The TP508 Solution for GI Damage

Novel regenerative peptide TP508 mitigates radiation-induced gastrointestinal damage by activating stem cells and preserving crypt integrity

Carla Kantara 1, Stephanie M Moya 1, Courtney W Houchen 2, Shahid Umar 3, Robert L Ullrich 4, Pomila Singh 5, Darrell H Carney 1 6

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Abstract

In recent years, increasing threats of radiation exposure and nuclear disasters have become a significant concern for the United States and countries worldwide. Exposure to high doses of radiation triggers a number of potentially lethal effects. Among the most severe is the gastrointestinal (GI) toxicity syndrome caused by the destruction of the intestinal barrier, resulting in bacterial translocation, systemic bacteremia, sepsis, and death. The lack of effective radioprotective agents capable of mitigating radiation-induced damage has prompted a search for novel countermeasures that can mitigate the effects of radiation post exposure, accelerate tissue repair in radiation-exposed individuals, and prevent mortality. We report that a single injection of regenerative peptide TP508 (rusalatide acetate, Chrysalin) 24 h after lethal radiation exposure (9 Gy, LD100/15) appears to significantly increase survival and delay mortality by mitigating radiation-induced intestinal and colonic toxicity. TP508 treatment post exposure prevents the disintegration of GI crypts, stimulates the expression of adherens junction protein E-cadherin, activates crypt cell proliferation, and decreases apoptosis. TP508 post-exposure treatment also upregulates the expression of DCLK1 and LGR5 markers of stem cells that have been shown to be responsible for maintaining and regenerating intestinal crypts. Thus, TP508 appears to mitigate the effects of GI toxicity by activating radioresistant stem cells and increasing the stemness potential of crypts to maintain and restore intestinal integrity. These results suggest that TP508 may be an effective emergency nuclear countermeasure that could be delivered within 24 h post exposure to increase survival and delay mortality, giving victims time to reach clinical sites for advanced medical treatment.

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Figures

Figure 1
Figure 1. Effects of TP508 on gastrointestinal colonic crypts integrity post-radiation exposure (A) Representative images taken at 10x and 40x magnifications of intact colonic crypts harvested at 48h, 5 days and 9 days post-RT from mice treated with either Saline or TP508, 24h post-radiation (0Gy or 9Gy). (Bi-ii) Representative H&E staining of colonic crypts sections harvested at 48h, day 5 and 9 days post-RT, from mice treated with the indicated treatments. Inset illustrating H&E images from colonic crypts isolated 5 days post-RT is shown in the right hand panel. White arrows depict change in crypt lengths. (C) Bar graphs showing the percent change in crypt lengths normalized to the control (0Gy+Saline) group, isolated 48h, 5 days and 9 days post-RT, respectively. Data=Mean±SEM from 6 mice/group/3 experiments. *=P<0.05 vs 9Gy+Saline values.
Figure 2
Figure 2. TP508 increases the expression of adherens junction E-cadherin and decreases apoptosis in gastrointestinal crypts post-radiation exposure (Ai) Representative immunofluorescent images of colonic crypt sections harvested 48h, 5 and 9 days post-RT and stained for E-cadherin. (Aii) Bar graphs illustrating the % change in the number of E-cadherin positive cells per crypt normalized to the control group (0Gy+Saline). (Bi) Immunofluorescent staining of colonic crypts sections harvested at 48h, 5and 9 days post-RT from mice treated with the indicated treatments for apoptotic marker activated-caspase-3. (Bii) Bar graphs showing the percent change in the number of activated caspase-3 positive cells per crypt normalized to the control (0Gy+Saline) group, isolated 48h, 5 days and 9 days post-RT, respectively. Data=Mean±SEM from 6 mice/group/3 experiments. *=P<0.05 vs 9Gy+Saline values.
Figure 3
Figure 3. TP508 stimulates proliferation of gastrointestinal crypt cells post-radiation exposure (Ai) Representative images of colonic crypts sections harvested 48h, 5 and 9 days post-RT from mice treated with the indicated treatments were stained for PCNA. (Aii) Bar graphs showing the percent change in the number of PCNA positive cells per crypt normalized to the control (0Gy+Saline) group, isolated 48h, 5 days and 9 days post-RT, respectively. Data=Mean±SEM from 6 mice/group/3 experiments. *=P<0.05 vs 9Gy+Saline values. Ratio of control samples (0Gy+Saline) were arbitrarily assigned 100% values; ratios of treated samples were expressed as a % of the control group. *=P<0.05 vs control (9Gy+Saline) values.
Figure 4
Figure 4. TP508 increases the stemness and proliferative potential of intact colonic crypts post-radiation exposure while decreasing apoptosis (A) Western blot analysis demonstrating the expression of the indicated markers in Saline vs TP508 treated groups at 48h and 9 days post-RT. (Bi-ii) Mean±SEM of WB data from 4 mice/group/3 experiments, presented as % change in ratio of target protein/β-actin from samples collected 48h (i) and 9 days (ii) post-RT. Ratio of control samples (0Gy+Saline) were arbitrarily assigned 100% values; ratios of treated samples were expressed as a % of control. *=P<0.05 vs control (9Gy+Saline) values.
Figure 5
Figure 5. TP508 increases the number of DCLK1+ve cells in gastrointestinal crypts as early as 48h post-radiation exposure (Ai) Representative images of colonic crypt sections harvested 48h, 5 and 9 days post-RT were stained by IF for DCLK1. White arrows depict positive staining for DCLK1. (Aii) Bar graph illustrating the % change in the number of DCLK1+ve cells per colonic crypt normalized to the control group (0Gy+Saline), 48h, 5 and 9 days post-RT. *=P<0.05 vs corresponding control (9Gy+Saline) values.
Figure 6
Figure 6. TP508 significantly delays mortality and increases survival post-radiation exposure (A) Scatter plot graph depicting the day of death of mice treated with the indicated treatments (n=34). (B) Graph illustrating the percent survival of mice treated with the indicated treatments, monitored for a total of 30 days.

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References

  1. Singh VK, Ducey EJ, Brown DS, et al. A review of radiation countermeasure work ongoing at the Armed Forces Radiobiology Research Institute. Int J Radiat Biol. 2012;88(4):296–310. – PubMed
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