0078] compound The compounds disclosed herein may include compounds of formula (I) or salts thereof. [ka] During the ceremony, R 1 C 1ー6 It is alkyl, R 2a and R 2b Each of them is independent of C 1ー6 It is alkyl, R 3 C 6ー12 It is aryl, R 3 This is an optional selection of 1 to 5 R's.X Replaced by, R X In each occurrence, independently, C 1ー6 Alkyl, C 1ー4 Haloalkyl, halogen, cyano, -N(R) Xa )2, -OR Xb , -SR Xb , -C(O)R Xb , -CO2R Xb ,-C(O)N(R Xa )2, -SO2R Xa , -L 2 -Y 2 ,-OL X -Y X -SL X -Y X , or -N(R Xa )-L X -Y X And, L X In each occurrence, independently, C 1ー6 Alkilen, C 2ー6 Alkenylene, or C 2ー6 It is alkynylene, Y X In each appearance, independently, hydrogen, cyano, halogen, haloalkyl, -OH, and -N(R) are present. Xa )2, -OR Xb , -SR Xb , -C(O)R Xb , -CO2R Xb ,-C(O)N(R Xa )2, or -SO2R Xa And, R Xa In each appearance, independently, hydrogen and C 1ー4 Alkyl, or -C(O)C 1ー4 It is alkyl, R Xb In each appearance, independently, hydrogen and C 1ー4 Alkyl, C 1ー2 Haloalkyl, or -C(O)C 1ー4 It is alkyl.

[0079] In some cases, R 1R may be methyl. In some cases, R 2a and R 2b Each of these may be methyl. In some cases, R 3 The compound may be an unsubstituted phenyl. In some cases, the compound of formula (I) may be pyrvinium or a salt thereof as shown below. [ka]

[0080] For the compounds described herein, the groups and substituents may be selected according to the allowable valencies of the atoms and substituents, and the selection and substitution result in stable compounds that do not undergo spontaneous transformations such as rearrangement, cyclization, or elimination.

[0081] It should be understood that the compound may have tautomers and geometric isomers, and these also constitute aspects of the present invention.

[0082] In the compounds of formula (I) and any subformula, any "hydrogen" or "H" is a hydrogen isotope, whether explicitly listed or implied in the structure. 1 H (protium) and 2 It contains H (deuterium).

[0083] salt In various examples, the compound of formula (I) may exist as a salt. A salt of the compound of formula (I) may be a pharmaceutically acceptable salt. As used herein, the term “pharmaceutically acceptable salt” refers to a salt or amphoteric ion of a compound that is water-soluble or oil-soluble, or water-dispersible or oil-dispersible, suitable for the treatment of a disorder without excessive toxicity, irritation, and allergic reactions, commensurate with a reasonable benefit / risk ratio, and effective for its intended use. Salts may be prepared during the final isolation and purification of the compound, or separately by reacting the amino group of the compound with a suitable acid. For example, the compound may be dissolved in a suitable solvent such as methanol and water, but not limited to these, and treated with an acid such as hydrochloric acid in at least one equivalent. The resulting salt may be precipitated, isolated by filtration, and dried under reduced pressure. Alternatively, the solvent and excess acid may be removed under reduced pressure to provide the salt. Typical salts include acetate, adipine, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphor sulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, isethionate, fumarate, lactate, maleate, methanesulfonate, naphthylenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, oxalate, maleate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, glutamate, para-toluenesulfonate, undecanoate, hydrochloride, hydrobromide, sulfate, and phosphate. The amino group of the compound may also be quaternized with alkyl chlorides, alkyl bromides, and alkyl iodides, such as methyl, ethyl, propyl, isopropyl, butyl, lauryl, myristyl, and stearyl.

[0084] Basic addition salts can be prepared during the final isolation and purification of the disclosed compounds by reaction of a carboxyl group with a suitable base such as a hydroxide, carbonate, or bicarbonate of a metal cation such as lithium, sodium, potassium, calcium, magnesium, or aluminum, or an organic primary, secondary, or tertiary amine. Quaternary amine salts may also be prepared, such as those derived from methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, etc.

[0085] In some cases, the salt of the compound of formula (I) is of formula (IA), [ka] Salt is also acceptable.

[0086] In some cases, the salt of formula (IA) may be pyrvinium pamoate as shown below. [ka]

[0087] Formulation and route of administration The disclosed compounds may be incorporated into pharmaceutical compositions suitable for administration to living organisms, such as patients, which may be human or non-human.

[0088] A pharmaceutical composition may contain a “therapeutic dose” or a “preventive dose” of the drug. The “therapeutic dose” refers to the effective amount in dosage and duration required to achieve the desired therapeutic outcome. The therapeutic dose of a composition may be determined by those skilled in the art and may vary depending on factors such as the individual’s disease state, age, sex, and weight, as well as the composition’s ability to induce the desired response in the individual. The therapeutic dose is also the amount in which the therapeutically beneficial effects of the compound of the present invention (e.g., the compound of formula (I)) outweigh any toxic or adverse effects. The “preventive dose” refers to the effective amount in dosage and duration required to achieve the desired preventive outcome. Typically, a preventive dose is administered to a subject before or earlier in the disease, and the preventive dose is less than the therapeutic dose.

[0089] When clinical application is intended, it will be necessary to prepare a pharmaceutical composition comprising the compound of formula (I), a salt thereof, or any additional therapeutic agent disclosed herein, in a form suitable for the intended application. Generally, this involves preparing a composition that is essentially free of pyrogens and other impurities that may be harmful to humans or animals.

[0090] In general, it would be desirable to use appropriate salts and buffers to stabilize the delivery vector and enable uptake by target cells. Buffers may also be used when introducing recombinant cells into a patient. An effective amount of the aqueous composition of this disclosure may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such a composition may also be called an inoculant. The terms “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that, when administered to animals or humans, do not cause adverse reactions, allergic reactions, or other undesirable reactions. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption retarders, etc.

[0091] Some examples of materials that can function as pharmaceutically acceptable carriers include sugars, e.g., lactose, glucose, and sucrose; starches, e.g., corn starch, and potato starch; cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, e.g., cocoa butter, and suppository waxes; oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and sesame oil. Soybean oil; glycols, such as propylene glycol; esters, such as but not limited to ethyl oleate and ethyl laurate; agar; buffers, such as but not limited to magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol and phosphate buffer solution; and other non-toxic compatible lubricants, such as but not limited to sodium lauryl sulfate and magnesium stearate. Similarly, colorants, release agents, coating agents, sweeteners, flavoring agents and fragrances, preservatives and antioxidants may also be present in the composition at the discretion of the compounder.

[0092] Therefore, compounds and their physiologically acceptable salts and solvates can be formulated for administration, for example, as solids, eye drops, topical oily preparations, injections, inhalations (either orally or nasally), implants, or by oral, oral, parenteral, or rectal administration. Techniques and formulations can generally be found in "Remington's Pharmaceutical Sciences" (Meade Publishing Co., Easton, Pa.). Therapeutic compositions typically must be sterile and stable under manufacturing and storage conditions.

[0093] The route by which the disclosed compound is administered and the form of the composition determine the type of carrier used. The composition may be in various forms and may be suitable for systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implant, or parenteral) or topical administration (e.g., skin, lung, nose, ear, eye, liposomal delivery system, or iontophoresis).

[0094] Carriers for systemic administration typically include at least one of diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, antioxidants, preservatives, glidants, solvents, suspending agents, wetting agents, surfactants, combinations thereof, and others. All carriers are optional in the composition. Suitable diluents include sugars such as glucose, lactose, dextrose, and sucrose; diols such as propylene glycol; calcium carbonate; sodium carbonate; sugar alcohols such as glycerin; mannitol; and sorbitol. The amount of diluent(s) in systemic or topical compositions is typically about 50% to about 90%.

[0095] Suitable lubricants include silica, talc, stearic acid and its magnesium and calcium salts, calcium sulfate, and liquid lubricants such as polyethylene glycol, as well as vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter. The amount of lubricant(s) in systemic or topical compositions is typically about 5% to about 10%.

[0096] Suitable binders include polyvinylpyrrolidone, magnesium aluminum silicate, starches such as corn starch and potato starch, gelatin, tragacanth, and cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, methyl cellulose, microcrystalline cellulose, and sodium carboxymethyl cellulose. The amount of binder(s) in systemic compositions is typically about 5% to about 50%.

[0097] Suitable disintegrants include agar, alginic acid and its sodium salts, effervescent mixtures, croscarmellose, crospovidone, sodium carboxymethyl starch, sodium starch glycolate, clays, and ion exchange resins. The amount of disintegrant(s) in a systemic or topical composition is typically from about 0.1% to about 10%. Suitable colorants include colorants such as FD&C dyes. When used, the amount of colorant in a systemic or topical composition is typically from about 0.005% to about 0.1%. Suitable flavorings (if any) include menthol, peppermint, and fruit flavors. When used, the amount of flavoring in a systemic or topical composition is typically from about 0.1% to about 1.0%.

[0098] Suitable sweeteners include aspartame and saccharin. The amount of sweetener(s) in a systemic or topical composition is typically from about 0.001% to about 1%. Suitable antioxidants include butylated hydroxyanisole ("BHA"), butylated hydroxytoluene ("BHT"), and vitamin E. The amount of antioxidant(s) in a systemic or topical composition is typically from about 0.1% to about 5%. Suitable preservatives include benzalkonium chloride, methylparaben, and sodium benzoate. The amount of preservative(s) in a systemic or topical composition is typically from about 0.01% to about 5%. Suitable glidants include silicon dioxide. The amount of glidant(s) in a systemic or topical composition is typically from about 1% to about 5%.

[0099] Suitable solvents include water, isotonic saline, ethyl oleate, glycerin, hydroxylated castor oil, alcohols such as ethanol, and phosphate buffer solutions. The amount of solvent(s) in a whole-body or topical composition is typically about 0 to about 100%. Suitable suspending agents include AVICEL RC-591 (from FMC Corporation in Philadelphia, PA) and sodium alginate. The amount of suspending agents(s) in a whole-body or topical composition is typically about 1 to about 8%. Suitable surfactants include lecithin, polysorbate 80, and sodium lauryl sulfate, as well as TWEENS from Atlas Powder Company in Wilmington, Delaware. Suitable surfactants include those disclosed in CTFA Cosmetic Ingredient Handbook, 1992, pp. 587–592, Remington's Pharmaceutical Sciences, 15th Ed. 1975, pp. 335–337, and McCutcheon's Volume 1, Emulsifiers & Detergents, 1994, North American Edition, pp. 236–239. The amount of surfactant(s) in a systemic or topical composition is typically about 0.1% to about 5%.

[0100] The amount of components in a systemic composition may vary depending on the type of systemic composition prepared, but generally, a systemic composition contains 0.01% to 50% of the active ingredient (e.g., the compound of formula (I)) and 50% to 99.99% of one or more carriers. Compositions for parenteral administration typically include a carrier containing 0.1% to 10% of the activator, and 90% to 99.9% of the diluent and solvent.

[0101] Oral administration compositions can have various dosage forms. For example, solid forms include tablets, capsules, granules, and bulk powders. These oral dosage forms contain a safe and effective amount of the active ingredient, usually at least about 5%, more specifically about 25% to about 50%. Oral administration compositions contain about 50% to about 95% of the carrier, more specifically about 50% to about 75% of the carrier.

[0102] Tablets can be compressed, crushed, enterically coated, sugar-coated, film-coated, or multiple-compressed. Tablets typically contain an active ingredient and a carrier containing components selected from diluents, lubricants, binders, disintegrants, colorants, flavorings, sweeteners, flow enhancers, and combinations thereof. Certain diluents include calcium carbonate, sodium carbonate, mannitol, lactose, and cellulose. Certain binders include starch, gelatin, and sucrose. Certain disintegrants include alginic acid and croscarmellose. Certain lubricants include magnesium stearate, stearic acid, and talc. Certain colorants are FD&C dyes, which may be added for appearance. Chewable tablets preferably contain sweeteners such as aspartame and saccharin, or flavorings such as menthol, peppermint, fruit flavors, or combinations thereof.

[0103] Capsules (including implants, sustained-release and sustained-release formulations) typically contain an active compound (e.g., a compound of formula (I)) and a carrier containing one or more diluents disclosed above, within a gelatin-containing capsule. Granules typically contain the disclosed compound and, preferably, a flow enhancer such as silicon dioxide to improve flow properties. Implants may be biodegradable or non-biodegradable.

[0104] The selection of components in a carrier for an oral composition depends on secondary considerations such as taste, cost, and shelf life, which are not important to the purposes of this invention. The solid composition may be coated by conventional methods, typically with a pH or time-dependent coating, so that the disclosed compound is released in the gastrointestinal tract near the desired application, or at various points and time points to extend the desired effect. The coating typically comprises one or more components selected from the group consisting of cellulose phthalate acetate, polyvinyl phthalate acetate, hydroxypropyl methylcellulose phthalate, ethylcellulose, EUDRAGIT coating (available from Rohm & Haas GmbH in Darmstadt, Germany), wax, and shellac.

[0105] Orally administered compositions may be in liquid form. For example, suitable liquid forms include aqueous solutions, emulsions, suspensions, solutions reconstituted from non-foaming granules, suspensions reconstituted from non-foaming granules, effervescent preparations reconstituted from effervescent granules, elixirs, tinctures, syrups, and the like. Liquid oral compositions typically comprise the disclosed compound and a carrier, i.e., a carrier selected from diluents, colorants, flavorings, sweeteners, preservatives, solvents, suspending agents, and surfactants. Oral liquid compositions preferably comprise one or more components selected from colorants, flavorings, and sweeteners.

[0106] Other compositions useful for achieving systemic delivery of the subject compound include sublingual, oral, and nasal formulations. Such compositions typically contain soluble filler substances such as diluents containing sucrose, sorbitol, and mannitol, as well as one or more binders such as acacia, microcrystalline cellulose, carboxymethylcellulose, and hydroxypropylmethylcellulose. Such compositions may further contain lubricants, colorants, fragrances, sweeteners, antioxidants, and flow enhancers.

[0107] The disclosed compounds can be administered topically. Topical compositions that can be applied topically to the skin may be in any form, including solids, solutions, oils, creams, ointments, gels, lotions, shampoos, leave-on and rinse-out hair conditioners, emulsions, cleansers, moisturizers, sprays, skin patches, and the like. A topical composition comprises the disclosed compounds (e.g., the compounds of formula (I)) and a carrier. The carrier of the topical composition preferably helps the compound to penetrate the skin. The carrier may further comprise one or more optional components.

[0108] The amount of carrier used in conjunction with the disclosed compound is sufficient to provide a practical amount of the dosing composition per unit dose of the pharmaceutical. Techniques and compositions for producing useful dosage forms in the method of the present invention are described in the following references: Modern Pharmaceutics, Chapters 9 and 10, Banker & Rhodes, eds. (1979), Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981), and Ansel, Introduction to Pharmaceutical Dosage Forms, 2 nd ed., (1976).

[0109] The carrier may comprise a single component or a combination of two or more components. In a topical composition, the carrier comprises a topical carrier. A suitable topical carrier comprises one or more components selected from phosphate-buffered saline, isotonic water, deionized water, monofunctional alcohol, symmetrical alcohol, aloe vera gel, allantoin, glycerin, vitamin A and E oils, mineral oil, propylene glycol, PPG-2 myristylpropionate, dimethyl isosorbide, castor oil, or combinations thereof. More specifically, a carrier for skin applications comprises propylene glycol, dimethyl isosorbide, and water, and even more specifically, phosphate-buffered saline, isotonic water, deionized water, monofunctional alcohol, and symmetrical alcohol. The carrier of a topical composition may further comprise one or more components selected from emollients, propellants, solvents, humectants, thickeners, powders, fragrances, pigments, and preservatives, all of which are optional.

[0110] Suitable softening agents include stearyl alcohol, glyceryl monoricinoleate, glyceryl monostearate, propane-1,2-diol, butane-1,3-diol, mink oil, cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecane-2-ol, isocetyl alcohol, cetyl palmitate, and sebacate. This includes n-butyl, isopropyl myristate, isopropyl palmitate, isopropyl stearate, butyl stearate, polyethylene glycol, triethylene glycol, lanolin, sesame oil, coconut oil, arachis oil, castor oil, acetylated lanolin alcohol, petroleum, mineral oil, butyl myristate, isostearic acid, palmitic acid, isopropyl linoleate, lauryl lactate, myristyl lactate, decyl oleate, myristyl myristate, and combinations thereof. Specific emollients for skin include stearyl alcohol and polydimethylsiloxane. The amount of emollient(s) in a skin-based topical composition is typically about 5% to about 95%.

[0111] Suitable propellants include propane, butane, isobutane, dimethyl ether, carbon dioxide, nitrous oxide, and combinations thereof. The amount of propellant(s) in the topical composition is typically about 0% to about 95%.

[0112] Suitable solvents include water, ethyl alcohol, methylene chloride, isopropanol, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethyl sulfoxide, dimethylformamide, tetrahydrofuran, and combinations thereof. Specific solvents include ethyl alcohol and homotopic alcohol. The amount of solvent(s) in the topical composition is typically about 0% to about 95%.

[0113] Suitable humectants include glycerin, sorbitol, sodium 2-pyrrolidone-5-carboxylate, soluble collagen, dibutyl phthalate, gelatin, and combinations thereof. Specific humectants include glycerin. The amount of humectants (multiple types) in a topical composition is typically 0% to 95%. The amount of thickeners (multiple types) in a topical composition is typically about 0% to about 95%. Suitable powders include beta-cyclodextrin, hydroxypropyl cyclodextrin, chalk, talc, fuller's earth, kaolin, starch, gum, colloidal silicon dioxide, sodium polyacrylate, tetraalkylammonium smectite, trialkylarylammonium mectite, chemically modified aluminum magnesium silicate, organically modified montmorillonite clay, hydrated aluminum silicate, fumed silica, carboxyvinyl polymer, sodium carboxymethylcellulose, ethylene glycol monostearate, and combinations thereof. The amount of powder(s) in the topical composition is typically 0% to 95%. The amount of fragrance in the topical composition is typically about 0% to about 0.5%, particularly about 0.001% to about 0.1%. A suitable pH adjusting additive contains an amount of HCl or NaOH sufficient to adjust the pH of the topical pharmaceutical composition.

[0114] Therapeutic amount of the composition The effective dose of the therapeutic agent(s) disclosed herein may be determined based on the intended purpose, e.g., (i) inhibition of tumor cell proliferation, or (ii) elimination of tumor cells. The term “unit dose” refers to a physically distinct unit suitable for use in the subject, each unit containing a predetermined amount of the therapeutic composition calculated to produce the desired response described above in relation to its administration, i.e., the appropriate route and treatment regimen. The amount administered according to both the number of treatments and the unit dose depends on the subject being treated, the condition of the subject, and the desired protection. The exact amount of the therapeutic composition may also depend on the judgment of the practitioner and may be individual-specific.

[0115] In some cases, the dose range of the compound of formula (I) or its salt may be about 0.5 mg / kg body weight to about 500 mg / kg body weight. The term “body weight” applies when animals are being treated. When isolated cells are being treated, “body weight” as used herein means “total cell weight.” The term “total weight” may be used to apply to both isolated cells and animal treatments. All concentrations and treatment levels expressed as “body weight” or “kg” in this application may be considered to cover similar “total cell weight” and “total weight” concentrations. However, those skilled in the art will recognize the usefulness of various dosage ranges, for example, 1 mg / kg body weight to 450 mg / kg body weight, 2 mg / kg body weight to 400 mg / kg body weight, 3 mg / kg body weight to 350 mg / kg body weight, 4 mg / kg body weight to 300 mg / kg body weight, 5 mg / kg body weight to 250 mg / kg body weight, 6 mg / kg body weight to 200 mg / kg body weight, 7 mg / kg body weight to 150 mg / kg body weight, 8 mg / kg body weight to 100 mg / kg body weight, or 9 mg / kg body weight to 50 mg / kg body weight. Furthermore, those skilled in the art can provide various dosage levels, for example, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 7.5 mg / kg, 10 mg / kg, 12.5 mg / kg, 15 mg / kg, 17.5 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 120 mg / kg, 140 mg / kg, 150 mg / kg, 160 mg / kg, 180 mg / kg, 2 You will be aware that 00 mg / kg, 225 mg / kg, 250 mg / kg, 275 mg / kg, 300 mg / kg, 325 mg / kg, 350 mg / kg, 375 mg / kg, 400 mg / kg, 450 mg / kg, 500 mg / kg, 550 mg / kg, 600 mg / kg, 700 mg / kg, 750 mg / kg, 800 mg / kg, 900 mg / kg, 1000 mg / kg, 1250 mg / kg, 1500 mg / kg, 1750 mg / kg, 2000 mg / kg, 2500 mg / kg, and / or 3000 mg / kg may be used.Any of the above dosage ranges or dosage levels can be used for the compounds of formula (I), or salts thereof, in combination with a second therapeutic agent.

[0116] As will be appreciated in the art, the specific dosage level of an active compound such as a compound of formula (I), or a salt thereof, for any particular patient will depend on a variety of factors including the activity of the specific compound being used, age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. The person responsible for administration will determine the appropriate dosage for the individual subject. Further, for human administration, the preparations must meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Biologics Standards Office.

[0117] In some embodiments, the compounds of formula (I), or salts thereof, may be administered alone or in combination with a second therapeutic agent. When the second therapeutic agent is administered in combination with a compound of formula (I) or a salt thereof, the effective amount of the second therapeutic agent can be defined as an amount effective to reduce pre-cancerous proliferation, provided that the second therapeutic agent dosage does not exceed the aforementioned toxicity levels. The amount effective to reduce pre-cancerous proliferation can be determined by monitoring an animal or patient and measuring physical and biochemical parameters of health and disease that indicate the success of a given treatment. Such methods are routine in animal experimentation and clinical practice.

[0118] In some embodiments, chemotherapy may be administered in regular cycles, as is typical. A cycle may consist of a single dose, followed by several days or weeks without treatment to allow normal tissue to recover from the side effects of the drug. The dose may be given over several consecutive days, followed by a rest period. If multiple drugs are used, the treatment plan specifies the frequency and exact timing of each drug's administration. The number of cycles a person will undergo may be determined before the start of treatment (based on the type and stage of precancerous disease) or may be flexible to take into account how quickly mucosal recovery is observed. Certain serious side effects may also necessitate adjustments to the chemotherapy plan by the physician to allow time for the patient to recover.

[0119] Combination therapy In some cases, the compound of formula (I), or a salt thereof, may be used in combination with a second therapeutic agent. Additional therapeutic agents intended to be used in combination with the compound of formula (I), or a salt thereof, may include, but are not limited to, anticancer agents. Anticancer agents may include, but are not limited to, radiation therapy, chemotherapy, gene therapy, hormone therapy, or immunotherapy that target cancer / tumor cells.

[0120] To kill cells, induce cell cycle arrest, inhibit cell proliferation, inhibit metastasis, inhibit angiogenesis, or otherwise reverse or reduce the malignant phenotype of precancerous cells using the methods and compositions of this disclosure, cells are generally contacted with a compound of formula (I) or a salt thereof in combination with a second therapeutic agent. These compositions are provided in compound amounts effective for killing cells or inhibiting their proliferation. This process may involve contacting cells with a compound of formula (I) or a salt thereof in combination with a second therapeutic agent or factor(s). This may be achieved by contacting cells with a single composition or pharmacological formulation containing both agents, or by contacting cells with two different compositions or formulations simultaneously, one of which contains a compound of formula (I) or a salt thereof, and the other containing the second agent.

[0121] Alternatively, treatment with the compound of formula (I) or a salt thereof may precede or follow additional drug treatment at intervals ranging from a few minutes to several weeks. In embodiments where the second drug is applied to cells separately, generally, no significant time should elapse between each delivery so that the drugs can still exert a beneficial synergistic effect on the cells. In such examples, it is assumed that the cells will be contacted by both modalities within approximately 12–24 hours of each other, or alternatively, within approximately 6–12 hours of each other. In some situations, it may be desirable to significantly extend the treatment period, with a few days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapsed between each administration.

[0122] Furthermore, it is considered desirable to administer pyrvinium or any of its salts more than once in combination with a second therapy, such as an anticancer drug or other treatment. Various combinations can be used, such as pyrvinium or its salt being "A" and the second therapeutic agent being "B," as illustrated below. [Table 1]

[0123] Other combinations may be considered. Similarly, in order to achieve cell death by inducing apoptosis, both drugs may be delivered to the cells in a combined amount effective in killing the cells.

[0124] Chemotherapy agents In some embodiments, the chemotherapeutic agent may be used in combination with a compound of formula (I) or a salt thereof to treat precancerous conditions. Examples of such chemotherapeutic agents include, but are not limited to, kinase inhibitors. Examples of kinase inhibitors include MEK inhibitors such as trametinib, binimetinib, cobimetinib, and / or selumetinib.

[0125] Immunotherapy agents In this disclosure, immunotherapeutic agents may also be used in combination with compounds of formula (I) or salts thereof in the treatment of precancerous cells. Generally, immunotherapeutic agents rely on the use of immune effector cells and molecules to target and destroy precancerous cells. Immune effectors may be, for example, antibodies specific to certain markers on the surface of tumor cells. Antibodies may function as effectors of therapy alone, or they may recruit other cells to cause cell death. Antibodies may also be conjugated with drugs or toxins (chemotherapeutic agents, radionuclides, lysine A chain, cholera toxin, pertussis toxin, etc.) and may simply function as targeting agents. Alternatively, effectors may be lymphocytes carrying surface molecules that interact directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells.

[0126] Generally, tumor cells must possess several markers that are suitable for targeting, i.e., not present in most other cells. Many tumor markers exist, and any of these may be suitable for targeting in the context of this disclosure. Common tumor markers include carcinoembryonic antigen, prostate-specific antigen, urinary tract tumor-associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B, and p155.

[0127] Checkpoint inhibitor therapy is another form of cancer immunotherapy. This therapy targets immune checkpoints, which are key regulators of the immune system that, when stimulated, can weaken the immune response to immunological stimuli. Some cancers may protect themselves from attack by stimulating immune checkpoint targets. Checkpoint therapy can block inhibitory checkpoints and restore the function of the immune system. Currently approved checkpoint inhibitors target the molecules CTLA4, PD-1, and PD-L1. PD-1 is a transmembrane programmed cell death 1 protein (also called PDCD1 and CD279) that interacts with PD-L1 (PD-1 ligand 1, or CD274). PD-L1 on the cell surface binds to PD-1 on the surface of immune cells, inhibiting immune cell activity. Among the functions of PD-L1 is an important regulatory role on T cell activity. Upregulation of PD-L1 on the cell surface (cancer-mediated) appears to inhibit T cells that could otherwise attack. Antibodies that bind to either PD-1 or PD-L1 and thus block their interaction may allow T cells to attack tumors.

[0128] Other drugs To improve the therapeutic efficacy of the treatment, other agents may be used in combination with the compound of formula (I) or a salt thereof. These additional agents include immunomodulators, agents that affect the upmodulation of cell surface receptors and GAP junctions, cell suppressors and differentiation agents, cell adhesion inhibitors, or agents that increase the sensitivity of hyperproliferating cells to apoptosis inducers. Immunomodulators include tumor necrosis factor, interferon alpha, beta and gamma, IL-2 and other cytokines, F42K and other cytokine analogs, or MIP-1, MIP-1 beta, MCP-1, RANTES, and other chemokines. Upmodulation of cell surface receptors or their ligands, such as Fas / Fas ligands, DR4 or DR5 / TRAIL, is further intended to enhance the apoptosis-inducing capacity of this disclosure by establishing autocrine or paracrine effects on hyperproliferating cells. Increased intercellular signaling by increasing the number of GAP junctions increases the anti-hyperproliferative effect on adjacent hyperproliferating cell populations. In other embodiments, cell inhibitors or differentiation agents may be used in combination with the disclosure to improve the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors may be intended to improve the efficacy of the disclosure. Examples of cell adhesion inhibitors are local adhesion kinase (FAK) inhibitors and lovastatin. It is further intended that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, may be used in combination with the compositions described herein to improve therapeutic efficacy.

[0129] Treatment method Cells, such as metaplastic or dysplastic cells, may be brought into contact with a compound of formula (I) or a salt thereof to kill cells, induce cell cycle arrest, inhibit cell proliferation, inhibit metastasis, inhibit angiogenesis, or otherwise reverse or reduce the malignant phenotype of precancerous cells, or to reduce the risk of developing cancer. The terms “contacted” and “exposed” are used herein to describe the process by which a therapeutic agent is delivered to a target or directly juxtaposed with target cells, when applied to cells. To achieve cell death or suppression, the therapeutic agent is delivered to cells in an amount effective in inducing cell cycle arrest, inhibiting cell growth, or inducing intracellular apoptosis.

[0130] In some cases, the methods described herein may reprogram precancerous mucosa to a noncancerous state, where the precancerous mucosa contains metaplastic or dysplastic cells. An exemplary method for reprogramming precancerous mucosa to a noncancerous state may include identifying precancerous mucosa containing metaplastic or dysplastic cells, and contacting the metaplastic or dysplastic cells with a compound of formula (I) or a salt thereof. Metaplastic or dysplastic cells may be located in vivo. Precancerous mucosa may contain dysplastic cells. The method may include killing the dysplastic cells. Dysplastic cells may be positive for CD133 expression, CD166 expression, and / or Trop2 expression. Dysplastic cells may be dysplastic gastric cells, dysplastic esophageal cells, dysplastic pancreatic cells, dysplastic colon cells, or dysplastic ovarian cells. Dysplastic gastric cells may be part of an organoid. Precancerous mucosa may contain metaplastic cells. Metaplastic gastric cells may be positive for CD133, CD166, and / or Trop2 expression. Metaplastic cells may be metaplastic gastric cells, metaplastic esophageal cells, metaplastic pancreatic cells, metaplastic colonic cells, or metaplastic ovarian cells. Metaplastic gastric cells may be part of organoids.

[0131] In some cases, the methods described herein may reduce the risk of cancer in a subject such as a non-human animal or human, containing metaplastic or dysplastic cells. An exemplary method for reducing the risk of cancer development in a subject containing metaplastic or dysplastic cells may include administering to the subject a compound of formula (I) or a salt thereof. The method may inhibit the STAT3 and MEK / ERK signaling pathways in metaplastic or dysplastic cells. In some cases, the subject may be a non-human animal. In other cases, the subject is human. Humans may be at risk of developing cancer. The method may further include administering to the subject a second agent. The second agent may include a kinase inhibitor. The kinase inhibitor may include a MEK inhibitor. The MEK inhibitor may include trametinib, binimetinib, cobimetinib, and / or selumetinib.

[0132] Compounds of formula (I), salts thereof, or pharmaceutical compositions containing the same may be administered to a subject once or more, at intervals ranging from a few minutes to several weeks. Generally, the intervals should not be interrupted within the same period so that the drug can not exert a favorable effect on the cells, and each delivery may be administered as a therapeutic time. However, in some situations, it may be desirable to extend the therapeutic period, with a few days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapsed between each administration.

[0133] The compound of formula (I) or a salt thereof may be administered to a subject by any method known in the art for delivering a therapeutic agent to a subject. For example, such methods may include, but are not limited to, oral, nasal, intramuscular, or intraperitoneal administration. Methods of administration are disclosed in detail elsewhere in this application.

[0134] Metaplasia and dysplasia Metaplasia is the transformation from one differentiated cell type to another. The change from one type of cell to another can be part of a normal maturation process or can be triggered by some abnormal stimulus. Simply put, it's like the original cell not being robust enough to withstand its environment and transforming into a different cell type better suited to that environment. Once the stimulus causing metaplasia is removed or stopped, the tissue returns to its normal differentiation pattern. Metaplasia is not synonymous with dysplasia and is not considered actual cancer. Today, metaplastic changes are generally considered an early stage of carcinogenesis, especially in individuals with a history of cancer or those known to be susceptible to oncogenic changes. Therefore, metaplastic changes are often considered a pre-malignant neoplastic condition.

[0135] Dysplasia is any abnormal growth or development of any type of cell (microscopic scale) or tissue (macroscopic scale), or any abnormal histological or anatomical structure(s) resulting from such growth.

[0136] In one of the modern histopathological meanings of this term, dysplasia can be distinguished from other categories of histological changes, including hyperplasia, metaplasia, and neoplasms. Dysplasia is generally considered to be precancerous.

[0137] Therefore, metaplasia represents the transformation of one mature cell type into another, while dysplasia often represents an increase in the amount of abnormal immature cell types. Both metaplasia and dysplasia typically result from chronic environmental stressors.

[0138] Cancer treated according to the method Compounds of formula (I) or salts thereof have been shown herein to inhibit metaplastic and dysplastic cells and may therefore be useful in the treatment of diseases of uncontrolled proliferation, particularly precancerous conditions. Accordingly, compounds of formula (I) or salts thereof may be used as therapeutic agents for the treatment of precancerous conditions of subjects such as hepatocellular carcinoma, Barrett's esophagus, bladder, breast, stomach, colon, head and neck, lung cell, mesothelioma, and / or cervical precancerous conditions. More specifically, in some cases, precancerous cells may be positive for CD133, CD166, and / or Trop2 expression.

[0139] It will be apparent to those skilled in the art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiment or its aspects. The compositions and methods provided are illustrative and are not intended to limit the scope of any of the specified embodiments. All the various embodiments, aspects, and options disclosed herein can be combined in any variation or iteration. The scope of the compositions, formulations, methods, and processes described herein includes all actual or potential combinations of the embodiments, aspects, options, examples, and preferences described herein. The exemplary compositions and formulations described herein may omit any components, substitute any components disclosed herein, or include any components disclosed elsewhere herein. The ratio of the mass of any component in any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or the total mass of the other components in the formulation is disclosed herein as if they were explicitly disclosed. If the meaning of any term in any of the patents or publications incorporated by reference conflicts with the meaning of a term used in this disclosure, the meaning of the term or phrase in this disclosure shall prevail. Furthermore, the foregoing discussion discloses and describes only exemplary embodiments. All patents and publications cited herein are incorporated herein by reference with respect to their specific teachings.

[0140] All compositions and / or methods disclosed and claimed herein can be prepared and performed without undue experimentation in light of this disclosure. While the compositions and methods of this disclosure are described in terms of preferred embodiments, it will be apparent to those skilled in the art that the compositions and / or methods described herein, as well as the steps or order of the steps of the methods, may be modified without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically relevant agents may be substituted with the agents described herein, and the same or similar results may be obtained. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope, and concept of this disclosure as defined by the appended claims.

[0141] The various embodiments and aspects of the invention described herein are summarized by the following clauses.

[0142] Article 1. A method for reprogramming a precancerous mucosa into a noncancerous state, Identifying precancerous mucosa containing metaplastic or dysplastic cells, The process involves contacting the metaplastic cells or dysplastic cells with a compound of formula (I) or a salt thereof. [ka] During the ceremony, R 1 However, C 1ー6 It is alkyl, R 2a and R 2b However, each is independent of C 1ー6 It is alkyl, R 3 However, C 6ー12 It is aryl, R 3 This is an optional selection of 1 to 5 R's. X Replaced by, R X However, in each occurrence, independently, C 1ー6Alkyl, C 1ー4 Haloalkyl, halogen, cyano, -N(R) Xa )2, -OR Xb , -SR Xb , -C(O)R Xb , -CO2R Xb ,-C(O)N(R Xa )2, -SO2R Xa , -L 2 -Y 2 ,-OL X -Y X -SL X -Y X , or -N(R Xa )-L X -Y X And, L X However, in each occurrence, independently, C 1ー6 Alkilen, C 2ー6 Alkenylene, or C 2ー6 It is alkynylene, Y X However, in each appearance, independently, hydrogen, cyano, halogen, haloalkyl, -OH, -N(R) Xa )2, -OR Xb , -SR Xb , -C(O)R Xb , -CO2R Xb ,-C(O)N(R Xa )2, or -SO2R Xa And, R Xa However, in each appearance, hydrogen and C appear independently. 1ー4 Alkyl, or -C(O)C 1ー4 It is alkyl, R Xb However, in each appearance, hydrogen and C appear independently. 1ー4 Alkyl, C 1ー2 Haloalkyl, or -C(O)C 1ー4 A method that is alkyl.

[0143] Clause 2. The method according to Clause 1, wherein the metaplastic cells or dysplastic cells are contacted with a salt of the compound of formula (I).

[0144] Clause 3. A salt of the compound of formula (I) is of formula (IA), [ka] The method described in Clause 2, which is a salt of the same.

[0145] Clause 4.R 1 The method according to any one of the clauses 1 to 3, wherein the substance is methyl.

[0146] Clause 5.R 2a and R 2b The method according to any one of the clauses 1 to 4, wherein each is methyl.

[0147] Clause 6.R 3 The method according to any one of the clauses 1 to 5, wherein the phenyl is unsubstituted.

[0148] Clause 7. The method according to any one of Clauses 1 to 6, wherein the compound of formula (I) is pyrvinium.

[0149] Clause 8. The method according to any one of Clauses 1 to 7, wherein the metaplastic cells or dysplastic cells are located in a living organism.

[0150] Clause 9. The method according to any one of Clauses 1 to 8, wherein the precancerous mucosa contains dysplastic cells.

[0151] Clause 10. The method according to Clause 9, wherein the method comprises killing the dysplastic cells.

[0152] Clause 11. The method according to Clause 9 or 10, wherein the dysplastic cells are positive for CD133 expression, CD166 expression, and / or Trop2 expression.

[0153] Clause 12. The method according to any one of Clauses 9 to 11, wherein the dysplastic cells are dysplastic gastric cells, dysplastic esophageal cells, dysplastic pancreatic cells, dysplastic colon cells, or dysplastic ovarian cells.

[0154] Clause 13. The method according to Clause 12, wherein the dysplastic gastric cells are part of an organoid.

[0155] Clause 14. The method according to any one of Clauses 1 to 13, wherein the precancerous mucosa includes metaplastic cells.

[0156] Clause 15. The method according to Clause 14, wherein metaplastic gastric cells are positive for CD133, CD166, and / or Trop2 expression.

[0157] Clause 16. The method according to any one of Clauses 1 to 15, wherein the metaplastic cells are metaplastic gastric cells, metaplastic esophageal cells, metaplastic pancreatic cells, metaplastic colonic cells, or metaplastic ovarian cells.

[0158] Clause 17. The method according to Clause 16, wherein the metaplastic gastric cells are part of an organoid.

[0159] Article 18. A method for reducing the risk of cancer development in a living organism containing metaplastic or dysplastic cells, The procedure involves administering a compound of formula (I) or a salt thereof to the living organism. [ka] During the ceremony, R 1 However, C 1ー6 It is alkyl, R 2a and R 2b However, each is independent of C 1ー6 It is alkyl, R 3 However, C 6ー12 It is aryl, R 3 However, you can choose 1 to 5 Rs at will. X Replaced by, R X However, in each occurrence, independently, C 1ー6 Alkyl, C 1ー4 Haloalkyl, halogen, cyano, -N(R) Xa )2, -OR Xb , -SR Xb , -C(O)R Xb, -CO2R Xb ,-C(O)N(R Xa )2, -SO2R Xa , -L 2 -Y 2 ,-OL X -Y X -SL X -Y X , or -N(R Xa )-L X -Y X And, L X However, in each occurrence, independently, C 1ー6 Alkilen, C 2ー6 Alkenylene, or C 2ー6 It is alkynylene, Y X However, in each appearance, independently, hydrogen, cyano, halogen, haloalkyl, -OH, -N(R) Xa )2, -OR Xb , -SR Xb , -C(O)R Xb , -CO2R Xb ,-C(O)N(R Xa )2, or -SO2R Xa And, R Xa However, in each appearance, hydrogen and C appear independently. 1ー4 Alkyl, or -C(O)C 1ー4 It is alkyl, R Xb However, in each appearance, hydrogen and C appear independently. 1ー4 Alkyl, C 1ー2 Haloalkyl, or -C(O)C 1ー4 A method that is alkyl.

[0160] Clause 19. The method according to Clause 18, wherein a salt of the compound of formula (I) is administered to the living organism.

[0161] Clause 20. A salt of the compound of formula (I) is of formula (IA), [ka] The method described in clause 18 or 19, which is a salt of the same.

[0162] Clause 21. The method according to any one of Clauses 18 to 20, wherein the compound of formula (I) is pyrvinium.

[0163] Clause 22. The method according to any one of Clauses 18 to 21, wherein the method inhibits the STAT3 and MEK / ERK signaling pathways in the metaplastic cells or dysplastic cells.

[0164] Clause 23. The method described in any one of Clauses 18 to 22, wherein the organism is a non-human animal.

[0165] Clause 24. The method described in any one of Clauses 18 to 23, wherein the organism is a human.

[0166] Clause 25. The method described in Clause 24, which puts a person at risk of developing cancer.

[0167] Clause 26. The method according to any one of Clauses 18 to 25, further comprising administering a second drug to the living organism.

[0168] Clause 27. The method according to Clause 26, wherein the second agent comprises a kinase inhibitor.

[0169] Clause 28. The method according to Clause 27, wherein the kinase inhibitor comprises a MEK inhibitor.

[0170] Clause 29. The method according to Clause 28, wherein the MEK inhibitor comprises trametinib, binimetinib, cobimetinib, and / or selumetinib.

[0171] Article 30. Use of pyrvinium or its salts in the manufacture of pharmaceuticals for reprogramming precancerous mucosa into a noncancerous state or for reducing the risk of developing cancer. [Examples]

[0172] material and method Organoid establishment, culture, and drug therapy To establish human precancerous organoids (hPCOs), fresh tissue was obtained from gastric cancer patients who underwent curative gastrectomy at SNUH (IRB No. H-1806-166-954). The organoid lineage was established as described in previous studies.

[0173] Meta3, Meta4, and hPCO organoids were cultured in Matrigel (ECM, Sigma) using mouse or human IntestiCult medium (StemCell Technology) supplemented with 1% penicillin / streptomycin (Gibco) in 48-well plates, with the medium replaced every 3 days. The organoids were divided every 5–7 days before budding structures formed.

[0174] Trametinib, pyrvinium pamoate, SSTC3, and three STAT3 inhibitors (STAT3-IN-1, Static, and cryptotancinone) (all from MedChemExpress) were dissolved in DMSO. Mouse or human organoids were divided and cultured in mouse or human IntestiCult medium for 1-2 days to form three-dimensional (3D) spherical structures, then the medium was switched to a DMSO vehicle or a medium containing a specific concentration of each drug, and cultured for 3 days. Phase-contrast images of the organoids were obtained using an EVOS M7000 inverted microscope. All experiments were repeated at least three times.

[0175] immunostaining To prepare formaldehyde-fixed paraffin-embedded (FFPE) organoids / spheres, Matrigel domes containing the organoids / spheres were fixed in 4% PFA at room temperature for 30 minutes. The fixed organoids were embedded in HistoGel® (Thermo Fisher Scientific), subsequently washed in PBS for 1 minute, and processed according to a standard histological protocol for paraffin embedding. Human tissues were fixed in 10% neutral buffered formalin (NBF) at room temperature for 1 week, and mouse tumor tissues were fixed in 4% paraformaldehyde (PFA) solution overnight at 4°C. The fixed tissues were transferred to 70% ethanol and processed according to a standard histological protocol for subsequent paraffin embedding. 4-5 micrometer organoids / spheres or tissue paraffin sections were deparaffinized in Histoclear solution (Electron Microscopy Services) and rehydrated through a series of ethanols (100%, 95%, and 75%).

[0176] Antigen was recovered in a pressure cooker for 15 minutes using pH 6 or pH 9 targeted recovery solution (Dako), followed by cooling for 1 hour. For immunofluorescence staining, each section was incubated in serum-free protein block solution (Dako) at room temperature for 1.5 hours. The primary antibody was diluted in antibody diluent containing background reduction component (Dako), applied to the sections, and incubated overnight at 4°C. The slides were washed three times in PBS for 5 minutes each, and the secondary antibody diluted in antibody diluent (Dako) was applied to each section. The sections were incubated at room temperature for 1 hour, and for nuclear counterstaining, the sections were applied to PBS containing Hoechst (1:5000) for 5 minutes. The slides were washed three times in PBS for 5 minutes each, mounted on a ProLong® Gold Antifade Mountant (ThermoFisher), and covered with a coverslip. All fluorescence images were acquired using a Zeiss Axio Imager M2 microscope with an Axiovision digital imaging system, or scanned using a Vanderbilt Digital Histology Shared Resource Aperio Versa 200 Fluorescent Slide Scanner (Leica). Fluorescence images were created and overlaid using Adobe Photoshop.

[0177] For immunohistochemical staining, sections were blocked in peroxidase blocking solution (Vector Laboratories) for 20 minutes at room temperature, followed by blocking in serum-free protein blocking solution (Dako) for 1.5 hours at room temperature. Primary antibodies were diluted in antibody diluents containing background reduction components (Dako), applied to sections, and incubated overnight at 4°C. Horseradish peroxidase (HRP)-conjugated secondary antibodies were applied to sections, incubated for 15 minutes at room temperature, and then washed three times in PBS for 5 minutes each. ImmPRESS polymer detection reagent and ImmPACT DAB substrate kit (Vector Laboratories) were used for antibody detection. Images were scanned at 20x magnification using an SCN400 slide scanner (Leica).

[0178] Quantitative Real-Time PCR (RT-PCR) Total RNA was extracted from Meta3 or Meta4 organoids using Trizol (Invitrogen) reagent. cDNA was synthesized using the iScript gDNA clear cDNA synthesis kit (Bio-Rad), and RT-PCR was performed using SsoAdvanced™ Universal SYBR Green supermix (Bio-Rad) and the CFX96 Real-Time PCR Detection System (Bio-Rad). Gene expression levels were normalized using the housekeeping gene GAPDH. All reactions were performed in triplicate, and relative gene expression levels were obtained by comparative ΔΔCt method using CFX Maestro software (Bio-Rad).

[0179] Western blot Total proteins were extracted from organoids using a radioimmunoprecipitation assay buffer containing a protease inhibitor (10 mmol / L Tris-HC, pH 7.2, 150 mmol / L NaCl, 5 mmol / L EDTA, 0.1% sodium dodecyl sulfate, 1.0% TritonX-100, 1% deoxycholate). Protein concentrations were measured using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). 5–10 μg of total protein was loaded onto a 12.5% ​​or 10% sodium dodecyl sulfate / polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA). The membrane was blocked at room temperature for 1 hour with 5% skim milk in 1× TBS (Corning) containing 0.1% Tween20 (Bio-Rad), and incubated overnight at 4°C with primary antibody diluted in 5% skim milk in TBS-T. The following day, the membrane was washed three times with TBS-T and incubated at room temperature for 1 hour with HRP-conjugated mouse secondary antibody (Promega) diluted in 5% skim milk in TBS-T. After washing with TBS-T, 1 mL of SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher) was applied to the membrane, and chemiluminescence was detected using an Amersham Imager 680 instrument (GE Healthcare).

[0180] Quantification and statistical analysis The number and diameter of drug-treated mouse and human organoids were manually counted and measured on days 0 and 3 or 6 post-treatment from four full-well images of each condition at 4x magnification. To count live or dead cells, organoids were stained with calcein AM for live cells and EthD-1 for dead cells three days after treatment, and the number of EthD-1 positive organoids was manually counted from images taken from three wells of each condition at 4x magnification. All experiments were performed in at least three consecutive sets. Mean values ​​from each condition were compared using Student's paired or unpaired t-tests with Graphpad Prism. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0181] result Pyrvinium selectively induces cell death in mouse dysplastic organoids. Using previously established metaplastic (Meta3) and dysplastic (Meta4) organoids, the combined drug effects of pyrvinium, a putative casein kinase 1α (CK1α) activator, and trametinib, a MEK inhibitor, were evaluated in Meta3 and Meta4 organoids. Meta3 expressed the SPEM cell markers aquaporin 5 (AQP5) and CD44v9, while Meta4 organoids were specifically positive for the dysplastic cell markers TROP2 and AQP5 (Figure 1A). Meta3 organoids showed a significant reduction in size compared to control organoids treated with DMSO in response to trametinib, pyrvinium, and both (Figures 1B and 1D). In contrast, Meta4 organoids showed a variety of responses to trametinib and pyrvinium. Trametinib treatment suppressed only dysplastic organoid proliferation and induced a significant reduction in organoid diameter in one Meta4 lineage, Meta4_2 (Figures 1C and 1E). Other Meta4 organoid lineages did not show a difference in size after trametinib treatment, but the height of the organoid cells was thinner compared to control organoids and showed lower cellularity as shown in the H&E image (Figure 1C). Pyrvinium treatment resulted in widespread cell death in all three Meta4 lineages, as well as a reduction in organoid size, as shown by the numerous nuclear-enriched cells in the phase-contrast image (Figure 1C) and confirmed by live / dead cell staining (Figure 1F). Interestingly, combined treatment with trametinib and pyrvinium did not produce any additive or synergistic effects on Meta4 organoids (Figures 1C and 1E). In summary, these results suggest that pyrvinium can induce cell death in dysplastic organoids and inhibit the growth of metaplastic organoids, while MEK inhibition can only lead to growth arrest in both metaplastic and dysplastic organoids.

[0182] It was hypothesized that pyrvinium may have acted as a downstream inhibitor of the Wnt pathway, regulating the survival and maintenance of dysplastic cells by activating CK1α. To further evaluate whether CK1α activation affects the survival of dysplastic organoids, Meta4 was treated with SSTC3, a CK1α-specific activator. Surprisingly, SSTC3 did not induce growth inhibition or cell death in Meta4 organoids (Figure 1G). Wnt target genes were not downregulated after pyrvinium treatment, and the expression of several genes, including Axin2, Cdca4, Rnf43, and Znrf3, increased 24 hours after treatment (Figure 9). Thus, the results indicate that pyrvinium does not adversely affect the Wnt signaling pathway in dysplastic gastric cells.

[0183] Pyrvinium affects both the MAPK and STAT pathways in mouse metaplastic and dysplastic organoids. To identify which signaling pathways are regulated by pyrvinium in Meta4 organoids, phosphoarray analysis was performed using Meta3 and Meta4 organoids treated with either DMSO vehicle or pyrvinium. Correspondence analysis between Meta3 and Meta4 organoids with and without pyrvinium treatment showed differential profiles of protein phosphorylation, including STAT, ERK signaling-related transcription factors, protein kinase enzymes, NF-κB, and the MAPK pathway (Figure 2A). In particular, the most significant effect of pyrvinium treatment was the dephosphorylation of proteins related to the STAT pathway (Figure 2B). To identify potential proteins involved in pyrvinium-induced cell death in Meta4 organoids, it was hypothesized that candidate phosphoproteins upregulated in Meta4 organoids would show a significant decrease in phosphorylation upon pyrvinium treatment. P-MYC T358 p-PKCtheta T538 pC-Raf S43 p-STAT3 Y705 , p-Pyk2 T580 p-histone H3.1 S10 p-CREB T100, p-Pyk2 T881 p-SRF S77 , and p-ERK1 / 2(p44 / 42) T202 We selected the top 10 protein candidates, including those shown in Figures 2C-2D. Since the organoids were derived from Mist1-Kras mice, it is not surprising that many of the proteins on the list, with the exception of STAT3 (Figure 2E), are directly related to the MAPK and PKC signaling pathways. Therefore, these results suggest that STAT3 signaling is upregulated in dysplastic cells and may be a crucial pathway for dysplastic cell survival.

[0184] Pyrvinium induces cell death in dysplastic organoids through a double blockade of the ERK and STAT3 signaling pathways. Dysregulated STAT3 signaling pathways are strongly involved in tumorigenesis through their effects on cell proliferation, angiogenesis, immune system evasion, and prevention of apoptosis. Therefore, we investigated how pyrvinium modulates STAT3 activity in dysplastic organoids. STAT3 activation is typically regulated by the tyrosine residue (pSTAT3). Y705 ) or serine residue (p-STAT3 S727 It is induced by phosphorylation at either of the following: Phosphoarray data is obtained from STAT3 after pyrvinium treatment. Y705 Loss of phosphorylation was revealed. Western blot analysis showed that in Meta4 organoids, STAT3 Y705 A significant decrease in phosphorylation was observed, but STAT3 S727 This was not confirmed (Figures 3A-3B). Notably, a significant decrease in p-ERK levels was observed after pyrvinium treatment, consistent with the results of phosphoarray analysis, and trametinib also resulted in a significant decrease in p-ERK levels, as expected (Figures 3A-3B). These findings suggest that STAT3 is necessary to induce cell death in dysplastic organoids. Y705 This suggests that a dual inhibition of ERK phosphorylation is necessary. Meta3 organoids also show that STAT3 Y705This showed a reduction in phosphorylation of both STAT3 and ERK (Figure 3C). However, as mentioned above, STAT3 in Meta3 organoids Y705 The basal phosphorylation levels of the organoids were significantly lower than those of the Meta4 organoids (Figure 3C-3E), and the organoids did not show cell death.

[0185] Furthermore, Meta3 and Meta4 organoids were treated with three different STAT3-specific inhibitors, including STAT3-IN-1, Sttatic, and cryptotancinone (CPT). All STAT3-specific inhibitors did not affect the survival and growth of Meta3 organoids (Figures 10A-10B), indicating that pyrvinium-induced growth arrest was mediated solely by the suppression of MEK / ERK signaling in metaplastic cells. Two STAT3-specific inhibitors, STAT3-IN-1 and Sttatic, induced growth arrest in Meta4 organoids but did not result in cell death (Figures 3F-3G). In contrast, combination treatment with trametinib and STAT3-IN-1 resulted in significant cell death in Meta4 organoids, comparable to the effect of pyrvinium (Figures 3H-3I). SSTC3 treatment was associated with p-STAT3 Y705 Alternatively, it was confirmed that p-ERK expression was not reduced, supporting the idea that pyrvinium action does not mediated through CK1α activation (Figures 3J-3K). Furthermore, changes in STAT3-related genes in response to pyrvinium treatment were examined. The expression of several STAT3 target genes, including cyclin B1 and Pim-1, was downregulated after pyrvinium treatment (Figures 3L and 11), which was also confirmed at the protein level (Figure 3M). Pim-1 is overexpressed in various human cancers and protects cells from apoptosis. Therefore, downregulation of Pim-1 may be associated with the induction of cell death in dysplastic cells. Thus, the results suggest that dual inhibition of both the ERK and STAT3 signaling pathways is important for dysplastic cell survival.

[0186] Pyrvinium target CD133 + CD166 + dysplastic stem cells To comprehensively assess transcriptome changes and identify subpopulations of dysplastic organoids affected by pyrvinium treatment, single-cell RNA sequencing was performed using cells from Meta4 organoids treated with either DMSO or pyrvinium for 1 day before the organoids showed morphological disruption. Uniform manifold projection (UMAP) showed near-perfect segregation between DMSO-treated and pyrvinium-treated cells, illustrating the dramatic transcriptional changes induced by pyrvinium (Figure 4A). Five subpopulations were identified by unsupervised clustering combined with lineage-specific markers including Stmn1 and Pcna for proliferating cells, TFF3 for differentiated intestinal cells, CD133 and CD166 for DSCs, Mal2 and Lgals3 for SPEM cells, and Ddit3 and Areg for damaged cells (Figure 4B). Gene ontology analysis combined with PANTHER classification showed distinct gene expression profiles between groups (Figure 4C). Pyrvinium treatment resulted in increased catalytic and transporter activity, but decreased binding and transcriptional regulatory factor activity. Many subpopulations, including proliferative cells, SPEM cells, and DSCs, were found in DMSO-treated cells, but over 73.9% of cells in pyrvinium-treated cells were damaged and primarily differentiated (Figure 4D-4E). Volcano plots and UMAP analyses showed that several STAT3 target genes observed in DMSO-treated cells, e.g., Fos, Fosb, and Jun, were downregulated after pyrvinium treatment (Figure 4F-4G), while damage-related genes, e.g., Dusp4, Ddit3, and Areg, were upregulated in pyrvinium-treated cells (Figure 4F-4H). Furthermore, increased expression of both 7-AAD and annexin V was observed in pyrvinium-treated cells, supporting increased activity in necrosis and apoptosis in response to pyrvinium (Figure 4I). It is important to note that the DSC subpopulation was not observed after pyrvinium treatment. qRT-PCR analysis confirmed the decrease in CD133 and CD166 gene expression (Figure 4J), and FACS analysis also confirmed the decrease in CD133. + CD166 +A significant reduction in DSCs was demonstrated (Figure 4K). These findings indicate that pyrvinium specifically targets DSCs and proliferating cells, inducing cell death in dysplastic organoids.

[0187] Pyrvinium treatment inhibits metaplasia progression in the mouse stomach. Next, we investigated whether the STAT3 signaling pathway is upregulated during the progression of metaplasia to dysplasia in vivo. Three months after tamoxifen injection into Mist1-Kras mice, the stomach predominantly showed metaplastic glands, and by four months, the appearance of dysplastic glands gradually became dominant. Therefore, we treated mice three months after tamoxifen injection to evaluate whether pyrvinium could improve the progression of metaplasia to dysplasia (Figure 5A). Gastric mucosa was examined in each group of mice one or two weeks after either DMSO or pyrvinium treatment. In mice treated with pyrvinium, acid-secreting parietal cells gradually refilled the mucosa (Figures 5B-5C). Conversely, the number of hyperplastic vesicular glands with UEA1 and Ki-67-positive proliferating cells was significantly reduced, along with a significant decrease in the number of dysplastic glands (Figures 5F-5G) (Figures 5D-5E). Furthermore, pyrvinium treatment resulted in downregulation of p-STAT3 and Pim-1 (Figure 5H), as observed in dysplastic organoids (Figure 3L-3M). In addition, comprehensive examination of major organs in wild-type mice treated with either DMSO or pyrvinium was performed to confirm that pyrvinium did not induce significant histopathological abnormalities (Figure 12). We further explored the changes in the immunomicroenvironment after pyrvinium treatment. Mice treated with DMSO showed infiltration of various inflammatory cells, including lymphocytes and macrophages, in the lamina propria. Pyrvinium treatment was associated with CD4 + It induced a dramatic decrease in the T cell population (Figure 5I), but CD8 + T or CD19 +No significant difference was observed in B cells (Figures 5I and 5J). M2 macrophages have been shown to promote the progression of SPEM and IM, and both NK cells and CD163-positive M2 macrophages were significantly reduced in pyrvinium-treated mice (Figures 5K-5M). In pyrvinium-treated mice, CD3 + The total number of T cells decreased, but CD3 + CD25 + The relative ratio of regulatory T cells increased significantly (Figures 13A-13B), and the expression of bone marrow-derived suppressor cell (MDSC)-related markers tended to increase, but the changes were not statistically significant (Figure 14). These results suggest that pyrvinium effectively inhibits metaplasia progression to dysplasia and promotes the regeneration of normal gastric mucosa in vivo by contributing to an anti-inflammatory microenvironment.

[0188] Pyrvinium inhibits the growth and survival of human precancerous cells with dysplastic characteristics. Non-tumor mucosa surrounding gastric cancer is often known to have metaplastic or dysplastic glands with varying degrees of genetic or epigenetic abnormalities, a phenomenon known as "field carcinogenesis." To investigate the inhibitory effect of pyrvinium on the growth and survival of these human precancerous lesions, 20 human precancerous organoid (hPCO) lineages were established from gastric mucosa adjacent to gastric cancer obtained from surgical specimens. The hPCO lineages were characterized based on the morphological and expression profiles of AQP5 and CD44v9 as metaplastic cell markers, and TROP2 as a dysplastic cell marker (Figure 6E). After 6 days of treatment with trametinib, pyrvinium, and combinations thereof, organoid diameter was measured and organoid death was examined and classified by a score ranging from 1 to 4 (0 indicates immortality, while 1, 2, 3, and 4 correspond to 0-10%, 10-50%, 50-90%, and >90% dead organoids, respectively). Based on these results, the hPCO lineages were classified into three categories: susceptible (score 4), moderate (score 2 or 3), and resistant (score 0 or 1) (Figure 6E). Five pyrvinium-susceptible hPCO lineages showed significant organoid death, while nine pyrvinium-resistant hPCO lineages were mostly viable with little to no dead cells, and six pyrvinium-moderate lineages showed mild to moderate cell death (Figure 6 and Figures 15A-15C). Three representative images from the pyrvinium-susceptible hPCO lineages (hPCO-2, hPCO-3, and hPCO-34) are shown in Figure 6A, where organoid death was confirmed by live / dead cell staining using the hPCO-34 lineage (Figure 16). Pyrvinium induced organoid death in the pyrvinium-sensitive series, but combination therapy with trametinib did not show any additional effect, as observed in mouse dysplastic organoids (Figure 1C). Interestingly, the pyrvinium-sensitive series did not respond to trametinib and continued to grow for 6 days, in contrast to the growth inhibition observed in mouse organoids after trametinib treatment (Figures 6A-6B).Furthermore, pyrvinium-resistant hPCO lineages (hPCO-12, hPCO-19, and hPCO31) did not die, but showed growth cessation after treatment with pyrvinium and / or trametinib (Figures 6C-6D). Additionally, we evaluated whether drug responses in different human organoids were related to their histological and molecular characteristics (Figure 6E). Notably, pyrvinium-sensitive hPCO lineages showed a positive correlation with dysplastic morphology and high levels of TROP2 expression, but a negative correlation with AQP5 and CD44v9 expression (Figures 6F-6H). In summary, these findings suggest that the pyrvinium effect is significantly related to the dysplastic characteristics of the hPCO lineage.

[0189] Pyrvinium blocks both the ERK and STAT3 signaling pathways in human dysplastic cells. The relationship between pyrvinium responsiveness in the hPCO series and activation of the STAT3 and ERK signaling pathways was further investigated. Y705 The changes in the phosphorylation levels of p-STAT3 in three pyruvium-sensitive and pyruvium-tolerant hPCO lines after pyruvium treatment were investigated. Pyruvium treatment affected all three pyruvium-sensitive hPCO lines. Y705 Both STAT3 and p-ERK levels were reduced (Figures 7A-7B), but no significant difference was observed in the pyrvinium-resistant group (Figures 7C-7D). Similar to the findings in mouse organoids, STAT3 Y705Upregulation of phosphorylation was observed in pyrvinium-sensitive hPCO lineages compared to pyrvinium-resistant groups (Figures 7E-7G) and in gastric tissue used to establish hPCOs (Figure 7H). Furthermore, hPCO lineages were treated with three STAT3-specific inhibitors, and their effects on growth and survival in the hPCO lineages were evaluated. Two STAT3 inhibitors, namely Static or CPT (but not STAT3-IN-1), halted organoid proliferation in the pyrvinium-sensitive hPCO-2 lineage, but the organoids survived up to 6 days post-treatment (Figures 7I-7J). In contrast, pyrvinium-resistant hPCO-31 did not respond to any of the STAT3 inhibitors (Figures 7K-7L). Therefore, these results suggest that pyrvinium can effectively suppress hPCO growth and survival by simultaneously inhibiting both the STAT3 and ERK signaling pathways.

[0190] conclusion Long-term tissue damage can lead to the accumulation of genetic changes over time, ultimately causing multifocal development of precancerous lesions that result in multiple types of cancer in the stomach. Both severe metaplastic and dysplastic lesions are considered irreversible (points of no return). Therefore, developing chemotherapy prophylactic measures that directly target these precancerous lesions is essential. However, due to the inherent difficulties in studying the proliferation and progression of gastric precancerous cells in humans, little research has been done in this area. In this study, we used mouse and human precancerous organoids to define changes in signaling pathways between the metaplastic and dysplastic phases. We observed that the STAT3 signaling pathway was dysregulated in the dysplastic phase, which led to different drug responses to pyrvinium. It is important to note that pyrvinium is known as a Wnt pathway inhibitor by activating CK1a and regulating colon cancer cell survival. In addition, several other signaling pathways, such as PI3K, Hedgehog, and Hippo, can also be regulated by pyrvinium in various cancers. This study reports that pyrvinium can also control MEK / ERK phosphorylation levels, and that dual blockade of both STAT3 and MEK / ERK signaling pathways, rather than CK1a activation, leads to widespread cell death in dysplastic organoids. Therefore, the appropriate use of pyrvinium should be carefully considered based on its targets and functions in different organs and diseases.

[0191] Numerous data demonstrate the therapeutic effects of MEK inhibitors in various cancers. Previous and current studies have shown the inhibitory effect of trametinib, a MEK inhibitor, on the proliferation of mouse dysplastic cells. However, most hPCO lineages did not respond to trametinib, and hPCO lineages with dysplastic characteristics were even larger in size compared to vehicle-treated organoids. Human precancerous organoid lineages were established from heterogeneous tissue from gastric cancer patients and showed relatively low levels of ERK phosphorylation. These findings suggest that MEK inhibition alone may not be sufficient to control the progression of human precancerous cells. Previously, dysplastic stem cells, Trop2 + CD133 + CD166 + The cells were identified as being the first to be present at the dysplastic stage in both mice and humans. Single-cell RNA-seq data analysis revealed that pyrvinium is present in CD133 in dysplastic organoids. + / CD166 + It was revealed that pyrvinium specifically targets the stem cell population. Furthermore, pyrvinium reversed the progression of dysplasia in vivo in Mist1-Kras stomachs. Metaplasia did not progress to dysplasia by targeting dysplastic stem cells, and normal gastric cells, particularly parietal cells, were observed two weeks after pyrvinium treatment. Therefore, the drug response to pyrvinium in human precancerous lesions depends not only on the phosphorylation levels of both ERK and STAT3, but also on the presence of dysplastic stem cells. This study also provides compelling evidence for pyrvinium as a potential therapeutic candidate that can target the stem cell population and prevent the progression of gastric precancerous lesions to gastric cancer.

[0192] Furthermore, potent activation of the STAT3 signaling pathway upon MEK inhibition has been reported in KRAS-mutated cancer cells, demonstrating that dual inhibition of MEK and STAT3 is more effective in treating oncogene-toxic cells. Similarly, high phosphorylation levels of STAT3 were observed in both mouse and human dysplastic organoids, which responded strongly to pyrvinium. The dramatic anticancer activity of pyrvinium has been suggested to be mediated by its inhibitory effect on the STAT3 pathway in KRAS-mutated lung cancer and leukemia. However, in this study, treatment with STAT3 inhibitors alone showed varied responses compared to pyrvinium treatment and was far less effective in inducing cell death in dysplastic organoids. All these findings suggest that dual blockade of MEK and STAT3 may be necessary to treat gastric dysplasia. Unlike dysplastic lesions, most dysplastic changes are benign regenerative adaptations to severe damage. Pyrvinium treatment did not induce apoptosis in metaplastic cells, but only resulted in growth arrest in both mouse metaplastic organoids and metaplastic hPCOs, suggesting a low potential for adverse effects on tissue regeneration. However, the MEK inhibitor trametinib and the STAT3 inhibitor did not limit the proliferation of metaplastic cells in the hPCO lineage. Since gastric metaplasia is considered a precancerous lesion associated with the development of dysplasia, it is important to reduce the risk of excessive metaplasia without compromising its protective effects.

[0193] The development of direct pharmacological interventions targeting precancerous lesions represents a promising strategy for cancer prevention. However, identifying specific signaling pathways essential for high-risk precancerous lesions is one of the most challenging problems in this field. This study concludes that the MEK / ERK and STAT3 signaling pathways are differentially activated in gastric precancerous lesions. Furthermore, pyrvinium exerted a dual blockade of MEK / ERK and STAT3, inducing significant cell death in STAT3-dysregulated dysplastic cells. Taken together, the reuse of pyrvinium may offer a meaningful therapeutic opportunity for patients with gastric precancerous lesions.

Claims

1. A method for reprogramming precancerous mucosa to a noncancerous state, Identifying precancerous mucosa containing metaplastic or dysplastic cells, The process involves contacting the metaplastic cells or dysplastic cells with a compound of formula (I) or a salt thereof. 【Chemistry 1】 During the ceremony, R 1 However, C 1ー6 It is alkyl, R 2a and R 2b However, each is independent of C 1ー6 It is alkyl, R 3 is C 6ー12 aryl, and R 3 is optionally substituted with 1 to 5 R X and R X However, in each occurrence, independently, C 1ー6 Alkyl, C 1ー4 Haloalkyl, halogen, cyano, -N(R) Xa ) 2 , -OR Xb , -SR Xb , -C(O)R Xb , -CO 2 R Xb , -C(O)N(R Xa ) 2 , -SO 2 R Xa , -L 2 -Y 2 , -O-L X -Y X , -S-L X -Y X , or -N(R Xa )-L X -Y X And, L X However, in each occurrence, independently, C 1ー6 Alkylene, C 2ー6 Alkenylene, or C 2ー6 It is alkynylene, Y X However, in each appearance, independently, hydrogen, cyano, halogen, haloalkyl, -OH, -N(R) Xa ) 2 , -OR Xb , -SR Xb , -C(O)R Xb , -CO 2 R Xb , -C(O)N(R Xa ) 2 , or -SO 2 R Xa And, R Xa However, in each appearance, hydrogen and C appear independently. 1ー4 Alkyl, or -C(O)C 1ー4 It is alkyl, R Xb However, in each appearance, hydrogen and C appear independently. 1ー4 Alkyl, C 1ー2 Haloalkyl, or -C(O)C 1ー4 A method that is alkyl.

2. The method according to claim 1, wherein the metaplastic cells or dysplastic cells are contacted with a salt of the compound of formula (I).

3. A salt of the compound of formula (I) is given by formula (I-A), 【Chemistry 2】 The method according to claim 2, wherein the salt is a salt of the salt.

4. R 1 The method according to claim 1, wherein the substance is methyl.

5. R 2a and R 2b The method according to claim 1, wherein each of them is methyl.

6. R 3 The method according to claim 1, wherein the phenyl is unsubstituted.

7. The method according to claim 1, wherein the compound of formula (I) is pyrvinium.

8. The method according to claim 1, wherein the metaplastic cells or dysplastic cells are located in a living organism.

9. The method according to claim 1, wherein the precancerous mucosa includes dysplastic cells.

10. The method according to claim 9, wherein the method comprises killing the dysplastic cells.

11. The method according to claim 9, wherein the dysplastic cells are positive for CD133 expression, CD166 expression, and / or Trop2 expression.

12. The method according to claim 9, wherein the dysplastic cells are dysplastic gastric cells, dysplastic esophageal cells, dysplastic pancreatic cells, dysplastic colon cells, or dysplastic ovarian cells.

13. The method according to claim 12, wherein the dysplastic gastric cells are part of an organoid.

14. The method according to claim 1, wherein the precancerous mucosa includes metaplastic cells.

15. The method according to claim 14, wherein the metaplastic gastric cells are positive for CD133, CD166, and / or Trop2 expression.

16. The method according to claim 14, wherein the metaplastic cells are metaplastic gastric cells, metaplastic esophageal cells, metaplastic pancreatic cells, metaplastic colonic cells, or metaplastic ovarian cells.

17. The method according to claim 16, wherein the metaplastic gastric cells are part of an organoid.

18. A method for reducing the risk of cancer development in a living organism containing metaplastic or dysplastic cells, The procedure involves administering a compound of formula (I) or a salt thereof to the living organism. 【Transformation 3】 During the ceremony, R 1 However, C 1ー6 It is alkyl, R 2a and R 2b However, each is independent of C 1ー6 It is alkyl, R 3 However, C 6ー12 It is aryl, R 3 However, you can choose 1 to 5 Rs at will. X Replaced by, R X is, in each occurrence, independently, C 1ー6 alkyl, C 1ー4 haloalkyl, halogen, cyano, -N(R Xa ) 2 , -OR Xb , -SR Xb , -C(O)R Xb , -COR 2 R Xb , -C(O)N(R Xa ) 2 , -SO 2 R Xa , -L 2 -Y 2 , -O-L X -Y X , -S-L X -Y X , or -N(R Xa )-L X -Y X wherein L X However, in each occurrence, independently, C 1ー6 Alkylene, C 2ー6 Alkenylene, or C 2ー6 It is alkynylene, Y X is, in each occurrence, independently, hydrogen, cyano, halogen, haloalkyl, -OH, -N(R Xa ), 2 , -OR Xb , -SR Xb , -C(O)R Xb , -COR 2 R Xb , -C(O)N(R Xa ), 2 , or -SO 2 R Xa and R Xa However, in each appearance, hydrogen and C appear independently. 1ー4 Alkyl, or -C(O)C 1ー4 It is alkyl, R Xb However, in each appearance, hydrogen and C appear independently. 1ー4 Alkyl, C 1ー2 Haloalkyl, or -C(O)C 1ー4 A method that is alkyl.

19. The method according to claim 18, wherein a salt of the compound of formula (I) is administered to the living organism.

20. A salt of the compound of formula (I) is given by formula (I-A), 【Chemistry 4】 The method according to claim 19, wherein the salt is a salt of the salt.

21. The method according to claim 18, wherein the compound of formula (I) is pyrvinium.

22. The method according to claim 18, wherein the method inhibits the STAT3 and MEK / ERK signaling pathway in the metaplastic cells or dysplastic cells.

23. The method according to claim 18, wherein the organism is a non-human animal.

24. The method according to claim 18, wherein the organism is a human.

25. The method according to claim 24, wherein a person is at risk of developing cancer.

26. The method according to claim 18, further comprising administering a second drug to the living organism.

27. The method according to claim 26, wherein the second agent comprises a kinase inhibitor.

28. The method according to claim 27, wherein the kinase inhibitor includes a MEK inhibitor.

29. The method according to claim 28, wherein the MEK inhibitor comprises trametinib, binimetinib, cobimetinib, and / or selumetinib.

30. The use of pyrvinium or its salts in the manufacture of pharmaceuticals for reprogramming precancerous mucosa into a noncancerous state or for reducing the risk of developing cancer.

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