阅读说明：本技术 预防或治疗由治疗诱导的胃肠损伤的方法 (Method for preventing or treating gastrointestinal damage induced by therapy ) 是由 余健 张�林 B·莱博维茨 于 2020-02-03 设计创作，主要内容包括：本公开提供了用于预防或治疗由化疗或放疗诱导的胃肠损伤的组合物和方法。(The present disclosure provides compositions and methods for preventing or treating gastrointestinal damage induced by chemotherapy or radiation therapy.)

1. A method for preventing or treating gastrointestinal damage induced by chemotherapy or radiotherapy, the method comprising:

administering to the subject a therapeutically effective amount of a compound selected from the group consisting of:

wherein the subject has received, is receiving, or will receive chemotherapy or radiation therapy.

2. The method of claim 1, wherein the subject has cancer.

3. The method of claim 2, wherein the cancer is a cancer of the skin, breast, pancreas, prostate, ovary, kidney, esophagus, gastrointestinal tract, colon, brain, liver, lung, head, and/or neck.

4. The method of claim 1, wherein administering the compound inhibits the activity of p53 up-regulated modulator of apoptosis (PUMA) in at least one non-cancer cell, thereby preventing apoptosis of the non-cancer cell induced by the chemotherapy or radiation therapy.

5. The method of claim 4, wherein the non-cancer cell is LGR5⁺A stem cell.

6. The method of claim 1, wherein administration of the compound does not protect cancer cells from the chemotherapy or radiation therapy.

7. The method of claim 1, wherein the chemotherapy is selected from the group consisting of: irinotecan hydrochloride (CPT-11) and 5-fluorouracil (5-FU).

8. The method of claim 1, wherein the compound is deuterated.

9. A method for treating cancer in a subject, the method comprising:

administering chemotherapy or radiation therapy to the subject; and

administering to the subject a therapeutically effective amount of a compound selected from the group consisting of:

10. the method of claim 9, wherein administering the compound prevents or treats gastrointestinal damage induced by the chemotherapy or radiation therapy.

11. The method of claim 9, wherein the cancer is a cancer of the skin, breast, pancreas, prostate, ovary, kidney, esophagus, gastrointestinal tract, colon, brain, liver, lung, head, and/or neck.

12. The method of claim 9, wherein administering the compound inhibits PUMA activity in at least one non-cancer cell, thereby preventing apoptosis of the non-cancer cell induced by the chemotherapy or radiation therapy.

13. The method of claim 12, wherein the non-cancer cell is LGR5⁺A stem cell.

14. The method of claim 9, wherein administration of the compound does not protect cancer cells from the chemotherapy or radiation therapy.

15. The method of claim 9, wherein the chemotherapy is selected from the group consisting of: irinotecan hydrochloride (CPT-11) and 5-fluorouracil (5-FU).

16. The method of claim 9, wherein the compound is deuterated.

17. A method for screening for an agent for preventing or treating gastrointestinal damage induced by chemotherapy or radiotherapy, the method comprising:

treating organoids prepared from gastrointestinal tissue with chemotherapy or radiotherapy; and

exposing the organoid to a candidate agent to identify an agent that prevents apoptosis of the organoid.

18. The method of claim 17, wherein the organoid is a colon organoid.

19. The method of claim 17, wherein the gastrointestinal tissue is obtained from a human subject.

20. The method of claim 17, wherein the organoid comprises at least one cancer cell.

21. The method of claim 17, wherein the chemotherapy is selected from the group consisting of: irinotecan hydrochloride (CPT-11) and 5-fluorouracil (5-FU).

22. The method of claim 17, wherein the agent prevents or treats gastrointestinal damage induced by chemotherapy or radiation therapy in vivo.

Technical Field

The present disclosure relates generally to the fields of medicine, oncology, and cancer therapy. More particularly, the present disclosure relates to compounds, compositions and methods for preventing or treating gastrointestinal damage induced by chemotherapy or radiotherapy.

Background

Gastrointestinal (GI) side effects are the major dose-limiting factor in chemotherapy and abdominal radiotherapy, and can cause long-term complications in cancer survivors. For example, 5-fluorouracil (5-FU), which is commonly used for the treatment of colorectal cancer, is reported to cause chemotherapy-induced diarrhea in 50% of patients. Although radiation and most chemotherapeutic agents damage multiple tissue and organ systems, irinotecan hydrochloride (CPT-11), which is commonly used in combination with 5-FU to treat colorectal cancer patients, causes selective GI damage, with severe diarrhea and nausea occurring in more than 50% of patients. This acute toxicity is caused by: high concentrations of SN-38 (i.e., the active metabolite of CPT-11) accumulate in the intestine, and nontoxic SN-38 glucuronide is converted back to SN-38 by intestinal bacteria, followed by intestinal reabsorption. SN-38 is an inhibitor of topoisomerase I (i.e., a key enzyme in both DNA replication and RNA transcription) and causes replication stress and DNA damage in proliferating cells. Anti-diarrhea drugs may help alleviate CPT-11-induced diarrhea, but have limited efficacy in reducing long-term GI dysfunction. There are currently no U.S. FDA-approved agents for the prevention or treatment of CPT-11-induced GI injury or complications. Therefore, there is an urgent need to develop new therapeutic agents and methods for preventing or treating GI damage induced by chemotherapy or radiotherapy.

Disclosure of Invention

In one aspect, the present disclosure provides a compound capable of preventing or treating gastrointestinal damage induced by therapy (e.g., induced by chemotherapy or radiotherapy). In one embodiment, the compound is selected from the group consisting of:

in certain embodiments, the compound is deuterated.

In another aspect, the present disclosure provides a method for preventing or treating gastrointestinal damage induced by a treatment. In one embodiment, the method comprises administering to a subject a therapeutically effective amount of a compound described herein, wherein the subject has received, is receiving, or will receive chemotherapy or radiation therapy.

In certain embodiments, the subject has cancer. In some embodiments, the cancer is a cancer of the skin, breast, pancreas, prostate, ovary, kidney, esophagus, gastrointestinal tract, colon, brain, liver, lung, head, and/or neck.

In certain embodiments, administration of the compound inhibits the activity of p53 up-regulated modulator of apoptosis (PUMA) in at least one non-cancer cell, thereby preventing chemotherapy or radiation therapy-induced apoptosis of the non-cancer cell. In certain embodiments, the non-cancerous cell is LGR5⁺A stem cell.

In certain embodiments, administration of the compound does not protect the cancer cells from chemotherapy or radiation therapy.

In certain embodiments, the chemotherapy is selected from the group consisting of: irinotecan hydrochloride (CPT-11) and 5-fluorouracil (5-FU).

In another aspect, the present disclosure provides a method for treating cancer in a subject. In certain embodiments, the method comprises administering chemotherapy or radiation therapy to a subject and administering to the subject a therapeutically effective amount of a compound described herein.

In another aspect, the present disclosure provides a method for screening for an agent that prevents or treats damage induced by chemotherapy or radiotherapy. In one embodiment, the method comprises: treating the organoid with chemotherapy or radiotherapy; and exposing the organoid to a candidate agent to identify an agent that prevents organoid apoptosis.

In certain embodiments, the organoid is derived from a tissue from a human subject. In certain embodiments, the organoid is derived from gastrointestinal tissue. In one embodiment, the organoid is a colon organoid. In certain embodiments, the organoid is derived from cancerous tissue. In certain embodiments, the organoid comprises at least one cancer cell.

In certain embodiments, the identified agent prevents or treats gastrointestinal damage induced by chemotherapy or radiotherapy in vivo.

It is contemplated that any method or composition described herein can be practiced with reference to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

Drawings

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIGS. 1A-1F show that PUMA mediates CPT-11 induced intestinal injury. Mice with the indicated genotype were treated once or as indicated with CPT-11(200mg/kg) and analyzed at the indicated times. (FIG. 1A) representative image of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nicked end labeling (TUNEL) staining in the intestinal crypts at 6 hours. 4', 6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. Scale bar, 50 μm. (FIG. 1B) quantification of TUNEL + columnar cells (CBC) and +4 to 9 cells at the bottom of crypts in FIG. 1A. (FIG. 1C) at 6 hours, the expression of the indicated protein was analyzed by Western blotting. Lysates were pooled from the intestinal mucosa of three mice. Representative results are shown, and similar results were obtained in at least three independent experiments. (FIG. 1D) at 6 hours, Puma RNA in crypts was hybridized In Situ (ISH). Scale bar, 20 μm. In (fig. 1A) and (fig. 1D), arrows indicate CBCs at positions 1 to 3 below the panne cell (Paneth cell), and asterisks indicate +4 to 9 cells at positions 4 to 9 above the CBCs. In (fig. 1A) to (fig. 1D), each group of n-3 mice. In (FIG. 1B), the values are mean + SEM. P <0.001 (two-tailed student t-test), gene knock-out (KO) versus wild-type (WT). (FIG. 1E) survival of mice treated with three consecutive daily doses of CPT-11 (215 mg/kg per day) on days 0, 1 and 2. WT for Puma KO, P ═ 0.0004; WT for P53 KO, P0.382 (log rank test). (FIG. 1F) hematoxylin and eosin (H & E) staining of the small intestine from mice at day 5 treated as in (FIG. 1E). Scale bar, 200 μm.

FIGS. 2A-2F show that PUMAi protects against CPT-11 induced intestinal injury. Mice were treated with CPT-11 with or without a PUMA inhibitor (PUMAI). PUMAi (10mg/kg) or vehicle was administered 2 hours prior to CPT-11 or intraperitoneally as indicated. The small intestine or designated tissue is analyzed at designated times. (FIG. 2A) representative images of TUNEL staining in intestinal crypts of mice treated with CPT-11(200 mg/kg). DAPI was used for nuclear staining. Scale bar, 50 μm. Arrows indicate CBC, and asterisks indicate +4 to 9 cells. (FIG. 2B) quantification of TUNEL + CBC and transit expanded cells from (FIG. 2A). Inh, inhibitor. (FIG. 2C) tissue distribution of PUMAi at different times after a single injection. (FIG. 2D) quantification of TUNEL + crypt cells in mice 6 hours after CPT-11(200mg/kg) treatment. PUMAi (10mg/kg) was administered 2 hours before or 1 hour after CPT-11 treatment. Each group of n-3 mice (fig. 2B to 2D). In (fig. 2B) and (fig. 2D), the values are mean + SEM. P <0.05 (two-tailed student t-test). Veh, vehicle. (FIG. 2E) survival of WT mice after three doses of CPT-11 (215 mg/kg per day) (performed with the control in FIG. 1E). PUMAi (10mg/kg) was administered 2 hours prior to each CPT-11 dose. P ═ 0.0047 (log rank test). (FIG. 2F) H & E staining of the small intestine from mice treated in (FIG. 2E) at days 0, 5 and 30. Scale bar, 200 μm.

FIGS. 3A-3G show that PUMA deficiency protects tumor-bearing mice from chemotherapy-induced GI damage. WT and Puma KO mice bearing Lewis Lung Carcinoma (LLC) tumors were treated six times with CPT-11(200mg/kg) over 15 of days 11, 14, 17, 19, 23, and 25. Mice or tumors were analyzed at the indicated times. (FIG. 3A) tumor volumes were measured every other day from day 11 to day 25. CT, vehicle control. (fig. 3B) body mass is expressed as a percentage of body mass per mouse at day 11 from day 11 to day 25. (FIG. 3C) H of Small intestine tissue at day 25&And E, dyeing. Scale bar, 100 μm. (FIG. 3D) pile height from (FIG. 3C). A minimum of 40 villi from each mouse was measured. (FIG. 3E) quantification of intestinal crypts from each field of view (FIG. 3C). (FIG. 3F) intestinal neutrophils detected by immunofluorescence on day 25. DAPI was used for nuclear staining. Scale bar, 100 μm. (FIG. 3G) for each slide from the same slide as (FIG. 3F)Quantification of neutrophils in the visual field. In fig. 3A, 3B, 3D, 3E and 3G, the values are mean + SEM; each group n was assigned (fig. 3A and 3B) or 3 to 4 mice (fig. 3D, 3E and 3G). P<0.01 and P<0.001 (two-tailed student t-test). Puma KO, CPT and control (fig. 3A); CPT-11 treatment, Puma KO vs. WT (FIG. 3B).

FIGS. 4A-4G show that PUMAi protects tumor-bearing mice from chemotherapy-induced GI damage. On day 15 of 11, 14, 17, 19, 23 and 25, CPT-11(200 m) was administeredg/kg) six treatments were performed on WT mice bearing LLC tumors. Vehicle or PUMAI was administered 2 hours before and 20 hours after each CPT-11 dose. Mice or tumors were analyzed at the indicated times. (FIG. 4A) tumor volume was measured starting on day 11. (FIG. 4B) body mass is expressed as a percentage of body mass from day 11 to day 25 for each mouse at day 11. (FIG. 4C) H of Small intestine tissue on day 25&And E, dyeing. Scale bar, 100 μm. (FIG. 4D) fluff height from (FIG. 4C). A minimum of 40 villi from each mouse was measured. (E) Quantification of intestinal crypts from each field (fig. 4C). (FIG. 4F) intestinal neutrophils detected by immunofluorescence on day 25. DAPI was used for nuclear staining. Scale bar, 100 μm. (FIG. 4G) for each slide from the same slide as (FIG. 4F)Quantification of neutrophils in the visual field. In fig. 4A, 4B, 4D, 4E and 4G, the values are mean + SEM; each group n was assigned (fig. 4A and 4B) or 3 to 4 mice (fig. 4D, 4E and 4G). P<0.05，**P<0.01 and P<0.001 (two-tailed student t-test). PUMAi, CPT and control (fig. 4A); CPT-11 treated mice, PUMAi and CT (FIG. 4B).

FIGS. 5A-5G show that targeted PUMA protected LGR5⁺Free from CPT-11. WT and Puma KO mice were treated with CPT-11(200mg/kg) and analyzed at the indicated times. The PUMAi (10mg/kg) was administered 2 hours before CPT-11 treatment. (FIG. 5A) immunofluorescent staining for Green Fluorescent Protein (GFP) (LGR5) and TUNEL in the intestinal crypts. Scale bar, 50 μm. Arrows indicate double positive cells. (FIG. 5B) quantification of GFP/TUNEL double positive cells from each GFP positive crypt (FIG. 5A). (FIG. 5C) immunofluorescence staining of CD166 in intestinal crypts. Scale bar, 50 μm. (FIG. 5D) quantification of CD166 cells in the +4 to 9 region of crypts. (FIG. 5E) the indicated mRNA was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Complementary DNA (cDNA) was synthesized from RNA pooled from three mice. Values were normalized to Gapdh expression and expressed relative to the 0 hour control for each gene by itself. (FIG. 5F) immunofluorescence staining of 53BP1 in the intestinal crypts. Scale bar, 50 μm. (FIG. 5G) 6 and 48 hours after a single CPT-11 treatment, orQuantification of 53BP1+ crypt cells at three daily doses of CPT-11 (215 mg/kg daily) #120 hours as in FIG. 2E. In (fig. 5C) and (fig. 5F), the arrows indicate CBC, and the asterisks indicate +4 to 9 cells. In FIGS. 5A, 5C and 5F, DAPI was used for nuclear staining. In fig. 5B, 5E and 5G, the values are mean + SEM; each group of n-3 mice. P<0.05，**P<0.01 and P<0.001[ one-way analysis of variance (ANOVA), post-hoc test of basis (Tukey post test) for each time point]. In (fig. 5D), the values are mean + SEM; each group of n-3 mice. P<0.01 (two-tailed student's t-test).

FIGS. 6A-6F show LGR5 after targeting PUMA to prevent repeated CPT-11 exposure⁺Exhaustion of stem cells. WT and Puma KO mice carrying the Lgr5-EGFP-IRES-creERT2 marker allele were treated with CPT-11 and PUMAi for 2 weeks as in FIGS. 5A-5G and analyzed for intestinal phenotype. (FIG. 6A) GFP (LGR5) immunofluorescence in intestinal crypts. Arrows indicate LGR5⁺(GFP) crypts. Scale bar, 100 μm. (FIG. 6B) for a probe containing at least one GFP⁺(LGR5⁺) Quantification of intestinal crypts of cells. (FIG. 6C) Top: olfm4 RNA ISH in intestinal crypts. Scale bar, 50 μm. Asterisks indicate Olfm4+ crypt cells. Bottom: percentage of crypts containing at least one Olfm4+ cell. (FIG. 6D) MMP7 immunofluorescence in the intestinal crypts. Scale bar, 100 μm. (FIG. 6E) quantification of MMP7+ crypt cells at the base of the crypt (CBC region). (FIG. 6F) Top: GFP (LGR5) and MMP7 in the intestinal crypts immunofluorescence. Scale bar, 20 μm. Asterisk indicates LGR5⁺/MMP7⁺A pair of cells. Bottom: LGR5 for each recess⁺/MMP7⁺Quantification of cell pairs. In (FIG. 6A), (FIG. 6D) and (FIG. 6F), DAPI was used to stain nuclei. In (fig. 6B), (fig. 6C), (fig. 6E), and (fig. 6F), the values are mean + SEM; n-3 mice. P<0.05，**P<0.01 and P<0.001 (one-way ANOVA with graph-based post-hoc test).

FIGS. 7A-7H show that PUMAi protects mouse and human colon cultures from CPT-induced damage. (FIG. 7A) representative image of mouse colon organoids 6 days after CPT treatment. Organoids were treated with vehicle Control (CT) or 500nM CPT (day 1) in the presence or absence of 50 μ M PUMAi for 24 hours and growth was monitored until day 7. Scale bar, 500 μm. (FIG. 7B) quantification of organoids with a diameter of 100 μm or more from each field of view (FIG. 7A). (FIG. 7C) Western blot of active (cleaved) caspase-3 (Casp3) from organoids 24 hours after CPT treatment. (FIG. 7D) CPT treatment was followed by qRTPCR analysis for indicated mouse mRNA at 24 hours. NS, not significant. (FIG. 7E) representative images of human colon organoids processed as in (FIG. 7A). Scale bar, 500 μm. (FIG. 7F) quantification of organoids with a diameter of 100 μm or more from each field of view (FIG. 7E). (FIG. 7G) Western blot of active (cleaved) caspase-3 from human organs 24 hours after CPT treatment. (FIG. 7H) CPT treatment was followed 24 hours by analysis to assign human mRNA. In (fig. 7C) and (fig. 7G), lysates were prepared from three wells. Actin was used as a protein loading control. In (FIG. 7D) and (FIG. 7H), cDNA was synthesized from RNA pooled from three cultured wells. Values were normalized to Gapdh and expressed relative to vehicle control. In (fig. 7B), (fig. 7D), (fig. 7F), and (fig. 7H), the values are mean + SEM. P <0.05, P <0.01 and P <0.001 (one-way ANOVA with post-graph-based examination).

FIGS. 8A-8F show that PUMA KO inhibits CPT-11-induced crypt apoptosis. Mice were treated with a single dose of CPT-11(200mg/kg) and analyzed at the indicated times. (FIG. 8A) quantification of TUNEL + crypt cells in WT mice at the indicated times. (FIG. 8B) quantification of TUNEL + crypt cells at 6 h. (FIG. 8C) representative image of active caspase-3 staining in crypts at 6 h. Scale bar 25 μm. (D) Quantification of active caspase 3+ CBC and +4-9 cells at 6 h. (FIG. 8E) mRNA was assigned by qRT-PCR analysis. cDNA was synthesized from RNA pooled from 3 mice. (FIG. 8F) the indicated proteins at 6h were analyzed by Western blotting. Lysates were pooled from the intestinal mucosa of 3 mice. Representative results are shown, and similar results were obtained in at least three independent experiments. Fig. 8A, 8B, 8D, 8E, mean ± SEM, with n-3 mice per group. P < 0.01; p <0.001 (student t-test, two-tailed).

FIG. 9 shows that CPT-11 causes dose-dependent lethality in mice. Survival of WT mice treated with three consecutive daily doses of 180, 215 or 250 mg/kg/day CPT-11 on days 0, 1 and 2. Each group of n-10 mice. P values were calculated by log rank test.

FIGS. 10A-10E show that PUMAi does not protect colon cancer cells from CPT-11-induced apoptosis. FIG. 10A shows the structure of a PUMA inhibitor lead compound (PUMAI). (FIG. 10B) lysates of 293 cells transfected with individual plasmids for 24 hours were mixed in the indicated combinations. Lysates were incubated with vehicle or PUMA inhibitor (PUMAi, 25 μ M) for 15min, followed by immunoprecipitation with anti-HA antibodies. HA binding protein and inputs were analyzed by western blotting. (FIG. 10C) lysates of 293 cells transfected with individual plasmids for 24 hours were mixed in the indicated combinations. Lysates were incubated with vehicle or PUMA inhibitor (PUMAi, 25 μ M) for 15min, followed by immunoprecipitation with anti-HA antibodies. HA binding protein and inputs were analyzed by western blotting. (FIG. 10D) designated colon cancer cell lines were treated with camptothecin (CPT, 500nM) for 24 h. HCT116 cells and PUMA KO HCT116 cells have WT p53, the other cells do not have (KO) or have mutant p 53. Apoptosis was measured by nuclear fragmentation analysis. Values are mean + SEM; n is 3 independent experiments. (FIG. 10E) cells were treated as in D for 24h and analyzed by Western blotting. Representative results are shown, and similar results were obtained in at least three independent experiments.

FIGS. 11A-11F show that PUMAi inhibits CPT-11-induced CBC apoptosis. Mice were treated with a single dose of CPT-11(200mg/kg) and analyzed at 6 h. PUMAI was given 2h before CPT-11. (FIG. 11A) representative images of TUNEL staining in the intestinal crypts of WT mice treated as indicated. Scale bar 25 μm. (FIG. 11B) quantification of TUNEL + crypt cells at positions 1-3 (CBC) or 4-9. (FIG. 11C) representative image of active caspase-3 staining in intestinal crypts. Scale bar 25 μm. (FIG. 11D) quantification of active caspase-3 + crypt cells at positions 1-3 (CBC) or 4-9. (FIG. 11E) quantification of TUNEL + crypt cells. (FIG. 11F) schematic representation of the treatment and tissue collection time course of the PUMAi pharmacokinetic experiments. CPT-11 was administered at 0 h. B. D and E, values mean ± SEM, n ═ 3 mice per group. A and C, asterisks indicate TA cells. P < 0.01. P <0.05 (student t-test, two-tailed).

FIGS. 12A-12D show that PUMAi protects against chemotherapy and radiation-induced lethality. (FIG. 12A) and (FIG. 12B) survival of WT mice treated with 3 daily doses of 180 mg/kg/day (A) and 250 mg/kg/day (B) CPT-11 on days 0, 1 and 2, respectively. PUMAi (10mg/kg) was administered 2 hours before each CPT-11 dose and 20 hours after the final dose. (FIG. 12C) and (FIG. 12D) survival of WT mice after whole body irradiation (TBI) at 9.5Gy (FIG. 12C) and 15Gy (FIG. 12D). PUMAi (10mg/kg) was administered 30min before and 30min after TBI, and then daily for 4 days. Survivors under 9.5Gy TBI in C were sacrificed on day 40 and had no apparent health problems. P values were calculated by log rank test.

FIGS. 13A-13E show that targeting PUMA does not impair tumor response to CPT-11. (FIG. 13A) schematic representation of tumor establishment and treatment. Four million Lewis Lung Cancer (LLC) cells were injected subcutaneously in the flank of the mice. Tumors were allowed to establish for 11 days and mice were treated 6 times with 200mg/kg CPT-11(C) over 2 weeks. Pumai (p) was administered 11 times, each 2 hours before and 20 hours after administration of CPT-11. Mice were sacrificed 4 hours after the last administration of CPT-11 dose. (FIG. 13B) representative images of tumors collected two weeks after treatment. Scale bar 1 cm. (FIG. 13C) representative images of PCNA, TUNEL and active (lytic) caspase-3 staining in LLC tumors two weeks after CPT-11 treatment. Scale bar 100 μm. (FIG. 13D) quantification of PCNA +, TUNEL + and cleaved caspase-3 + cells for each field in C. Values are mean ± SEM, with n ═ 3 tumors per group. P < 0.01. P <0.001 (student t-test, two-tailed). (FIG. 13E) Western blot of indicated proteins from tumors as treated in A. Each lane represents a single tumor.

FIGS. 14A-14F show 5-FU induced LGR5⁺Apoptosis is PUMA dependent. WT and Puma KO LGR5-EGFP mice were treated with 200mg/kg 5-FU for 24 h. (FIG. 14A) representative images of TUNEL staining in intestinal crypts. Scale bar 25 μm. (FIG. 14B) quantification of TUNEL + crypt cells. (FIG. 14C) representative image of cleaved caspase-3 staining in intestinal crypts. Scale bar 25 μm. (FIG. 14D) quantification of active (lytic) caspase-3 + crypt cells. (FIG. 14E) LGR5(GFP)/TUNEL double immunization in intestinal cryptsRepresentative images of fluorescence. (FIG. 14F) quantification of GFP/TUNEL double positive cells in GFP positive crypts. B. D and F, values mean ± SEM, each group of n-3 mice. P<0.01 (student's t-test, two-tailed).

FIG. 15 shows that Puma KO and PUMAi inhibit CPT-11-induced expression of WNT and NOTCH targets. mRNA was assigned by qRT-PCR analysis. cDNA was synthesized from RNA pooled from 3 mice. The values for each gene were normalized to Gapdh and expressed relative to its own 0h control. P <0.05, P <0.01 (one-way ANOVA, graph-based post-hoc testing was performed separately for each time point).

Detailed Description

The following description of the present disclosure is intended to be merely illustrative of various embodiments of the present disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of the disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the disclosure, and it is to be understood that such equivalent embodiments are to be included herein. All references, including publications, patents, and patent applications, cited herein are hereby incorporated by reference in their entirety.

I. Definition of

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. In this disclosure, the term "or" is used to mean "and/or" unless explicitly indicated to refer only to alternatives or alternatives are mutually exclusive. As used herein, "another" may mean at least a second or more. Furthermore, the terms "include" and other forms of use, such as "includes" and "including," are not limiting. Furthermore, unless specifically stated otherwise, terms such as "element" or "component" encompass elements and components comprising one unit as well as elements and components comprising more than one subunit. Further, use of the term "portion" can include a portion of a portion or an entire portion.

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, the term "apoptosis" refers to a form of programmed cell death that occurs in a multicellular organism. Apoptosis is a highly regulated and controlled process that can be initiated by an intrinsic pathway when a cell senses cell pressure, or by an extrinsic pathway due to signals from other cells. During the apoptotic process, cells undergo a series of characteristic changes, including blebbing, cell contraction, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, and overall mRNA degradation.

As used herein, the term "cancer" refers to any disease involving abnormal cell growth, and includes all stages and all forms of disease affecting any tissue, organ or cell in the body. The term includes all known cancers and neoplastic conditions (whether characterized by malignancy, benign, soft tissue, or solid mass), as well as all stages and grades of cancer, including pre-and post-metastatic cancer. In general, cancers can be classified according to the tissue or organ in which the cancer is located or originates and the morphology of the cancerous tissue and cells. As used herein, cancer types include Acute Lymphoblastic Leukemia (ALL), acute myelogenous leukemia, adrenocortical carcinoma, anal carcinoma, astrocytoma, childhood cerebellar or cerebral basal cell carcinoma, bile duct carcinoma, bladder carcinoma, bone tumor, brain cancer, breast cancer, Burkitt's lymphoma (Burkitt's lymphoma), cerebellar astrocytoma, cerebral astrocytoma/glioblastoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, emphysema, endometrial cancer, ependymoma, esophageal cancer, Ewing's family tumor (Ewing's tumors), Ewing's sarcoma, gastric (gastic/stomach) cancer, glioma, head and neck cancer, cardiac cancer, Hodgkin's lymphoma (Hodgkin's lymphoma), islet cell carcinoma (pancreas), Kaposi sarcoma, kidney cancer (renal cell carcinoma), pancreatic islet cell carcinoma (endocrine carcinoma), Kaposi sarcoma, renal carcinoma (renal cell carcinoma), and methods of cancer, Laryngeal cancer, leukemia, liver cancer, lung cancer, medulloblastoma, melanoma, neuroblastoma, non-hodgkin's lymphoma, ovarian cancer, pancreatic cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), retinoblastoma, skin cancer, supratentorial primitive neuroectodermal tumors, testicular cancer, laryngeal cancer, thyroid cancer, vaginal cancer, optic way and hypothalamic glioma.

As used herein, a "cell" can be a prokaryotic or eukaryotic cell. Prokaryotic cells include, for example, bacteria. Eukaryotic cells include, for example, fungi, plant cells, and animal cells. Types of animal cells (e.g., mammalian cells or human cells) include, for example, cells from the circulatory/immune system or organ (e.g., B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, T helper cells), natural killer cells, granulocytes (e.g., basophils, eosinophils, neutrophils, and multilobal neutrophils), monocytes or macrophages, red blood cells (e.g., reticulocytes), mast cells, platelets or megakaryocytes, and dendritic cells); cells from the endocrine system or organ (e.g., thyroid cells (e.g., thyroid epithelial cells, parafollicular cells), parathyroid cells (e.g., parathyroid chief cells, eosinophils), adrenal cells (e.g., pheochromocytes), and pineal cells (e.g., pineal gland cells)); cells from the nervous system or organ (e.g., glial cells (e.g., astrocytes and oligodendrocytes), microglia, large cell neurosecretory cells, astrocytes, burtech cells, and pituitary cells (e.g., gonadotropic, corticotropic, thyroid, somatotropin, and prolactin)); cells from the respiratory system or organ (e.g., lung cells (type I and type II), clara cells, goblet cells, and alveolar macrophages); cells from the circulatory system or organ (e.g., cardiomyocytes and pericytes); cells from the digestive system or organ (e.g., gastral host cell, parietal cell, goblet cell, panne cell, G cell, D cell, ECL cell, I cell, K cell, S cell, enteroendocrine cell, enterochromaffin cell, APUD cell, and liver cell (e.g., hepatocyte and Kupffer cell)), (e.g., skeletal cells (e.g., osteoblasts, osteocytes, and osteoclasts), odontoblasts (e.g., cementoblasts and amelogues), chondrocytes (e.g., chondroblasts and chondrocytes), skin/hair cells (e.g., hair cells, keratinocytes, and melanocytes (nevus)), muscle cells (e.g., myocytes), adipocytes, fibroblasts, and tendon cells), and cells from the urinary system or organ (e.g., podocytes, pararenal cells, paraglomerular cells, endothelial cells, and endothelial cells, and endothelial cells, and endothelial cells, and endothelial cells, and endothelial cells, endothelial cells, Mesangial cells, renal proximal tubule brush border cells, and dense plaque cells); and cells from the reproductive system or organ (e.g., sperm, Sertoli cell, leydig cell, ovum, oocyte). The cell may be a normal healthy cell; or diseased or unhealthy cells (e.g., cancer cells). Cells further include mammalian zygotes or stem cells, including embryonic stem cells, fetal stem cells, induced pluripotent stem cells, and adult stem cells. A stem cell is a cell that is capable of undergoing a cell division cycle while maintaining an undifferentiated state and differentiating into a specialized cell type. The stem cell may be a pluripotent stem cell, a multipotent stem cell, an oligopotent stem cell, or a unipotent stem cell, any of which may be induced by a somatic cell. The stem cells may also include cancer stem cells. The mammalian cell can be a rodent cell, e.g., a mouse, rat, hamster cell. The mammalian cell may be a cell of the order Leporiformes, such as a rabbit cell. The mammalian cell can also be a primate cell, such as a human cell.

As used herein, the term "chemotherapy" refers to the treatment of cancer with one or more anti-cancer drugs. Anti-cancer drugs include, but are not limited to Avastin (Avastin), Bevacizumab (Bevacizumab), kemptorin (Camptosar) (irinotecan hydrochloride), Capecitabine (Capecitabine), Cetuximab (Cetuximab), cerazan (Cyramza), lexadine (Eloxatin) (Oxaliplatin)), Erbitux (Erbitux), 5-FU (fluorouracil), fossile (Fusilev) (calcium formyltetrahydrofolate), Ipilimumab (Ipilimumab), curitan (keyruda), lansfurf (lonesurf) (trifluridine and tipepidine hydrochloride), Nivolumab (Nivolumab) (oldivo (opsivo)), Panitumumab (panitumab), Pembrolizumab (pemetrolizumab), ramucirumab (ramuciflor), ramuciflor (ramuciflor), ravirkub (regia), argentum (regia).

As used herein, the term "organoid" refers to an in vitro three-dimensional culture that resembles a miniaturized and simplified organ and shows actual microdissection. Organoids are derived from one or several cells from a tissue, embryonic stem cells or induced pluripotent stem cells by self-renewal and differentiation.

As used herein, the term "radiotherapy" refers to a therapy that uses ionizing radiation to control or kill malignant cells. Radiotherapy is typically delivered by a linear accelerator. If multiple types of cancer are located in one region of the body, radiation therapy may be used to treat the cancer. Radiotherapy may also be used as part of adjuvant therapy to prevent tumor recurrence following surgery to remove the primary malignancy. Radiotherapy may act synergistically with chemotherapy and is used before, during and after chemotherapy of sensitive cancers.

As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Humans include prenatal and postnatal forms. In many embodiments, the subject is a human. The subject may be a patient, which is a person who is referring to a medical provider for disease diagnosis or treatment. The term "subject" is used interchangeably herein with "individual" or "patient". The subject may be suffering from or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

As used herein, the term "therapeutically effective amount" or "effective dose" refers to a dose or concentration of a drug effective to treat a disease or condition. For example, with respect to the use of the small molecule compounds disclosed herein to treat GI damage induced by chemotherapy or radiotherapy, a therapeutically effective amount is a dose or concentration of the compound capable of preventing or ameliorating GI damage induced by chemotherapy or radiotherapy.

As used herein, "treating" of a condition includes preventing or alleviating the condition, slowing the onset or rate of progression of the condition, reducing the risk of developing the condition, preventing or delaying the progression of symptoms associated with the condition, reducing or ending symptoms associated with the condition, producing a complete or partial regression of the condition, curing the condition, or some combination thereof.

Treatment of induced gastrointestinal injury

The Gastrointestinal (GI) epithelium is the fastest renewing adult tissue and is maintained by tissue-specific stem cells. Treatment-induced GI side effects are the major dose-limiting factor for chemotherapy and abdominal radiotherapy, and can reduce the quality of life of cancer patients and survivors. For example, 5-fluorouracil (5-FU), which is commonly used for the treatment of colorectal cancer, is reported to cause chemotherapy-induced diarrhea in 50% of patients. Although radiation and most chemotherapeutic agents damage multiple tissue and organ systems, irinotecan hydrochloride (CPT-11), which is commonly used in combination with 5-FU to treat colorectal cancer patients, causes selective GI damage, with severe diarrhea and nausea occurring in more than 50% of patients. This acute toxicity is caused by: high concentrations of SN-38, an active metabolite of CPT-11, accumulate in the intestine, and nontoxic SN-38 glucuronide is converted back to SN-38 by intestinal bacteria, followed by intestinal reabsorption. SN-38 is an inhibitor of topoisomerase I, a key enzyme in both DNA replication and RNA transcription, and causes replication stress and DNA damage in proliferating cells. Anti-diarrhea drugs may help alleviate CPT-11-induced diarrhea, but have limited efficacy in reducing long-term GI dysfunction. There are currently no U.S. FDA-approved agents for the prevention or treatment of CPT-11-induced GI injury or complications.

Chemotherapy or radiation-induced acute bowel disease is characterized by loss of proliferating crypt cells, epithelial barrier damage, and inflammation during or shortly after treatment. In many patients, late-onset bowel disease can occur months or later after therapy and is characterized by intestinal dysfunction associated with pathological changes in the epithelial and mesenchymal compartments, such as vascular sclerosis and progressive intestinal wall fibrosis. Emerging evidence suggests that chronic bowel injury may be the result of early toxicity. Enterotoxicity caused by chemotherapeutic drugs, such as CPT-11 and 5-FU, is associated with rapid crypt apoptosis in mice and humans. Small intestinal epithelial cells are the fastest renewing adult tissue, and Intestinal Stem Cells (ISCs) located at the bottom of crypts drive renewal and regeneration following injury. ISCs comprise columnar cells (CBCs) at the bottom of the crypt, and some cells (+4 cells) at position 4 relative to the bottom of the crypt. Gene ablation of G protein containing leucine rich repeats (heterotrimeric guanine nucleotide binding protein) coupled receptor 5 expression (LGR5+) CBC is well tolerated in healthy mice but strongly exacerbates radiation-induced intestinal injury, although the underlying mechanism is still unknown.

Activation of p53 following DNA damage leads to tissue and target specific consequences such as tumor suppression or acute toxicity. p53 activation is a double-edged sword that controls bowel regeneration after high dose radiation. In one aspect, p 53-dependent PUMA induction is responsible for most radiation-induced apoptosis and acute loss of intestinal and hematopoietic stem and progenitor cells, and PUMA deficiency or downregulation protects against radiation-induced lethality and lymphopoiesis. However, p 53-dependent induction of p21 and possibly other targets is critical for productive intestinal regeneration by preventing DNA damage accumulation, replication stress and delayed non-apoptotic cell death, and the lack of p53/p21 exacerbates intestinal damage. The role of p53 in chemotherapy-induced GI injury is not well understood. The p53 inhibitor, pivithrin) - α, reduced CPT-11-induced apoptosis in rats within the first few hours, but did not affect delayed cell death or mucositis onset.

Therapeutic compounds

As disclosed herein, the inventors surprisingly found that pharmacological inhibition of Puma, rather than p53 deficiency, by a panel of small molecule compounds provides potent protection against GI damage induced by single and repeated exposures to CPT-11, but does not compromise the in vivo anti-tumor activity of CPT-11. This protection and selective preservation of LGR5⁺Stem cells, stem cell microenvironment (niche), and genomic integrity are related.

In certain embodiments, the small molecule compound is selected from the group consisting of:

in certain embodiments, the above compounds are deuterated. As used herein, "deuterated compound" refers to a compound in which one or more of the hydrogen atoms has been replaced with a deuterium isotope. It will be appreciated that there is some variation in the abundance of natural isotopes in the synthesized compounds depending on the source of the chemical materials used in the synthesis. Thus, a preparation of a compound will inherently contain a small amount of deuterated compounds. The concentration of naturally abundant stable hydrogen and carbon isotopes is small and unimportant compared to the degree of stable isotopic substitution of deuterated compounds described herein. In the deuterated compounds described herein, when a particular position is designated as having deuterium, it is understood that the abundance of deuterium at that position is significantly greater than the natural abundance of deuterium (which is 0.015%). A position designated as having deuterium typically has a minimum isotopic enrichment factor of at least 3000 (45% deuterium incorporation), such as at least 3500 (52.5% deuterium incorporation), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation).

In certain embodiments, the deuterated compounds disclosed herein have the following structure:

wherein "D" refers to deuterium.

The synthesis of the small molecule compounds described herein can be readily accomplished by synthetic chemists of ordinary skill using methods known in the art. Such methods can be performed using the corresponding deuterated and optionally other isotopically-containing reagents and/or intermediates to synthesize the compounds described herein, or by resorting to standard synthetic protocols known in the art for introducing isotopic atoms into chemical structures.

Formulation and administration

The present disclosure provides pharmaceutical compositions for preventing or treating GI damage induced by treatment. Such compositions comprise a prophylactically or therapeutically effective amount of a compound disclosed herein and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia (u.s.pharmacopeia) or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Physiological saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

The compositions may also contain minor amounts of wetting or emulsifying agents or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable Pharmaceutical agents are described in Remington's Pharmaceutical Sciences. Such compositions will contain a prophylactically or therapeutically effective amount of the compound, together with a suitable amount of carrier so as to provide a form for proper administration to a patient. The formulation should be adapted to the mode of administration, which may be oral, intravenous, intra-arterial, intra-buccal, intranasal, nebulized, bronchoinhaled, or delivered by mechanical ventilation.

As described herein, the compounds of the present disclosure can be formulated for parenteral administration, e.g., formulated for injection by the intradermal, intravenous, intramuscular, subcutaneous, intratumoral, or even intraperitoneal routes. Alternatively, the compounds may be administered directly to the mucosa by a topical route, for example by nasal drops, inhalation or by nebuliser. Pharmaceutically acceptable salts include acid salts and salts formed with inorganic acids such as hydrochloric or phosphoric acids or with organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine (procaine), and the like.

Typically, the ingredients of the compositions of the present disclosure are supplied separately or mixed together in unit dosage form, e.g., as a dry lyophilized powder or water-free concentrate, in a sealed container (e.g., ampoule or sachet) that indicates the amount of active agent. In the case of administration of the composition by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. In administering the composition by injection, an ampoule of sterile water for injection or physiological saline may be provided so that the ingredients may be mixed prior to administration.

The compositions of the present disclosure may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like; and salts formed with cations such as those derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The compounds or pharmaceutical compositions disclosed herein can be administered to a subject in a therapeutically effective amount such that treatment-induced damage is prevented or treated. In certain embodiments, a therapeutically effective amount is from about 0.001 to about 10mg/kg of body weight, wherein the composition is administered once a day, twice a day, three times a day, once a week, twice a week, three times a week, or once every two weeks. In certain embodiments, the effective amount is about 0.01mg/kg body weight, 0.1mg/kg body weight, about 0.5mg/kg body weight, about 1mg/kg body weight, about 1.5mg/kg body weight, about 2mg/kg body weight, about 2.5mg/kg body weight, about 3mg/kg body weight, about 3.5mg/kg body weight, about 4mg/kg body weight, about 4.5mg/kg body weight, about 5mg/kg body weight, about 5.5mg/kg body weight, or about 6mg/kg body weight. In certain embodiments, the unit dose of the composition is between about 0.001mg to about 10mg (e.g., about 0.005mg, about 0.01mg, about 0.05mg, about 0.1mg, about 0.5mg, about 1mg, about 2mg, about 3mg, about 4mg, about 5mg, about 6mg, about 7mg, about 8mg, about 9mg, about 10 mg). In certain embodiments, the effective amount is 0.01mg/kg body weight, 0.1mg/kg body weight, 0.5mg/kg body weight, or 1mg/kg body weight, wherein the composition is administered once a day. In certain embodiments, the effective amount is 5mg/kg body weight, wherein the composition is administered once a week.

V. examples

The following examples are included to demonstrate illustrative embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute only illustrative modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 materials and methods

Compound screening and preparation

Candidate PUMA inhibitor structures and analogs were identified using in silico screening of the ZINC 8.0 database and in silico ADME/toxicity profiles as previously described (Mustata g, et al current topics of medicinal chemistry (Curr Top Med Chem) (2011)11: 281-. Compounds related to PUMAi were obtained from commercial suppliers.

Design of research

The goal of this study was to determine whether PUMA and p 53-dependent apoptosis mediated chemotherapy-induced intestinal stem cell loss and intestinal disease, and whether its genetic and pharmacological inhibition provided gut chemoprotection. The effect of Puma inhibition on intestinal injury and regeneration, as well as tumor response following chemotherapy, was measured using non-tumor-bearing and tumor-bearing mice, p53 and Puma KO mice, small molecule PUMAi, and in vitro mouse and human colon organoids. Sample size was determined using the disclosed work and test efficacy calculations. During data acquisition, the experimenter was blinded to the treatment groups.

Mice and treatments

All Animal experimental procedures were approved by the Institutional Animal Care and Use Committee (Institutional Animal Care and Use Committee) of the University of Pittsburgh. Mice were generated internally by heterozygote breeding for Puma +/+ (WT) and Puma-/- (Puma KO) (52). As described (52), WT and Puma KO alleles were genotyped from genomic DNA isolated from tail snips (tail snip). p53-/- (p53 KO) mice were purchased from Jackson laboratories (Jackson Laboratory). LGR5 has been described to label mouse Lgr5-EGFP (Lgr5-EGFP-IRES-creERT2) (53). All lines were in the C57BL/6 background or had been back crossed (backcross) with it for more than 10 generations (F10). Mice were housed in miniature isolation cages in rooms illuminated at 07:00 to 19:00 hours (12:12 hours light/dark cycle), with water and food ad libitum. Unless otherwise indicated, 200mg/kg CPT-11 (irinotecan hydrochloride; piotupol, Pfizer) in physiological saline was administered by intraperitoneal injection (200. mu.l for 20g mice). PUMAi was custom synthesized by chemcridge with a minimum purity of 95% according to HPLC/Mass Spectrometry (MS), prepared as a stock solution in dimethyl sulfoxide (50mg/ml), followed by fresh dilution in Phosphate Buffered Saline (PBS) (2mg/ml), and given intraperitoneally at 10mg/kg (200 μ Ι for 20g mice).

For tumor experiments, 400 million LLC cells [ American Type Culture Collection (ATCC) ] were injected into the flanks of WT and Puma KO mice and allowed to grow for 11 days. Mice were then treated three times weekly with CPT-11(200mg/kg) for 2 weeks. Either PUMAi (10mg/kg) or vehicle control was administered by intraperitoneal injection 2 hours before and 20 hours after each dose of CPT-11. Mice were sacrificed 4 hours after the final CPT-11 treatment.

Unless otherwise indicated, CPT-11 (irinotecan hydrochloride; topotecan, pyroxene) was treated with an i.p. administration of 200 mg/kg. Unless otherwise indicated, PUMA inhibitor (PUMAi) was prepared as a 50mg/ml stock in DMSO, then freshly diluted to 2mg/ml in PBS and given at 10mg/kg i.p. 2 hours prior to chemotherapy treatment. For survival experiments, mice were treated with CPT-11 for three consecutive days. PUMAi was administered 4 times, each 2 hours before and 22 hours after the last chemotherapy.

For tumor experiments, mice were treated three times weekly with CPT-11 for two weeks. Mice receiving PUMAI were treated 2 hours before and 20 hours after each CPT-11 dose. Mice were sacrificed four hours after the final CPT-11 treatment (fig. 13A). Tumor volume was measured with a caliper and calculated as volume (length × width 2)/2. Mice are 7-9 weeks old, and approximately equal numbers of male and female animals are used. To account for initial weight changes, the values in fig. 3 and 4 are expressed as a percentage of the corresponding mouse weight at the first CPT-11 treatment.

5-Fluorouracil (5-FU) (APP Pharmaceuticals) treatment 200mg/kg i.p.. For the whole body irradiation experiment, PUMAi (10mg/kg) was administered 30 minutes before and 30 minutes after irradiation, followed by daily administration for four days.

Tissue processing and histological analysis

Immediately after sacrifice, approximately 10cm sections of jejunum were removed and carefully rinsed with ice cold saline. The tissues were opened longitudinally and fixed to foam plates for fixation in 10% formalin overnight. The tissue was then rolled up into a "swiss roll" and embedded in paraffin. For tumor analysis, flank tumors were removed, measured, and sectioned for formalin fixation or snap frozen in a dry ice/ethanol bath. Tissue sections (5 μm) were deparaffinized and rehydrated through gradient ethanol. Histological analysis was performed by hematoxylin and eosin (H & E) staining.

The mean villus height was determined by measuring 40 to 50 villi from different sites of the small intestine of at least three different animals per group and reported as the meanSEM. Use of～H&The E-images and SPOT 5.1Advanced software (Diagnostic Instruments Inc.) were measured from the top of the crypt to the tip of the villus.

Immunohistochemistry and immunofluorescence

Reconstituted sections were treated with 3% hydrogen peroxide (immunochemistry only) followed by antigen retrieval in boiling 0.1M citrate buffer (pH 6.0) containing 1mM EDTA for 10 min. Apoptosis was analyzed by TUNEL staining using the ApopTag peroxidase in situ apoptosis detection kit (Chemicon International) according to the manufacturer's instructions.

Active caspase 3 IHC. Sections were dewaxed and rehydrated through gradient ethanol. The rehydrated slices were then treated with 3% hydrogen peroxide followed by antigen retrieval in boiling 0.1M citrate buffer (pH 6.0) containing 1mM EDTA for 10 minutes. Nonspecific antibody binding was blocked for 30 minutes at room temperature using 20% goat serum (Invitrogen). Sections were incubated overnight in a humidified chamber at 4 ℃ with 1:100 dilution of rabbit anti-caspase-3 (cleaved, Asp 175) (9661; Cell Signaling Technologies). The sections were then incubated with biotin-labeled goat anti-rabbit secondary antibody (# 31822; Pierce) for 1 hour at room temperature and developed with ABC kit and DAB (vector laboratories).

LGR5(GFP) IF. Sections were dewaxed and rehydrated through gradient ethanol. Antigen retrieval was performed by boiling for 10 minutes in 0.1M citrate buffer (pH 6.0) containing 1mM EDTA. Nonspecific antibody binding was blocked for 30min at room temperature using 20% goat serum (invitrogen). Sections were incubated overnight at 4 ℃ in a humidified chamber with 1:50 diluted mouse anti-GFP (sc-9996; Santa Cruz Biotechnology). The sections were then incubated with AlexaFluor 488-conjugated goat anti-mouse secondary antibody (1: 200; AA 11001; Invitrogen) for 1 hour at room temperature (58). Sections were then washed in PBS and mounted with VectaShield + dapi (vector labs).

LGR5(GFP)/TUNEL IF. Sections were prepared as described above. Nonspecific antibody binding was blocked for 30min at room temperature using 20% goat serum (invitrogen). Sections were incubated overnight at 4 ℃ in a humidified chamber with 1:50 diluted mouse anti-GFP (sc-9996; Santa Cruz Biotechnology). Sections were then incubated with AlexaFluor594 conjugated goat anti-mouse secondary antibody (1: 200; AA 11005; Invitrogen) for 1 hour at room temperature. Sections were then washed in PBS and TUNEL staining was performed with ApopTag fluorescein in situ apoptosis detection kit (Chemicon) according to the manufacturer's instructions (37, 46).

MMP7 IF. Sections were prepared as described above. Nonspecific antibody binding was blocked for 30min at room temperature using 20% chicken serum (invitrogen). Sections were washed in PBS and incubated overnight in a humidified chamber at 4 ℃ with 1:100 dilution of goat anti-MMP 7(AF 2967; Andy biologies (R & D Systems)). Sections were then incubated with AlexaFluor594 conjugated rabbit anti-goat secondary antibody (1: 200; AA 11080; Invitrogen) for 1 hour at room temperature. Sections were then washed in PBS and mounted with VectaShield + dapi (vector labs) for visualization.

LGR5(GFP)/MMP7 IF. Sections were prepared as described above. Nonspecific antibody binding was blocked for 30min at room temperature using 20% chicken serum (invitrogen). The sections were then stained for GFP as described above, followed by staining for MMP7 as described.

PCNA IF. Sections were prepared as described above. Nonspecific antibody binding was blocked for 30min at room temperature using 20% goat serum (invitrogen). The sections were incubated overnight at 4 ℃ in a humidified chamber with 1:100 dilution of mouse anti-PCNA (sc-56; Santa Cruz). Sections were then incubated with AlexaFluor594 conjugated goat anti-mouse secondary antibody (1: 200; Invitrogen) for 1 hour at room temperature. Sections were then washed in PBS and mounted with VectaShield + dapi (vector labs).

CD166 IF. Sections were prepared as described above. Nonspecific antibody binding was blocked for 30min at room temperature using 20% rabbit serum (Pierce). Sections were incubated overnight at 4 ℃ in a humidified chamber with goat anti-mouse CD166 antibody (1: 100; AF 1172; Andy Biopsis). Sections were then incubated with AlexaFluor594 rabbit anti-goat secondary antibody (1: 200; A11080; Invitrogen) for 1 hour at room temperature and counterstained with VectaShield plus DAPI (59).

p53 BP1 IF: sections were prepared as described above. Nonspecific antibody binding was blocked for 30min at room temperature using 20% goat serum (invitrogen). Sections were incubated overnight in a humidified chamber at 4 ℃ with rabbit anti-mouse 53BP1 antibody (1: 100; IHC-00001; Bethy Laboratories). Sections were then incubated with AlexaFluor594 goat anti-rabbit secondary antibody (1: 200; A11012; Invitrogen) for 1 hour at room temperature and counterstained with VectaShield plus DAPI.

Neutrophil IF. Sections were prepared as described above. Nonspecific antibody binding was blocked for 30min at room temperature using 20% goat serum (invitrogen). Sections were incubated overnight at 4 ℃ in a humidified chamber with rat anti-mouse Ly-6B.2(1: 100; MCA771 GT; AbD Serotec) (56). Sections were then incubated with AlexaFluor-594 goat anti-rat secondary antibody (1: 200; A11007; Invitrogen) for 1 hour at room temperature and counterstained with VectaShield plus DAPI.

LC-MS/MS quantification of PUMAi

Acetonitrile and water (both HPLC grade) were purchased from Fisher Scientific (Fisher Scientific). Formic acid and trifluoroacetic acid were purchased from Sigma-Aldrich (Sigma-Aldrich). Stock solutions of the internal standards of PUMAI and [ D5] -PUMAI were prepared at 1mg/mL in DMSO, diluted 10-fold in acetonitrile, and then stored at-80 ℃. On the day of analysis, the solution was serially diluted (in 10-fold steps) in acetonitrile to obtain lower calibration working solutions of 0.01 and 0.001 mg/mL. These calibration working solutions were diluted in mouse plasma (Lampire Biological Laboratories, Inc.) to yield the following analyte concentrations: 10. 30, 50, 100, 300, 500, 1000, 3000 ng/mL. For each calibration series, null (containing 10ng/mL internal standard) and blank samples were also prepared from 50 μ L of Lampire plasma. Quality Control (QC) stock solutions were diluted in Lampire mouse plasma to generate the following QC samples: QC Low (QCL)20 ng/mL; QC at 200ng/mL (QCM) and QC high (QCH) at 2500 ng/mL.

To extract PUMAI, we added 10. mu.L of internal standard solution (1000ng/mL [ D5] -PUMAI in acetonitrile) to 50. mu.L of plasma sample in a microfuge tube. Add 250 μ Ι _ of acetonitrile and vortex the sample for 30 s. The sample was centrifuged at 12,000 Xg for five minutes and the resulting supernatant was evaporated to dryness under nitrogen in a 12X 75mm glass tube at 34 ℃. The dried residue was reconstituted with 100 μ L acetonitrile/water (v/v) and 10 μ L was injected into the LC-MS/MS system.

The LC-MS/MS system consisted of an Agilent 1100 autosampler and binary pump and a watt (Waters) Quattromicro tandem mass spectrometer. The method comprises the following steps of A: acetonitrile/0.1% formic acid (v/v) and B: liquid chromatography was performed on a gradient mobile phase of water/0.1% formic acid (v/v). The mobile phase was pumped at a flow rate of 0.3mL/min and separation was achieved using a Philomenex (Phenomenex) Luna 3 μm PFP (100a 150X 2mm) column. The gradient mobile phase was as follows: 0 to 5min, 15 percent of solvent A. Solvent a was then increased linearly to 80% at 12min and maintained at 80% until 16 min. At 16.1min, solvent a was reduced to 15% and maintained at 15% until 22 min. The operation time is 22 min.

The mass spectrometer was operated in ESI positive mode using MRM detection. The parameters of the mass spectrometer were as follows: capillary 1.0kV, taper hole voltage 40V, desolvation temperature 400 ℃, desolvation gas flow 550L/h, taper hole blowback gas flow 50L/h, LM and HM resolution 12, collision energy 25V, and air chamber pirani (pirani) pressure 1.5 x 10^-3Millibar. The monitored mass transition was m/z 331 for PUMAI>143 and for internal standard 345>157。

To assess recovery from plasma, we compared the area of control plasma plus 1000ng/mL of PUMAi prepared as described above with the area obtained with a pure solution of PUMAi in water. Intestinal mucosa was analyzed after dilution to within the calibration range in control plasma. The intestinal mucosa was collected by scraping from a10 cm jejunal section and stored at-80 ℃ until use. Three mice were used per treatment group.

In situ hybridization

ISH of Olfm4 was performed with RNAscope 2.0 browse kit (Advanced Cell Diagnostics) according to the manufacturer's instructions. Briefly, the tissue is sectioned atLower bake for 60min, followed by dewaxing in xylene, and passage through a gradientEthanol is rehydrated. The sections were then subjected to three pretreatment steps, probe hybridization, six amplification steps, development and counterstaining. ISH for Puma proceeds as previously described.

Western blotting method

Fresh mucosal scrape or minced tumor tissue was washed in 1ml ice-cold PBS and precipitated at 400 g. The pellet was resuspended in 700 μ l homogenization buffer (0.25M sucrose, 10mM Hepes and 1mM EGTA) supplemented with protease inhibitors (clomplete EDTA-free micro, Roche) and homogenized with 50 pestle strokes in a dunnes (Dounce) homogenizer. After removal by centrifugation at 16,000g, the protein concentration in the supernatant was determined by spectrophotometer (NanoDrop 2000, zemer Fisher Scientific).

Proteins (30 μ g) were separated by SDS-polyacrylamide gel electrophoresis using a NuPAGE system (Invitrogen) and transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore). Representative results are shown, and similar results were obtained in at least three independent experiments. Antibodies used included PUMA (ab9643, Ebos (Abcam)), p53, p21, Hemagglutinin (HA) (sc-6243, sc-397 and sc-805; Santa Cruz Biotechnology), V5(R960-25, Invitrogen), tubulin (CP06, Oncogene Science) and actin (A5541, Sigma-Aldrich).

Quantitative real-time polymerase chain reaction

Fresh mucosal scrapers from 10cm jejunum were washed in cold PBS, resuspended in 700 μ l RNA lysis buffer, and homogenized in a dunnes homogenizer. RNA was isolated using the Quick-RNA MiniPrep kit (Zymo Research) according to the manufacturer's instructions. As described, organoids were released from Matrigel (Matrigel) using a cell recovery solution (Corning) prior to RNA isolation. Complementary DNA was generated from 2. mu.g total RNA from mice or about 100ng total RNA from colon organoids and pooled from wells of three mice or three organoid cultures of each treatment group using SuperScript III reverse transcriptase (Invitrogen) and random primers. Gene expression was normalized to Gapdh. Representative results are shown, and similar results were obtained in at least three independent experiments. Details of mouse and human primers are found in tables 1 and 2.

TABLE 1 mouse specific primers for real-time reverse transcription polymerase chain reaction.

TABLE 2 human specific primers for real-time reverse transcription polymerase chain reaction.

Gene	Primer and method for producing the same	Sequence of
			LGR5	Forward direction	5′-AACAGTCCTGTGACTCAACTCAAG-3’
	Reverse direction	5’-TTAGAGACATGGGACAAATGCCAC-3’
			OLFM4	Forward direction	5′-CTGCCAGACACCACCTTTCC-3′
	Reverse direction	5′-CTCGAAGTCCAGTTCAGTGTAAG-3′
			CD44	Forward direction	5′-GACAAGTTTTGGTGGCACG-3’
	Reverse direction	5’-CACGTGGAATACACCTGCAA-3’
			ACTIN	Forward direction	5′-GACCTCACAGACTACCTCAT-3′
	Reverse direction	5′-AGACAGCACTGTGTTGGCTA-3′

Cells and treatments

HCT116, DLD1, SW480 and HT29 human colon cancer cells (ATCC) as well as HCT116 p53 KO and PUMAKO were maintained in McCoy's 5A medium (Invitrogen) supplemented with 10% extra-fetal bovine serum (HyClone), penicillin (100U/m1) and streptomycin (100. mu.g/ml) (Invitrogen) at 37 ℃ and 5% CO 2. Human embryonic kidney 293 cells (ATCC) were maintained in Dulbecco's modified Eagle's medium (Invitrogen) with the same supplements. For treatment, cells were seeded at approximately 30% density in 12-well plates for 24 hours, and then treated with CPT (sigma-aldrich). 25 μ M of PUMAI was added to the cells simultaneously with CPT.

Measurement of apoptosis

After treatment, the suspended and adherent cells were collected and placed in PBS containing 3.7% formaldehyde, 0.5% NP-40, and Hoechst (Hoechst)33258 (10. mu.g/ml) (Molecular Probes) for staining. Apoptosis was assessed by microscopic visualization of condensed and fragmented nuclei, as previously described (Yu J. et al, Proc Natl Acad Sci U S A (2003)100:1931-36, incorporated herein by reference). A minimum of 300 cells were analyzed in triplicate per treatment. Representative results are shown, and similar results were obtained in at least three independent experiments.

Immunoprecipitation

HEK293 cells were transfected with HA-tagged PUMA (HA-PUMA), HA-tagged inactive PUMA (Δ BH3), or V5-tagged Bcl-xL (V5-Bcl-xL) plasmids previously described using Lipofectamine (Lipofectamine)2000 according to the manufacturer's instructions. To test for PUMAI, 293 cell lysates containing HA-PUMA or HA-. DELTA.BH 3 were incubated with 25. mu.M PUMAI for 15min, followed by mixing with V5-Bcl-xL lysates for 1 h and immunoprecipitation with an anti-HA antibody (sc-805, Santa Cruz Biotechnology). BIM/Mcl-1 interaction was tested in a similar manner using lysates containing HA-BIM and V5-Mcl-1. Representative results are shown, and similar results were obtained in at least three independent experiments.

Measurement of PUMAI in tissue

PUMAi {1- [4- (2-hydroxyethyl) -1-piperazinyl ] -3- (2-naphthoxy) -2-propanol dihydrochloride } was synthesized by chemcridge with a purity of 95% according to HPLC-MS and the internal isotope standard PUMAih7(D5, five internal hydrogens replaced by deuterium) was synthesized by Alsachim with a purity of approximately 100% according to HPLC-MS. Nuclear magnetic resonance confirmed isotopic enrichment of 99.4% or more. Pooled plasma from untreated mice was used as control plasma. Detailed methods for liquid chromatography-tandem MS (LC-MS/MS) quantification of PUMAi are found in supplementary materials and methods.

Colon organoids of mouse and human

Preparation of WRN conditioned Medium

Mixing L-WRN cells (CRL-3276) were cultured in DMEM (invitrogen) supplemented with 1 xpicillin/streptomycin (invitrogen), 0.5mg/mL G418(ant-gn-1, InvivoGen), 0.5mg/mL hygromycin B (10687010, invitrogen), and 10% FBS (vol/vol) until confluent. Cells were then harvested with trypsin-EDTA (invitrogen) and split into DMEM supplemented with 1 x penicillin/streptomycin and 10% fbs (hyclone) (vol/vol) in three T75 cell culture flasks (Sarstedt) until the cells became over confluent. The cells were then plated with 5ml of primary medium (supplemented with 1 XPicillin/streptomycin, 2mM GlutaMAX (ThermoFisher)), 20% FBS [ vol/vol ]]Was washed with Advanced DMEM/F12 (Invitrogen)) and cultured in the same medium (15 ml/flask). Every 24 hours, primary medium (conditioned medium) was collected and fresh medium was added to the flask. Conditioned medium was centrifuged at 2,000g for 5min at room temperature, and the supernatant was decanted and stored in aliquots at-20 ℃ until use.

Mouse crypt isolation, organoids and treatment

Mouse crypt isolation, organoid development and passaging were performed as previously described with minor modifications. Briefly, the complete medium for mouse intestinal organoids was slightly modified as follows: advanced Dartbox Modified Eagle Medium (DMEM)/F12(12634-010, invitrogen) was supplemented with 100 units/ml penicillin/0.1 mg/ml streptomycin (invitrogen), 2mM GlutaMAX (sermer femtols), 20% (vol/vol) FBS (S11150, ATLANTA Biologicals) and WRN conditioned medium (50%, vol/vol). Freshly isolated crypts were incubated with the same medium plus 10. mu. M Y-27632(Y0503 Sigma) and 100. mu.g/ml Primocin (ant-pm-1, InvivoGen). For CPT and PUMAi treatment, gut organoids were passaged and reseeded in 25 μ L matrigel (356231, corning) in 24-well plates and treated 24 hours later with CPT (0.5 μ M, C9911, Sigma-Aldrich) and PUMAi (50 μ M). The medium containing CPT was replaced with complete medium containing PUMAi (50. mu.M). Three groups were set (treatment control, CPT only and CPT plus PUMAi). On day 6 after the CPT treatment, organoids with a diameter of 100 μm or more are listed. Similar results were obtained from at least three independent experiments, with three replicate wells in each experiment.

Human crypt isolation, organoid development and management

Surgically excised intestinal Tissue was obtained from the Health Science Tissue Bank (HSTB) at the UPMC sharyside hospital and the UPMC presbytean hospital. All samples were obtained with informed consent and approval from the Ethics Committee of university of pittsburgh (Ethics Committee). Human normal colon crypt isolation and organoid development was performed as previously described with modifications. Complete medium for normal human colon organoids contains high-grade DMEM/F12(12634-010, Invitrogen), it was supplemented with 1 XPcillin/streptomycin (15140-122, Invitrogen), 10mM HEPES (15630-106, Invitrogen), 2mM GlutaMAX (Invitrogen), 1 XB 27(17504-044, Invitrogen), 1 XN 2(17502-048, Invitrogen), 1mM N-acetylcysteine (A0737, Sigma), 10nM [ leu-15] -gastrin (G9145, Sigma), 10mM nicotinamide (N0636, Sigma), 10. mu.M SB 190(S7067, Sigma), 50ng/ml recombinant murine EGF (315-09, Peprotech), 0.5. mu. M A83-01(2939, Tocrisis Bioscience), 10nM PGE2(22-961-0, Tocrisis Bioscience) and 50% Wolv/Vol conditioned medium (Wolvol). Freshly isolated crypts were cultured with the same medium plus 10. mu. M Y-27632 and 100. mu.g/ml Primocin. On day 6 after the CPT treatment, organoids with a diameter of 100 μm or more are listed. Similar results were obtained from at least three independent experiments, with three replicate wells in each experiment.

Statistical analysis

Statistical analysis was performed using GraphPad Prism IV software. Multiple comparisons of responses were analyzed by one-way analysis of variance (ANOVA) and graph-based post-hoc tests, and comparisons between the two groups were performed by two-tailed unpaired t-tests. Survival data were analyzed by log rank test. Differences are considered significant if the chance of accidental differences is less than 5/100(P < 0.05). Sample size was determined using a combination of the disclosed work and assay performance calculations. For ANOVA, the test efficiency of the interactive test is calculated by constructing a two-way factorial design applied by a mixed linear growth model, and the required sample size is calculated. It is estimated that typically 5 to 10 per group will provide a standardized interaction of 80% assay potency test at 1.5 SD.

Example 2 PUMA-mediated chemotherapy-induced crypt apoptosis and lethality

To investigate the potential role of p 53-dependent apoptosis in chemotherapy-induced acute intestinal injury, the inventors treated wild-type (WT), p53 knock-out (KO) and Puma KO mice with a single dose of irinotecan (200 mg/kg; CPT-11). CPT-11 induced robust crypt apoptosis in WT mice, as measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) and active caspase-3 staining, which peaked at 6 hours and gradually decreased by 48 hours. Apoptosis was observed in both stem cells at the crypt floor (CBC at positions 1 to 3 relative to the crypt floor) and +4 to 9 cells (relative to the crypt floor), but apoptosis did not occur in p53 KO and Puma KO mice (fig. 1A and 1B, and fig. 8A-8D). CPT-11 treatment induced p53 and its targets PUMA and p21 within 6 hours in the intestinal mucosa of WT but not p53 KO mice (fig. 1C and 8E). Puma KO did not significantly affect p53 or p21 expression (fig. 1C). BIM and NOXA, the BH3 proteins only, were not induced by CPT-11 or affected by Puma KO (FIG. 8F). RNA In Situ Hybridization (ISH) confirmed that Puma mRNA was undetectable in the crypt of untreated WT mice and was strongly induced in CBCs and some +4 cells by CPT-11 treatment (fig. 1D). CPT-11 caused dose-dependent lethal GI injury in mice (24). To determine the sensitivity of C57BL/6J mice, survival was monitored after three consecutive daily doses of CPT-11. Doses of 180, 215 and 250mg/kg daily caused 30%, 90% and 100% mortality, respectively, and all deaths occurred between day 5 and day 8, characteristic of lethal GI injury (fig. 9). At a 90% lethal dose (LD90) of WT mice (215 mg/kg per day for 3 days), all Puma KO mice survived for 30 days, while all p53 KO mice died between day 6 and day 9 (FIG. 1E). Histological analysis confirmed lethal GI injury in WT and p53 KO mice at day 5, showing severe loss of crypts and villus shortening, a phenomenon that did not occur in KO mice (fig. 1F). Consistent with the dual role of p53 in intestinal recovery from radiation (20), our data demonstrates that selective inhibition of p 53-dependent induction of PUMA and apoptosis, but not p21 or other functions, results in intestinal chemoprotection.

Example 3 Small molecule PUMAI protects against chemotherapy-induced crypt apoptosis and lethality

The inventors identified a variety of potential PUMAi using a pharmacophore model of the key interaction of PUMA BH3 domain with BCL-2 family proteins, computerized compound screening, and apoptosis analysis. It was confirmed that one of these compounds (PUMAi) inhibited the interaction of PUMA/BCL-xL but not BIM/MCL-1 (FIGS. 10A-10C). In colon cancer cell lines with different p53 or PUMA genotypes, including p53 WT (HCT 116), null (p53 KO), mutant (HT29, SW480, and DLD1), or PUMA null (PUMA KO), PUMAi did not reduce Camptothecin (CPT) -induced apoptosis (fig. 10D). PUMA was induced by CPT treatment only in p53 WT HCT116 cells, but not any p 53-deficient cell line (fig. 10E). The basal amount of PUMA in HCT116 cells was much higher compared to very low or undetectable expression in mouse intestinal mucosa (fig. 1C and 1D) and in normal human small intestine and colon.

To examine the in vivo efficacy of PUMAI, WT mice were treated with vehicle or PUMAI (10mg/kg, empirical dose) 2 hours prior to a single dose of CPT-11(200mg/kg), and crypt apoptosis was analyzed 6 and 24 hours after CPT-11 administration. Compared to the transitional expansion (TA), +4 to 9 cells, the tubal and active caspase-3 staining decreased by PUMAi was preferentially in CBC (fig. 2A and 2B, and fig. 11A-11D). PUMAi treatment did not further reduce crypt apoptosis in Puma KO mice (fig. 11E). High Performance Liquid Chromatography (HPLC) analysis was then developed to assess PUMAi distribution in intestinal mucosa and plasma at three time points, i.e., -1.5, +1, and +6 hours of CPT-11 administration (0 hours) (fig. 11F). Compared to-1.5 hours, the PUMAi concentration remained 67% and 30% in the intestinal mucosa at +1 and +6 hours, respectively, but declined more rapidly in plasma (fig. 2C). CPT-11 is rapidly cleared from plasma with a half-life of approximately 36 min. The inventors found that pamai administered 1 hour after CPT-11 treatment was still effective in reducing apoptosis, indicating that pamai did not affect CPT-11 conversion or intestinal absorption (fig. 2D).

In addition, PUMAi reduced CPT-11-induced lethality at three different doses (fig. 2E and fig. 12A and 12B). Under LD90, PUMAi improved survival from 10% to 70%. Histological analysis confirmed a reduction in GI damage at day 5 in the PUMAi and Puma KO groups compared to WT mice (fig. 1F and 2F). Pamai also increased survival of 9.5 and 15 ge mice after systemic irradiation (fig. 12C and 12D), further supporting the idea of targeting p 53-dependent apoptosis rather than other functions to selectively reduce therapy-induced normal tissue toxicity.

Example 4 PUMA inhibition does not reduce the antitumor Activity of CPT-11

To determine whether PUMA inhibition provides selective gut chemoprotection, the inventors established subcutaneous Lewis Lung Cancer (LLC) tumors in immunocompetent WT and PUMA KO mice. After tumors reached an average of 100mm3 (day 11), mice received six administrations of CPT-11(200mg/kg, i.p.) over 2 weeks (FIG. 13A). Tumor engraftment and CPT-11 responses were indistinguishable in WT and Puma KO hosts (fig. 3A and 13B). In contrast, weight loss induced by CPT-11 was reduced in Puma KO mice (fig. 3B), associated with preserved intestinal structure and barrier apparent by higher villus height, crypt number, and lower neutrophil infiltration (fig. 3C-3G).

The effect of PUMAI was then determined in tumor-bearing mice. Either PUMAi (10mg/kg) or vehicle was administered 2 hours before and 20 hours after each CPT-11 dose. The PUMAi group showed comparable tumor responses (fig. 4A and 13B) compared to the vehicle group, but was highly resistant to CPT-11-induced weight loss, intestinal injury (fig. 4B-4G), and lethality (table 3). Furthermore, based on daily examination, the PUMAi or PUMA KO groups exhibited improved combing and physical activity throughout the study. LLC tumors in all three CPT-11 treated groups showed similar effects on proliferation or apoptosis (fig. 13C and 13D), but lack induction of p53, PUMA, or p21 (fig. 13E), consistent with the mutation p 53. These studies demonstrated that PUMA inhibition attenuated CPT-11-induced intestinal toxicity, but did not attenuate the establishment or therapeutic response of p53 mutant tumors in immunocompetent hosts.

Table 3 treatment-related lethality in LLC tumor experiments.

Genotype(s)	Treatment group	Mice that completed the treatment%
			WT	Vehicle control	100％(8/8)
WT	CPT-11	73％(8/11)
			PUMA KO	Vehicle control	100％(6/6)
PUMA KO	CPT-11	100％(6/6)
			WT	PUMAi	100％(5/5)
WT	CPT-11+PUMAi	100％(10/10)

Example 5 targeting PUMA strongly protected LGR5⁺Stem cells and microenvironment protection from chemotherapy

Little is known about the role of ISC microenvironment in injury-induced regeneration. The inventors tested whether the loss of CBC, but not the more chemosensitive TA cells, sent a "damage" signal through microenvironment "emptying" to activate the remaining stem cells. PUMAI provides a pharmacological means of testing this because of its choiceSexual blockade of CPT-11-induced CBC apoptosis (FIG. 2B). Using LGR5-EGFP (enhanced Green fluorescent protein) reporter mice, the inventors found that Puma KO or PUMAi, respectively, reduced CPT-11-induced LGR5⁺Apoptosis was greater than 90% or 70% (fig. 5A and 5B). Puma KO also blocks LGR5 induced by another commonly used chemotherapeutic drug, 5-FU⁺Apoptosis (fig. 14). CD166 is a WNT target that labels the steady-state ISC microenvironment, including CBC and pangolin cells at the bottom of the crypt. CPT-11 treatment induced significant expansion of CD166+ cells from 48 to 96 hours in WT mice, which was completely suppressed by 96 hours of PUMAi treatment (FIGS. 5C and 5D). This was preceded by a short but strong activation of WNT (Lgr5, CD44 and WNT3A) as early as 6 hours as well as NOTCH (Olfm4, Math1, Hes1, Hes5 and Dll1) responsive genes, which were largely suppressed in Puma KO and PUMAi treated mice (fig. 15). The only exception was TA manufacturer CD44, which was strongly suppressed by Puma KO but not by PUMAi (fig. 5E). The inventors then monitored crypt DNA damage caused by the 53BP1 foci, which was comparable in Puma KO, PUMAi and control at 6 hours. However, DNA damage in the Puma KO and PUMAi groups decreased 48 hours after the single dose of CPT-11, and decreased more 120 hours after the three doses of CPT-11 (FIGS. 5F and 5G). These data strongly suggest that LGR5 is present after chemotherapy⁺Cell loss disrupts the microenvironment and triggers WNT and Notch signaling and intestinal regeneration, and blocks LGR5⁺Cell loss may delay compensatory proliferation and improve DNA repair.

Example 6 LGR5⁺Cells are key targets in gut chemoprotection by PUMAi

The inventors further tested LGR5⁺WNT and NOTCH activation triggered by cell loss would likely sensitize the remaining stem cells to repeated exposure and stem cell depletion. LGR5 after six doses of CPT-11 treatment⁺Crypt cells and LGR5⁺Crypt fraction decreased by more than 90% in WT mice compared to only 40% to 60% in the Puma KO and PUMAi groups (fig. 6A and 6B). Olfm4 RNA ISH indicated a 93% decrease in Olfm4+ crypts in WT mice compared to 8% and 49% in Puma KO and PUMAi groups, respectively (fig. 6C). The Pan's cell is LGR5⁺Microenvironment of cells andMMP7 expression is characteristic. Repeated CPT-11 treatment, rather than single treatment, reduced MMP7+ cells within the crypts with significant dislocation by epithelial spreading, as well as MMP7+ and LGR5⁺Almost complete loss of close contact of cells, which was suppressed in the Puma KO and PUMAi groups (fig. 6D to 6F). Collectively, these data demonstrate LGR5⁺Cell depletion, which is the underlying key mechanism of chemotherapy-induced enterotoxicity, can be effectively targeted by PUMAi.

Example 7 protection of mouse and human Colon organoids from CPT-induced injury by PUMAi

To determine whether PUMAi acts directly on crypt cells, the inventors used the PUMA-dependent LGR5 previously used to exhibit radiation induction⁺Three-dimensional epithelial organoids or gut-like systems of apoptosis. CPT induced growth inhibition and caspase-3 activation in mouse colon organoids, which was strongly blocked by PUMAi (fig. 7A to 7C). PUMAi also inhibited CPT-induced WNT and NOTCH target (Lgr5, CD44, and Olfm4) activation (fig. 7D). Inhibition of the TA marker CD44 was less pronounced compared to Lgr5 or Olfm4, similar to the results observed by the inventors in vivo.

To demonstrate the transformation potential of PUMAi, the inventors used primary human colon organoids. PUMAi enhanced organoid growth (fig. 7E and 7F) and inhibited CPT-induced caspase-3 activation (fig. 7G). PUMAI inhibited CPT-induced WNT and NOTCH target expression (FIG. 7H), establishing p53 and PUMA-dependent LGR5⁺Cell loss triggers significant activation of ISC-related pathways within the epithelial compartment.

Example 8 additional Compounds that inhibit PUMA-induced apoptosis.

In addition to PUMAI, several compounds exhibiting inhibition of PUMA-induced apoptosis were identified using in silico screening of the ZINC 8.0 database and in silico ADME/toxicity profiles as previously described (Mustata G. et al, Current topics in medicinal chemistry (2011)11:281-290, incorporated herein by reference). The inventors also tested a group of PUMAi-related compounds for their ability to reduce PUMA-induced apoptosis in colon cancer cell lines DLD1 and HCT-116. The structures and apoptosis inhibitory effects of these additional compounds are listed in the table below.

TABLE 4 Structure and apoptosis inhibition of Compounds

All of the compositions and methods disclosed or claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of illustrative examples, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.