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Effect of 8 Gy irradiation on NO production in response to TP508 and VEGF. Panel A: Two-day post-confluent cultures of HCAEC were sham-irradiated (solid bars) or 8 Gy gamma irradiated (hatched bars). After 24 h, media was replaced with EBM containing 200 lM L-arginine and cells were treated for 1 h with saline (vehicle; white bars), TP508 (50 lg/ml; dark gray bars) or VEGF (50 ng/ml; light gray bars). Supernatants were collected and analyzed for NO, as described in Materials and Methods. To determine if TP508 could mitigate effects of radiation, HCAEC were treated 1 h postirradiation with TP508 (50 lg/ml; cross-hatched bars) and then assayed for NO production 24 h later, as described above. *P , 0.01 vs. saline alone; # P , 0.01 vs. VEGF-treated; and ^P , 0.05 vs. 8 Gy irradiated cells treated with VEGF without TP508. Panel B: Western blot analysis of eNOS expression from HCAEC 24 h after sham irradiation or 8 Gy irradiation and treated at 1 h postirradiation with saline (vehicle) or TP508 (50 lg/ml; TP). Blot was reprobed with GAPDH as protein loading control.
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There is increasing evidence that radiation-induced damage to endothelial cells and loss of endothelial function may contribute to both acute radiation syndromes and long-term effects of whole-body nuclear irradiation. Therefore, several drugs are being developed to mitigate the effects of nuclear radiation, most of these drugs will target and prot...
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... show in Fig. 1A, VEGF treatment of sham-irradiated HCAEC stimulated a fivefold increase in NO production. In the same sham-irradiated cells, TP508 treatment caused a ninefold increase in NO production. Irradiation (8 Gy) of HCAEC caused a complete loss of VEGF-stimulated NO production over sham-irradiated HCAEC when measured 24 h postirradiation (Fig. ...
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... show in Fig. 1A, VEGF treatment of sham-irradiated HCAEC stimulated a fivefold increase in NO production. In the same sham-irradiated cells, TP508 treatment caused a ninefold increase in NO production. Irradiation (8 Gy) of HCAEC caused a complete loss of VEGF-stimulated NO production over sham-irradiated HCAEC when measured 24 h postirradiation (Fig. 1A). In contrast, when 8 Gy irradiated cells were TP508 treated, NO production was still significantly stimulated (.sixfold) at 24 h postirradiation. Thus, TP508 treatment was able to stimulate NO production in the irradiated cells, while VEGF treatment did not stimulate NO ...
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... then investigated whether TP508 treatment of cells 1 h postirradiation could prevent or diminish radiation- induced attenuation of NO production in response to VEGF or TP508. As shown in Fig. 1A, when cells were treated with TP508 1 h postirradiation (8 Gy), VEGF stimulation of NO production measured at 24 h postirradiation was partially restored relative to saline-alone treated cells, resulting in a significant increase in NO production (2.8 fold over controls). Moreover, in these cells that were treated with TP508 1 h ...
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... that irradiation of HCAEC may have reduced eNOS expression or the activation of eNOS and that TP508 treatment 1 h postirradiation, mitigated or diminished this radiation-induced effect. Indeed, Western blot analysis showed that there was approximately a 50% reduction of eNOS protein expression in endothelial cells 24 h postirradiation (8 Gy) (Fig. 1B). However, TP508 treatment 1 h postirradiation (8 Gy) prevented the radiation-induced downregulation of eNOS (Fig. 1B). These results suggest that TP508 treatment reversed radiation- induced ED and loss of NO signaling by attenuating the downregulation of eNOS ...
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... postirradiation, mitigated or diminished this radiation-induced effect. Indeed, Western blot analysis showed that there was approximately a 50% reduction of eNOS protein expression in endothelial cells 24 h postirradiation (8 Gy) (Fig. 1B). However, TP508 treatment 1 h postirradiation (8 Gy) prevented the radiation-induced downregulation of eNOS (Fig. 1B). These results suggest that TP508 treatment reversed radiation- induced ED and loss of NO signaling by attenuating the downregulation of eNOS ...
Citations
... To establish a robust model amenable to systematic analysis, we first isolated mouse embryonic NSPCs (E12.5) and OPCs (E17) from wildtype, outbred CD-1 mice that are an established model for brain and radiation research (38,48,49), and differentiated these ex vivo into the main neural cell types (27) outlined in Figure 1A. In this system, the cultures antigenically and morphologically recapitulated their in vivo counterparts when examined by immunofluorescence (for further rationale for using this model system, please see Supplemental Information). ...
Cranial irradiation is part of the standard of care for treating pediatric brain tumors. However, ionizing radiation can trigger serious long-term neurologic sequelae, including oligodendrocyte and brain white matter loss enabling neurocognitive decline in children surviving brain cancer. Oxidative stress-mediated oligodendrocyte precursor cell (OPC) radiosensitivity has been proposed as a possible explanation for this. Here, however, we demonstrate that antioxidants fail to improve OPC viability after irradiation, despite suppressing oxidative stress, suggesting an alternative etiology for OPC radiosensitivity. Using systematic approaches, we find that OPCs have higher irradiation-induced and endogenous γH2AX foci compared to neural stem cells, neurons, astrocytes and mature oligodendrocytes, and these correlate with replication-associated DNA double strand breakage. Furthermore, OPCs are reliant upon ATR kinase and Mre11 nuclease-dependent processes for viability, are more sensitive to drugs increasing replication fork collapse, and display synthetic lethality with PARP inhibitors after irradiation. This suggests an insufficiency for homology-mediated DNA repair in OPCs—a model that is supported by evidence of normal RPA but reduced RAD51 filament formation at resected lesions in irradiated OPCs. We therefore propose a DNA repair-centric mechanism of OPC radiosensitivity, involving chronically-elevated replication stress combined with ‘bottlenecks’ in RAD51-dependent DNA repair that together reduce radiation resilience.
... The peptide is capable of increasing vascularization processes both grossly and microscopically compared with wounds treated only with saline. These effects are caused by enhancing the number and average size of vessels as well as developing their functional properties [45][46][47]. After applying the TP508, the healing processes of radiationinduced injuries improved. ...
... Furthermore, the action of TP508 is completely independent to PDGF and TGF-β [46]. This peptide can be used for protection of intestinal mucosa against radiation-induced damage by improving crypt stem cell proliferation and maintaining the integrity of crypts and the gastrointestinal barrier; furthermore, it can be applied as radiation mitigators 24 h after radiation exposure (9 Gy, LD100/15) [47]. Mitigative effects of TP508 inhibit disintegration of gastrointestinal crypts, improve the length of crypt, stimulate the expression of adheres junction protein E-cadherin, activate the crypt cell proliferation and finally reduce the apoptosis [47]. ...
... This peptide can be used for protection of intestinal mucosa against radiation-induced damage by improving crypt stem cell proliferation and maintaining the integrity of crypts and the gastrointestinal barrier; furthermore, it can be applied as radiation mitigators 24 h after radiation exposure (9 Gy, LD100/15) [47]. Mitigative effects of TP508 inhibit disintegration of gastrointestinal crypts, improve the length of crypt, stimulate the expression of adheres junction protein E-cadherin, activate the crypt cell proliferation and finally reduce the apoptosis [47]. Further, when TP508 is applied as postradiation treatment in mice stimulates the stem cell hematopoietic processes in bone marrow and prevents gastrointestinal damage [47]. ...
Radioprotection is the process whereby biological systems are aided against undesirable radiation hazards. Primitive radioprotectors suffered from either having crucial side effects or low efficacy in clinical applications. Therefore, the search for less toxic but more capable radioprotectants has continued for decades. Peptides have been investigated as radioprotectants in a variety of preclinical models both in vitro and in vivo. Peptides exert their influence through scavenging free radicals, modifying cell signaling and inhibiting cell apoptosis. Demonstrating potential in vivo properties, peptide radiation countermeasures might find enough credit for use in humans in the future. This article reviews the potential therapeutic value of currently known radioprotective peptides and attempts to provide a comprehensive source for further scientific research in this area.
... TP508 stimulates cell signaling and endothelial cell nitric oxide (NO) production to activate progenitor stem cells, stimulate regeneration and revascularization, and mitigate effects of radiation. Its mode of action includes restoration of endothelial function and vascular epidermal growth factor, NOdependent signaling, reducing ischemia and decreasing inflammation (162,163), stimulation of progenitor stem cell proliferation (164), and increasing survival and maintenance of GI crypt integrity (165,166). An RCI animal model was also developed by Chrysalis, in which mice received either 3 or 8 Gy of gamma radiation, followed by full-dermal excisional wounds 24 h later. ...
Many cases of human exposures to high-dose radiation have been documented, including individuals exposed during the detonation of atomic bombs in Hiroshima and Nagasaki, nuclear power plant disasters (e.g., Chernobyl), as well as industrial and medical accidents. For many of these exposures, injuries to the skin have been present and have played a significant role in the progression of the injuries and survivability from the radiation exposure. There are also instances of radiation-induced skin complications in routine clinical radiotherapy and radiation diagnostic imaging procedures. In response to the threat of a radiological or nuclear mass casualty incident, the U.S. Department of Health and Human Services tasked the National Institute of Allergy and Infectious Diseases (NIAID) with identifying and funding early- to mid-stage medical countermeasure (MCM) development to treat radiation-induced injuries, including those to the skin. To appropriately assess the severity of radiation-induced skin injuries and determine efficacy of different approaches to mitigate/treat them, it is necessary to develop animal models that appropriately simulate what is seen in humans who have been exposed. In addition, it is important to understand the techniques that are used in other clinical indications (e.g., thermal burns, diabetic ulcers, etc.) to accurately assess the extent of skin injury and progression of healing. For these reasons, the NIAID partnered with two other U.S. Government funding and regulatory agencies, the Biomedical Advanced Research and Development Authority (BARDA) and the Food and Drug Administration (FDA), to identify state-of-the-art methods in assessment of skin injuries, explore animal models to better understand radiation-induced cutaneous damage and investigate treatment approaches. A two-day workshop was convened in May 2019 highlighting talks from 28 subject matter experts across five scientific sessions. This report provides an overview of information that was presented and the subsequent guided discussions.
... Des études précliniques soulignent que l'administration de la prothrombine permet une revascularisation et une régénération des tissus notamment intestinaux suite à une irradiation en corps entier chez la souris (Kantara et al., 2015;Olszewska-Pazdrak et al., 2016). ...
Malgré l’évolution de la radiothérapie (RT), la toxicité aux tissus sains reste une limite en clinique. Les mesures d’Efficacité Biologique Relative (EBR) permettent de prédire les effets biologiques d’un rayonnement d’intérêt par rapport à celui de référence. Elles sont principalement basées sur le test de survie clonogénique qui ne peut suffire à lui seul à prédire le devenir de tissus sains exposés. Les nouveaux appareils de RT utilisent des débits de dose plus élevés sans que les effets biologiques soient bien connus. Le but de ces travaux est d’acquérir des mesures biologiques multiparamétriques à intégrer dans un futur modèle prédictif pour mieux prédire les effets biologiques des protocoles de RT émergents. Pour les irradiations (IR) en dose unique, la modélisation des données in vitro a mis en évidence un effet plus délétère du débit de dose le plus élevé sur la survie clonogénique, la morphologie, la viabilité et le cycle cellulaire, la sénescence et l’expression de gènes signant une dysfonction cellulaire. Ces résultats ont été confirmés in vivo sur un modèle d’IR intestinale. Contrairement au postulat de la CIPR, l’EBR des photons n’est pas de 1 et dépend du débit de dose. Pour les IR fractionnées selon différents protocoles, un impact du débit de dose sur un continuum de “dose biologique équivalente” (BED) a également été démontré in vitro. En revanche, la réponse in vitro et in vivo est différente pour des protocoles à BED équivalente ce qui montre une limite son utilisation pour comparer des protocoles. L’utilisation de mesures biologiques multiples pourrait permettre à terme de mieux prédire les risques potentiels des pratiques actuelles et futures en RT.
... The public Internet search yielded 3 published abstracts and 6 manuscripts that were germane to the aims of the SBIR contracts, details of which are provided in Figure 1. Current progress and pertinent phase 1 and 2 milestones for the SBIR-funded research contracts that advanced to phase 2 are summarized in Table 2. [4][5][6][7][8][9][10][11][12] In the overall cohort of SBIR contracts for protectors and mitigators, the rate of transitioning from phase 1 to phase 2 funding was 42% (5 of 12). All 3 groups that submitted Fast-Track applications successfully completed phase 1 milestones and transitioned to SBIR phase 2 funding, whereas 22% (2 of 9) of the standard phase 1 contracts advanced to phase 2 funding. ...
... 13 In addition, on September 12, 2017, Humanetics Pharmaceuticals (Edina, MN) was awarded a US patent for BIO 300 for use in patients with cancer who had solid tumors. 12 Humanetics is also developing BIO 300 as a radiation countermeasure and a radioprotector for astronauts and has received funding from the National Institute of Allergy and Infectious Diseases (NIAID), the National Aeronautics and Space Administration, the Biomedical Advanced Research and Development Authority (BARDA), and the Department of Defense (DoD). ...
Purpose:
The use of radioprotectors and radiomitigators could improve the therapeutic index of radiation therapy. With the intention of accelerating translation of radiation-effect modulators (radioprotectors and mitigators), the Radiation Research Program and SBIR (Small Business Innovation Research) Development Center within the National Cancer Institute issued 4 Requests for Proposals (RFPs) from 2010 to 2013. Twelve SBIR contract awards in total were made in response to the 4 RFPs from September 2011 through September 2014. Here, we provide an update on the status of SBIR contract projects for the development of radiation-effect modulators.
Methods and materials:
To assess the status of research and development efforts under the 4 RFPs on radiation-effect modulators, we searched PubMed for research articles, google.com for published abstracts, clinicaltrials.gov for ongoing or completed clinical trials, and company websites for press releases and other news. All information obtained and reported here is publicly available and thus protects the intellectual property of the investigators and companies.
Results:
Of the 12 SBIR projects funded, 5 (42%) transitioned successfully from phase 1 to phase 2 SBIR funding, and among the Fast-Track contracts, this rate was 100% (3 of 3). The Internet search identified 3 abstracts and 6 publications related to the aims of the SBIR contracts. One-third of the companies (4 of 12) have successfully launched a total of 8 clinical trials to demonstrate the safety and efficacy of their investigational agents. Two drugs are in clinical trials for their indication as a radioprotector, and 2 drugs are under evaluation for their anticancer properties (an immunomodulator and a small molecule inhibitor).
Conclusions:
The National Cancer Institute's SBIR has provided pivotal funding to small businesses for the development of radioprotectors and radiomitigators, which resulted in multiple early-phase clinical trials. Longer follow-up is needed to determine the full impact of these novel therapeutics that enter clinical practice.
... The suggestion has been made that TP508 mitigates GI injury by activating radioresistant stem cells and increasing the stemness of intestinal crypts to restore and maintain GI integrity. One recent study confirmed the radiomitigative potential of TP508 when administered to CD-1 outbred mice 24 h after 8.5 Gy (0.458 Gy/min) 137 Cs c-irradiation (Olszewska-Pazdrak et al. 2016). This study, along with other data, suggests that increased survival following TP508 treatments may be due in part to the agent's effect on vascular endothelial cells. ...
Purpose: Terrorist attacks, with their intent to maximize psychological and economic damage as well as inflicting sickness and death on given targeted populations, are an ever-growing worldwide concern in government and public sectors as they become more frequent, violent, and sensational. If given the chance, it is likely that terrorists will use radiological or nuclear weapons. To thwart these sinister efforts, both physical and medical countermeasures against these weapons are currently being researched and developed so that they can be utilized by the first responders, military, and medical providers alike. This is the third article of a three-part series in which we have reviewed additional radiation countermeasures that are currently under early preclinical phases of development using largely animal models and have listed and discussed clinical support measures, including agents used for radiation-induced emesis, as well as countermeasures not requiring Food and Drug Administration approval.
Conclusions: Despite the significant progress that has been made in this area during the last several years, additional effort is needed in order to push promising new agents, currently under development, through the regulatory pipeline. This pipeline for new promising drugs appears to be unreasonably slow and cumbersome; possible reasons for this inefficiency are briefly discussed.
Significant and continued effort needs to be afforded to this research and development area, as to date, there is no approved radioprotector that can be administered prior to high dose radiation exposure. This represents a very significant, unmet medical need and a significant security issue. A large number of agents with potential to interact with different biological targets are under development. In the next few years, several additional radiation countermeasures will likely receive Food and Drug Administration approval, increasing treatment options for victims exposed to unwanted ionizing irradiation.
... Recent preclinical investigations demonstrate that TP508 also counteracts the effects of radiation exposure, possessing both radioprotective and radiomitigating properties (20,21). This suggests that TP508 may be used a nuclear countermeasure and to prevent radiation therapy damage to normal tissue (22,23). ...
... We therefore used male CD-1 outbred mice in a model of radiation combined with injury to determine whether the extended plasma half-life of the PEGylated TP508 derivatives results in enhanced wound healing as compared to TP508 when injected systemically. CD-1 mice were chosen because they have previously been used to demonstrate radiomitigation properties of TP508 (20,21). A Wound Zoom camera, which makes use of four positioning lasers (seen as red lighting at corners of images in Figs. 4 and 5) to determine scale, was used for wound imaging, and its onboard software was used to calculate wound areas based on the user-defined periphery of each wound. ...
... To determine whether the PEG20k-TP508 and PEG20k-Cys14-TP508 derivatives had lost biological activity due to PEGylation or were simply at concentrations outside the therapeutic window, the derivatives were also evaluated for radioprotective activity in vitro. Our previous studies have shown that TP508 accelerates DNA DSB repair in irradiated HDMECs (21). Therefore, we chose to evaluate DNA repair in HDMECs using the γ-H2AX assay for quantifying the repair of DNA DSBs. ...
TP508 is a synthetically derived tissue repair peptide that has previously demonstrated safety and potential efficacy in phase I/II clinical trials for the treatment of diabetic foot ulcers. Recent studies show that a single injection of TP508 administered 24 h after irradiation significantly increases survival and delays mortality in murine models of acute radiation mortality. Thus, TP508 is being developed as a potential nuclear countermeasure. Because of the short plasma half-life of TP508, we hypothesize that increasing the peptide bioavailability would increase TP508 efficacy or reduce the dosage required for therapeutic effects. We, therefore, evaluated the covalent attachment of various sizes of polyethylene glycol to TP508 at either its N-terminus or at an internal cysteine. A size-dependent increase in TP508 plasma half-life due to PEGylation was observed in blood samples from male CD-1 mice using fluorescently labeled TP508 and PEGylated TP508 derivatives. Biological activity of PEGylated TP508 derivatives was evaluated using a combination of biologically relevant assays for wound closure, angiogenesis, and DNA repair. PEG5k-TP508 enhanced wound closure after irradiation and enhanced angiogenic sprouting in murine aortic ring segments relative to equimolar dosages of TP508 without enhancing circulating half-life. PEG30k-TP508 extended the plasma half-life by approximately 19-fold while also showing enhanced biological activity. Intermediate-sized PEGylated TP508 derivatives had enhanced plasma half-life but were not active in vivo. Thus, increased half-life does not necessarily correlate with increased biological activity. Nevertheless, these results identify two candidates, PEG5k-TP508 and PEG30k-TP508, for potential development as second-generation TP508 injectable drugs.
Altered by ionizing radiation, the vascular network is considered as a prime target to limit normal tissue damage and improve tumour control in radiotherapy. Irradiation damages and/or activates endothelial cells, which then participate in the recruitment of circulating cells, especially by overexpressing cell adhesion molecules, but also by other as yet unknown mechanisms. Radiation-induced lesions are associated with infiltration of immune-inflammatory cells from the blood and/or the lymph circulation. Damaged cells from the tissues and immune-inflammatory resident cells release factors that attract cells from the circulation, leading to the restoration of tissue balance by fighting against infection, elimination of damaged cells and healing of the injured area. In normal tissues that surround the tumours, the development of an immune-inflammatory reaction in response to radiation-induced tissue injury can turn out to be chronic and deleterious for the organ concerned, potentially leading to fibrosis and/or necrosis of the irradiated area. Similarly, tumours can elicit an immune-inflammation reaction, which can be initialized and amplified by cancer therapy such as radiotherapy, although immune checkpoints often allow many cancers to be protected by inhibiting the T cell signal. Herein, we have explored the involvement of vascular endothelium in the fate of healthy tissues and tumours undergoing radiotherapy. This review also covers current investigations that take advantage of the radiation-induced response of the vasculature to spare healthy tissue and/or target tumours better.
