Brachytherapy at the nanoscale with protein functionalized and intrinsically radiolabeled [169Yb]Yb2O3 nanoseeds.

PURPOSE: Classical brachytherapy of solid malignant tumors is an invasive procedure which often results in an uneven dose distribution, while requiring surgical removal of sealed radioactive seed sources after a certain period of time. To circumvent these issues, we report the synthesis of intrinsically radiolabeled and gum Arabic glycoprotein functionalized [169Yb]Yb2O3 nanoseeds as a novel nanoscale brachytherapy agent, which could directly be administered via intratumoral injection for tumor therapy. METHODS: 169Yb (T½ = 32 days) was produced by neutron irradiation of enriched (15.2% in 168Yb) Yb2O3 target in a nuclear reactor, radiochemically converted to [169Yb]YbCl3 and used for nanoparticle (NP) synthesis. Intrinsically radiolabeled NP were synthesized by controlled hydrolysis of Yb3+ ions in gum Arabic glycoprotein medium. In vivo SPECT/CT imaging, autoradiography, and biodistribution studies were performed after intratumoral injection of radiolabeled NP in B16F10 tumor bearing C57BL/6 mice. Systematic tumor regression studies and histopathological analyses were performed to demonstrate therapeutic efficacy in the same mice model. RESULTS: The nanoformulation was a clear solution having high colloidal and radiochemical stability. Uniform distribution and retention of the radiolabeled nanoformulation in the tumor mass were observed via SPECT/CT imaging and autoradiography studies. In a tumor regression study, tumor growth was significantly arrested with different doses of radiolabeled NP compared to the control and the best treatment effect was observed with ~ 27.8 MBq dose. In histopathological analysis, loss of mitotic cells was apparent in tumor tissue of treated groups, whereas no significant damage in kidney, lungs, and liver tissue morphology was observed. CONCLUSIONS: These results hold promise for nanoscale brachytherapy to become a clinically practical treatment modality for unresectable solid cancers.

Sex as a Factor in Murine Radiation Research: Implications for Countermeasure Development.

There is an increased threat of exposure to ionizing radiation; in the event of such exposure, the availability of medical countermeasures will be vital to ensure the protection of the population. Effective countermeasures should be efficacious across a varied population and most importantly amongst both males and females. Radiation research must be conducted in animal models which act as a surrogate for the human response. Here, we identify differences in survival in male and female C57BL/6 in both a total body irradiation (TBI) model using the Armed Forces Radiobiology Research Institute (AFRRI) 60Co source and a partial body irradiation (PBI) model using the AFRRI Linear Accelerator (LINAC) with 4 MV photons and 2.5% bone marrow shielding. In both models, we observed a higher degree of radioresistance in female animals and a corresponding radiosensitivity in males. One striking difference in male and female rodents is body size/weight and we investigated the role of pre-irradiation body weight on survivability for animals irradiated at the same dose of irradiation (8 Gy TBI, 14 Gy PBI). We found that weight does not influence survival in the TBI model and that heavier males but lighter females have increased survival in the PBI model. This incongruence in survival amongst the sexes should be taken into consideration in the course of developing radiation countermeasures for response to a mass casualty incident.

Podocyte Integrin-ß3 and Activated Protein C Coordinately Restrict RhoA Signaling and Ameliorate Diabetic Nephropathy.

BACKGROUND: Diabetic nephropathy (dNP), now the leading cause of ESKD, lacks efficient therapies. Coagulation protease-dependent signaling modulates dNP, in part via the G protein-coupled, protease-activated receptors (PARs). Specifically, the cytoprotective protease-activated protein C (aPC) protects from dNP, but the mechanisms are not clear. METHODS: A combination of in vitro approaches and mouse models evaluated the role of aPC-integrin interaction and related signaling in dNP. RESULTS: The zymogen protein C and aPC bind to podocyte integrin-ß3, a subunit of integrin-αvß3. Deficiency of this integrin impairs thrombin-mediated generation of aPC on podocytes. The interaction of aPC with integrin-αvß3 induces transient binding of integrin-ß3 with G α13 and controls PAR-dependent RhoA signaling in podocytes. Binding of aPC to integrin-ß3via its RGD sequence is required for the temporal restriction of RhoA signaling in podocytes. In podocytes lacking integrin-ß3, aPC induces sustained RhoA activation, mimicking the effect of thrombin. In vivo, overexpression of wild-type aPC suppresses pathologic renal RhoA activation and protects against dNP. Disrupting the aPC-integrin-ß3 interaction by specifically deleting podocyte integrin-ß3 or by abolishing aPC's integrin-binding RGD sequence enhances RhoA signaling in mice with high aPC levels and abolishes aPC's nephroprotective effect. Pharmacologic inhibition of PAR1, the pivotal thrombin receptor, restricts RhoA activation and nephroprotects RGE-aPChigh and wild-type mice.Conclusions aPC-integrin-αvß3 acts as a rheostat, controlling PAR1-dependent RhoA activation in podocytes in diabetic nephropathy. These results identify integrin-αvß3 as an essential coreceptor for aPC that is required for nephroprotective aPC-PAR signaling in dNP.

Impairment of IGF-1 Signaling and Antioxidant Response Are Associated with Radiation Sensitivity and Mortality.

Following exposure to high doses of ionizing radiation, diverse strains of vertebrate species will manifest varying levels of radiation sensitivity. To understand the inter-strain cellular and molecular mechanisms of radiation sensitivity, two mouse strains with varying radiosensitivity (C3H/HeN, and CD2F1), were exposed to total body irradiation (TBI). Since Insulin-like Growth Factor-1 (IGF-1) signaling pathway is associated with radiosensitivity, we investigated the link between systemic or tissue-specific IGF-1 signaling and radiosensitivity. Adult male C3H/HeN and CD2F1 mice were irradiated using gamma photons at Lethal Dose-70/30 (LD70/30), 7.8 and 9.35 Gy doses, respectively. Those mice that survived up to 30 days post-irradiation, were termed the survivors. Mice that were euthanized prior to 30 days post-irradiation due to deteriorated health were termed decedents. The analysis of non-irradiated and irradiated survivor and decedent mice showed that inter-strain radiosensitivity and post-irradiation survival outcomes are associated with activation status of tissue and systemic IGF-1 signaling, nuclear factor erythroid 2-related factor 2 (Nrf2) activation, and the gene expression profile of cardiac mitochondrial energy metabolism pathways. Our findings link radiosensitivity with dysregulation of IGF-1 signaling, and highlight the role of antioxidant gene response and mitochondrial function in radiation sensitivity.

Dysregulated Cardiac IGF-1 Signaling and Antioxidant Response Are Associated with Radiation Sensitivity.

Acute exposure to ionizing radiation leads to Hematopoietic Acute Radiation Syndrome (H-ARS). To understand the inter-strain cellular and molecular mechanisms of radiation sensitivity, adult males of two strains of minipig, one with higher radiosensitivity, the Gottingen minipig (GMP), and another strain with comparatively lower radiosensitivity, the Sinclair minipig (SMP), were exposed to total body irradiation (TBI). Since Insulin-like Growth Factor-1 (IGF-1) signaling is associated with radiation sensitivity and regulation of cardiovascular homeostasis, we investigated the link between dysregulation of cardiac IGF-1 signaling and radiosensitivity. The adult male GMP; n = 48, and SMP; n = 24, were irradiated using gamma photons at 1.7-2.3 Gy doses. The animals that survived to day 45 after irradiation were euthanized and termed the survivors. Those animals that were euthanized prior to day 45 post-irradiation due to severe illness or health deterioration were termed the decedents. Cardiac tissue analysis of unirradiated and irradiated animals showed that inter-strain radiosensitivity and survival outcomes in H-ARS are associated with activation status of the cardiac IGF-1 signaling and nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated induction of antioxidant gene expression. Our data link H-ARS with dysregulation of cardiac IGF-1 signaling, and highlight the role of oxidative stress and cardiac antioxidant response in radiation sensitivity.

Cytoprotective activated protein C averts Nlrp3 inflammasome-induced ischemia-reperfusion injury via mTORC1 inhibition.

Cytoprotection by activated protein C (aPC) after ischemia-reperfusion injury (IRI) is associated with apoptosis inhibition. However, IRI is hallmarked by inflammation, and hence, cell-death forms disjunct from immunologically silent apoptosis are, in theory, more likely to be relevant. Because pyroptosis (ie, cell death resulting from inflammasome activation) is typically observed in IRI, we speculated that aPC ameliorates IRI by inhibiting inflammasome activation. Here we analyzed the impact of aPC on inflammasome activity in myocardial and renal IRIs. aPC treatment before or after myocardial IRI reduced infarct size and Nlrp3 inflammasome activation in mice. Kinetic in vivo analyses revealed that Nlrp3 inflammasome activation preceded myocardial injury and apoptosis, corroborating a pathogenic role of the Nlrp3 inflammasome. The constitutively active Nlrp3A350V mutation abolished the protective effect of aPC, demonstrating that Nlrp3 suppression is required for aPC-mediated protection from IRI. In vitro aPC inhibited inflammasome activation in macrophages, cardiomyocytes, and cardiac fibroblasts via proteinase-activated receptor 1 (PAR-1) and mammalian target of rapamycin complex 1 (mTORC1) signaling. Accordingly, inhibiting PAR-1 signaling, but not the anticoagulant properties of aPC, abolished the ability of aPC to restrict Nlrp3 inflammasome activity and tissue damage in myocardial IRI. Targeting biased PAR-1 signaling via parmodulin-2 restricted mTORC1 and Nlrp3 inflammasome activation and limited myocardial IRI as efficiently as aPC. The relevance of aPC-mediated Nlrp3 inflammasome suppression after IRI was corroborated in renal IRI, where the tissue protective effect of aPC was likewise dependent on Nlrp3 inflammasome suppression. These studies reveal that aPC protects from IRI by restricting mTORC1-dependent inflammasome activation and that mimicking biased aPC PAR-1 signaling using parmodulins may be a feasible therapeutic approach to combat IRI.

Signal integration at the PI3K-p85-XBP1 hub endows coagulation protease activated protein C with insulin-like function.

Coagulation proteases have increasingly recognized functions beyond hemostasis and thrombosis. Disruption of activated protein C (aPC) or insulin signaling impair function of podocytes and ultimately cause dysfunction of the glomerular filtration barrier and diabetic kidney disease (DKD). We here show that insulin and aPC converge on a common spliced-X-box binding protein-1 (sXBP1) signaling pathway to maintain endoplasmic reticulum (ER) homeostasis. Analogous to insulin, physiological levels of aPC maintain ER proteostasis in DKD. Accordingly, genetically impaired protein C activation exacerbates maladaptive ER response, whereas genetic or pharmacological restoration of aPC maintains ER proteostasis in DKD models. Importantly, in mice with podocyte-specific deficiency of insulin receptor (INSR), aPC selectively restores the activity of the cytoprotective ER-transcription factor sXBP1 by temporally targeting INSR downstream signaling intermediates, the regulatory subunits of PI3Kinase, p85α and p85ß. Genome-wide mapping of condition-specific XBP1-transcriptional regulatory patterns confirmed that concordant unfolded protein response target genes are involved in maintenance of ER proteostasis by both insulin and aPC. Thus, aPC efficiently employs disengaged insulin signaling components to reconfigure ER signaling and restore proteostasis. These results identify ER reprogramming as a novel hormonelike function of coagulation proteases and demonstrate that targeting insulin signaling intermediates may be a feasible therapeutic approach ameliorating defective insulin signaling.

Farnesoid X Receptor Agonism Protects against Diabetic Tubulopathy: Potential Add-On Therapy for Diabetic Nephropathy.

Established therapies for diabetic nephropathy (dNP) delay but do not prevent its progression. The shortage of established therapies may reflect the inability to target the tubular compartment. The chemical chaperone tauroursodeoxycholic acid (TUDCA) ameliorates maladaptive endoplasmic reticulum (ER) stress signaling and experimental dNP. Additionally, TUDCA activates the farnesoid X receptor (FXR), which is highly expressed in tubular cells. We hypothesized that TUDCA ameliorates maladaptive ER signaling via FXR agonism specifically in tubular cells. Indeed, TUDCA induced expression of FXR-dependent genes (SOCS3 and DDAH1) in tubular cells but not in other renal cells. In vivo, TUDCA reduced glomerular and tubular injury in db/db and diabetic endothelial nitric oxide synthase-deficient mice. FXR inhibition with Z-guggulsterone or vivo-morpholino targeting of FXR diminished the ER-stabilizing and renoprotective effects of TUDCA. Notably, these in vivo approaches abolished tubular but not glomerular protection by TUDCA. Combined intervention with TUDCA and the angiotensin-converting enzyme inhibitor enalapril in 16-week-old db/db mice reduced albuminuria more efficiently than did either treatment alone. Although both therapies reduced glomerular damage, only TUDCA ameliorated tubular damage. Thus, interventions that specifically protect the tubular compartment in dNP, such as FXR agonism, may provide renoprotective effects on top of those achieved by inhibiting angiotensin-converting enzyme.

A Literature Review on Current and Proposed Technologies of Noninvasive Blood Pressure Measurement.

BACKGROUND: Noninvasive continuous blood pressure (BP) measurement has become an evolving topic in the field of remote healthcare. The classical noninvasive BP measurement techniques provide spontaneous values of systolic and diastolic BP. On the other hand, intrusive type BP measurement techniques provide continuous values of systolic and diastolic BP. However, these techniques are very painful, cannot be used for long-term monitoring, and are obtainable only in an intensive care unit environment. With the advancement of the remote healthcare industry, there is a growing demand for noninvasive continuous BP monitoring. OBJECTIVE: The objective of this research was to present a compact literature review on the various prospective approaches of noninvasive continuous BP measurement techniques. MATERIALS & METHODS: The most contemporary and advanced technologies on noninvasive continuous BP measurement are Tactile Sensing, Vascular Unloading Technique, Pulse Transit Time, Photoplethysmography, Ultrasound-based BP measurement, BP measurement from image processing, etc. The literature search based on these technologies was conducted in EMBASE, Web of Science, IEEE, PubMed, and Ovid MEDLINE databases. In this study, each selected approach was evaluated and characterized using the following criteria: (1) accuracy; (2) cost; (3) portability; (4) comfort and convenience of use; (5) clinical health and safety; and (6) ability to integrate with the remote healthcare system. RESULTS: A detailed technical analysis was done to determine the advantages and limitations of each technique in the context of the abovementioned parameters. It was observed that BP measurement, using photoplethysmography (using camera or sensor or both), perhaps was the most promising technique among all. CONCLUSION: The study emphasized the fact that the noninvasive, continuous BP measurement technique needs to evolve further to make it reliable, accurate, and user-friendly. Lastly, a possible direction toward a more reliable and comfortable noninvasive continuous BP measurement technique has been discussed.

A Universal Noninvasive Continuous Blood Pressure Measurement System for Remote Healthcare Monitoring.

BACKGROUND: The effectiveness of any remote healthcare monitoring system depends on how much accurate, patient-friendly, versatile, and cost-effective measurement it is delivering. There has always been a huge demand for such a long-term noninvasive remote blood pressure (BP) measurement system, which could be used worldwide in the remote healthcare industry. Thus, noninvasive continuous BP measurement and remote monitoring have become an emerging area in the remote healthcare industry. INTRODUCTION: Photoplethysmography-based (PPG) BP measurement is a continuous, unobtrusive, patient-friendly, and cost-effective solution. However, BP measurements through PPG sensors are not much reliable and accurate due to some major limitations like pressure disturbance, motion artifacts, and variations in human skin tone. MATERIALS AND METHODS: A novel reflective PPG sensor has been developed to eliminate the abovementioned pressure disturbance and motion artifacts during the BP measurement. Considering the variations of the human skin tone across demography, a novel algorithm has been developed to make the BP measurement accurate and reliable. The training dataset captured 186 subjects' data and the trial dataset captured another new 102 subjects' data. RESULTS AND DISCUSSION: The overall accuracy achieved by using the proposed method is nearly 98%. Thus, demonstrating the efficacy of the proposed method. CONCLUSIONS: The developed BP monitoring system is quite accurate, reliable, cost-effective, handy, and user friendly. It is also expected that this system would be quite useful to monitor the BP of infants, elderly people, patients having wounds, burn injury, or in the intensive care unit environment.

Caspase-1, but Not Caspase-3, Promotes Diabetic Nephropathy.

Glomerular apoptosis may contribute to diabetic nephropathy (dNP), but the pathophysiologic relevance of this process remains obscure. Here, we administered two partially disjunct polycaspase inhibitors in 8-week-old diabetic (db/db) mice: M-920 (inhibiting caspase-1, -3, -4, -5, -6, -7, and -8) and CIX (inhibiting caspase-3, -6, -7, -8, and -10). Notably, despite reduction in glomerular cell death and caspase-3 activity by both inhibitors, only M-920 ameliorated dNP. Nephroprotection by M-920 was associated with reduced renal caspase-1 and inflammasome activity. Accordingly, analysis of gene expression data in the Nephromine database revealed persistently elevated glomerular expression of inflammasome markers (NLRP3, CASP1, PYCARD, IL-18, IL-1ß), but not of apoptosis markers (CASP3, CASP7, PARP1), in patients with and murine models of dNP. In vitro, increased levels of markers of inflammasome activation (Nlrp3, caspase-1 cleavage) preceded those of markers of apoptosis activation (caspase-3 and -7, PARP1 cleavage) in glucose-stressed podocytes. Finally, caspase-3 deficiency did not protect mice from dNP, whereas both homozygous and hemizygous caspase-1 deficiency did. Hence, these results suggest caspase-3-dependent cell death has a negligible effect, whereas caspase-1-dependent inflammasome activation has a crucial function in the establishment of dNP. Furthermore, small molecules targeting caspase-1 or inflammasome activation may be a feasible therapeutic approach in dNP.

The Vitamin E Analog Gamma-Tocotrienol (GT3) Suppresses Radiation-Induced Cytogenetic Damage.

PURPOSE: Ionizing radiation (IR) generates reactive oxygen species (ROS), which cause DNA double-strand breaks (DSBs) that are responsible for cytogenetic alterations. Because antioxidants are potent ROS scavengers, we determined whether the vitamin E isoform γ-tocotrienol (GT3), a radio-protective multifunctional dietary antioxidant, can suppress IR-induced cytogenetic damage. METHODS: We measured DSB formation in irradiated primary human umbilical vein endothelial cells (HUVECs) by quantifying the formation of γ-H2AX foci. Chromosomal aberrations (CAs) were analyzed in irradiated HUVECs and in the bone marrow cells of irradiated mice by conventional and fluorescence-based chromosome painting techniques. Gene expression was measured in HUVECs with quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). RESULTS: GT3 pretreatment reduced DSB formation in HUVECS, and also decreased CAs in HUVECs and mouse bone marrow cells after irradiation. Moreover, GT3 increased expression of the DNA-repair gene RAD50 and attenuated radiation-induced RAD50 suppression. CONCLUSIONS: GT3 attenuates radiation-induced cytogenetic damage, possibly by affecting RAD50 expression. GT3 should be explored as a therapeutic to reduce the risk of developing genetic diseases after radiation exposure.

The Vitamin E Analog Gamma-Tocotrienol (GT3) and Statins Synergistically Up-Regulate Endothelial Thrombomodulin (TM).

Statins; a class of routinely prescribed cholesterol-lowering drugs; inhibit 3-hydroxy-3-methylglutaryl-coenzymeA reductase (HMGCR) and strongly induce endothelial thrombomodulin (TM); which is known to have anti-inflammatory; anti-coagulation; anti-oxidant; and radioprotective properties. However; high-dose toxicity limits the clinical use of statins. The vitamin E family member gamma-tocotrienol (GT3) also suppresses HMGCR activity and induces TM expression without causing significant adverse side effects; even at high concentrations. To investigate the synergistic effect of statins and GT3 on TM; a low dose of atorvastatin and GT3 was used to treat human primary endothelial cells. Protein-level TM expression was measured by flow cytometry. TM functional activity was determined by activated protein C (APC) generation assay. Expression of Kruppel-like factor 2 (KLF2), one of the key transcription factors of TM, was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). TM expression increased in a dose-dependent manner after both atorvastatin and GT3 treatment. A combined treatment of a low-dose of atorvastatin and GT3 synergistically up-regulated TM expression and functional activity. Finally; atorvastatin and GT3 synergistically increased KLF2 expression. These findings suggest that combined treatment of statins with GT3 may provide significant health benefits in treating a number of pathophysiological conditions; including inflammatory and cardiovascular diseases.

Development of Orally Administered γ-Tocotrienol (GT3) Nanoemulsion for Radioprotection.

The purpose of this study was two-fold: (1) to formulate γ-tocotrienol (GT3) in a nanoemulsion formulation as a prophylactic orally administered radioprotective agent; and (2) to optimize the storage conditions to preserve the structural integrity of both the formulation and the compound. γ-tocotrienol was incorporated into a nanoemulsion and lyophilized with lactose. Ultra performance liquid chromatography-mass spectroscopy (UPLC-MS) was used to monitor the chemical stability of GT3 over time, the particle size and ζ potential, and scanning electron microscopy (SEM) were used to study the physical stability of the nanoemulsion. Radioprotective and toxicity studies were performed in mice. The liquid formulation exhibited GT3 degradation at all storage temperatures. Lyophilization, in the presence of lactose, significantly reduced GT3 degradation. Both the liquid and lyophilized nanoemulsions had stable particle size and ζ potential when stored at 4 °C. Toxicity studies of the nanoemulsion resulted in no observable toxicity in mice at an oral dose of 600 mg/kg GT3. The nano-formulated GT3 (300 mg/kg) demonstrated enhanced survival efficacy compared to GT3 alone (200 and 400 mg/kg) in CD2F1 mice exposed to total body gamma radiation. The optimal long-term storage of formulated GT3 is as a powder at -20 °C to preserve drug and formulation integrity. Formulation of GT3 as a nanoemulsion for oral delivery as a prophylactic radioprotectant shows promise and warrants further investigation.

Liver metabolomics reveals increased oxidative stress and fibrogenic potential in gfrp transgenic mice in response to ionizing radiation.

Although radiation-induced tissue-specific injury is well documented, the underlying molecular changes resulting in organ dysfunction and the consequences thereof on overall metabolism and physiology have not been elucidated. We previously reported the generation and characterization of a transgenic mouse strain that ubiquitously overexpresses Gfrp (GTPH-1 feedback regulatory protein) and exhibits higher oxidative stress, which is a possible result of decreased tetrahydrobiopterin (BH4) bioavailability. In this study, we report genotype-dependent changes in the metabolic profiles of liver tissue after exposure to nonlethal doses of ionizing radiation. Using a combination of untargeted and targeted quantitative mass spectrometry, we report significant accumulation of metabolites associated with oxidative stress, as well as the dysregulation of lipid metabolism in transgenic mice after radiation exposure. The radiation stress seems to exacerbate lipid peroxidation and also results in higher expression of genes that facilitate liver fibrosis, in a manner that is dependent on the genetic background and post-irradiation time interval. These findings suggest the significance of Gfrp in regulating redox homeostasis in response to stress induced by ionizing radiation affecting overall physiology.

Acute toxicity of subcutaneously administered vitamin E isomers delta- and gamma-tocotrienol in mice.

The toxicity of parenterally administered vitamin E isomers, delta-tocotrienol (DT3) and gamma-tocotrienol (GT3), was evaluated in male and female CD2F1 mice. In an acute toxicity study, a single dose of DT3 or GT3 was administered subcutaneously in a dose range of 200 to 800 mg/kg. A mild to moderately severe dermatitis was observed clinically and microscopically in animals at the injection site at doses above 200 mg/kg. The severity of the reaction was reduced when the drug concentration was lowered. Neither drug produced detectable toxic effects in any other tissue at the doses tested. Based on histopathological analysis for both DT3 and GT3, and macroscopic observations of inflammation at the injection site, a dose of 300 mg/kg was selected as the lowest toxic dose in a 30-day toxicity study performed in male mice. At this dose, a mild skin irritation occurred at the injection site that recovered completely by the end of the experimental period. At a dose of 300 mg/kg of DT3 or GT3, no adverse effects were observed in any tissues or organs.

Molecular dynamics guided design of tocoflexol: a new radioprotectant tocotrienol with enhanced bioavailability.

There is a pressing need to develop safe and effective radioprotector/radiomitigator agents for use in accidental or terrorist-initiated radiological emergencies. Naturally occurring vitamin E family constituents, termed tocols, that include the tocotrienols, are known to have radiation-protection properties. These agents, which work through multiple mechanisms, are promising radioprotectant agents having minimal toxicity. Although α-tocopherol (AT) is the most commonly studied form of vitamin E, the tocotrienols are more potent than AT in providing radioprotection and radiomitigation. Unfortunately, despite their very significant radioprotectant activity, tocotrienols have very short plasma half-lives and require dosing at very high levels to achieve necessary therapeutic benefits. Thus, it would be highly desirable to develop new vitamin E analogues with improved pharmacokinetic properties, specifically increased elimination half-life and increased area under the plasma level versus time curve. The short elimination half-life of the tocotrienols is related to their low affinity for the α-tocopherol transfer protein (ATTP), the protein responsible for maintaining the plasma level of the tocols. Tocotrienols have less affinity for ATTP than does AT, and thus have a longer residence time in the liver, putting them at higher risk for metabolism and biliary excretion. We hypothesized that the low-binding affinity of tocotrienols to ATTP is due to the relatively more rigid tail structure of the tocotrienols in comparison with that of the tocopherols. Therefore, compounds with a more flexible tail would have better binding to ATTP and consequently would have longer elimination half-life and, consequently, an increased exposure to drug, as measured by area under the plasma drug level versus time curve (AUC). This represents an enhanced residence of drug in the systemic circulation. Based on this hypothesis, we developed a new class of vitamin E analogues, the tocoflexols, which maintain the superior bioactivity of the tocotrienols with the potential to achieve the longer half-life and larger AUC of the tocopherols.

FSL-1: A Synthetic Peptide Increases Survival in a Murine Model of Hematopoietic Acute Radiation Syndrome.

In the current geopolitical climate there is an unmet need to identify and develop prophylactic radiation countermeasures, particularly to ensure the well-being of warfighters and first responders that may be required to perform on radiation-contaminated fields for operational or rescue missions. Currently, no countermeasures have been approved by the U.S. FDA for prophylactic administration. Here we report on the efficacious nature of FSL-1 (toll-like receptor 2/6 agonist) and the protection from acute radiation syndrome (ARS) in a murine total-body irradiation (TBI) model. A single dose of FSL-1 was administered subcutaneously in mice. The safety of the compound was assessed in non-irradiated animals, the efficacy of the compound was assessed in animals exposed to TBI in the AFRRI Co-60 facility, the dose of FSL-1 was optimized, and common hematological parameters [complete blood cell (CBC), cytokines, and bone marrow progenitor cells] were assessed. Animals were monitored up to 60 days after exposure and radiation-induced damage was evaluated. FSL-1 was shown to be non-toxic when administered to non-irradiated mice at doses up to 3 mg/kg. The window of efficacy was determined to be 24 h prior to 24 h after TBI. FSL-1 administration resulted in significantly increased survival when administered either 24 h prior to or 24 h after exposure to supralethal doses of TBI. The optimal dose of FSL-1 administration was determined to be 1.5 mg/kg when administered prior to irradiation. Finally, FSL-1 protected the hematopoietic system (recovery of CBC and bone marrow CFU). Taken together, the effects of increased survival and accelerated recovery of hematological parameters suggests that FSL-1 should be developed as a novel radiation countermeasure for soldiers and civilians, which can be used either before or after irradiation in the aftermath of a radiological or nuclear event.

Development of a Multi-Organ Radiation Injury Model with Precise Dosimetry with Focus on GI-ARS.

The goal of this study was to establish a model of partial-body irradiation (PBI) sparing 2.5% of the bone marrow (BM2.5-PBI) that accurately recapitulates radiological/nuclear exposure scenarios. Here we have reported a model which produces gastrointestinal (GI) damage utilizing a clinical linear accelerator (LINAC) with precise dosimetry, which can be used to develop medical countermeasures (MCM) for GI acute radiation syndrome (ARS) under the FDA animal rule. The PBI model (1 hind leg spared) was developed in male and female C57BL/6 mice that received radiation doses ranging from 12-17 Gy with no supportive care. GI pathophysiology was assessed by crypt cell loss and correlated with peak lethality between days 4 and 10 after PBI. The radiation dose resulting in 50% mortality in 30 days (LD50/30) was determined by probit analysis. Differential blood cell counts in peripheral blood, colony forming units (CFU) in bone marrow, and sternal megakaryocytes were analyzed between days 1-30, to assess the extent of hematopoietic ARS (H-ARS) injury. Radiation-induced GI damage was also assessed by measuring: 1. bacterial load (16S rRNA) by RT-PCR on days 4 and 7 after PBI in liver, spleen and jejunum, 2. liposaccharide binding protein (LBP) levels in liver, and 3. fluorescein isothiocyanate (FITC)-dextran, E-selectin, sP-selectin, VEGF, FGF-2, MMP-9, citrulline, and serum amyloid A (SAA) levels in serum. The LD50/30 of male mice was 14.3 Gy (95% confidence interval 14.1-14.7 Gy) and of female mice was 14.5 Gy (95% confidence interval 14.3-14.7 Gy). Secondary endpoints included loss of viable crypts, higher bacterial loads in spleen and liver, higher LBP in liver, increased FITC-dextran and SAA levels, and decreased levels of citrulline and endothelial biomarkers in serum. The BM2.5-PBI model, developed for the first time with precise dosimetry, showed acute radiation-induced GI damage that is correlated with lethality, as well as a response to various markers of inflammation and vascular damage. Sex-specific differences were observed with respect to radiation dose response. Currently, no MCM is available as a mitigator for GI-ARS. This BM2.5-PBI mouse model can be regarded as the first high-throughput PBI model with precise dosimetry for developing MCMs for GI-ARS under the FDA animal rule.

Development of a Radiation-induced Pulmonary Fibrosis Partial Body Irradiation Model in C57BL/6 Mice.

With the current volatile geopolitical climate, the threat of nuclear assault is high. Exposure to ionizing radiation from either nuclear incidents or radiological accidents often lead to major harmful consequences to human health. Depending on the absorbed dose, the symptoms of the acute radiation syndrome and delayed effects of acute radiation exposure (DEARE) can appear within hours, weeks to months. The lung is a relatively radiosensitive organ with manifestation of radiation pneumonitis as an acute effect, followed by apparent fibrosis in weeks or even months. A recently developed, first-of-its-kind murine model for partial-body irradiation (PBI) injury, which can be used to test potential countermeasures against multi-organ damage such as gastrointestinal (GI) tract and lungs was used for irradiation, with 2.5% bone marrow spared (BM2.5-PBI) from radiation exposure. Long-term damage to lungs from radiation was evaluated using µ-CT scans, pulmonary function testing, histopathological parameters and molecular biomarkers. Pulmonary fibrosis was detected by ground glass opacity observed in µ-CT scans of male and female C57BL/6J mice 6-7 months after BM2.5-PBI. Lung mechanics assessments pertaining to peripheral airways suggested fibrotic lungs with stiffer parenchymal lung tissue and reduced inspiratory capacity in irradiated animals 6-7 months after BM2.5-PBI. Histopathological evaluation of the irradiated lungs revealed presence of focal and diffuse pleural, and parenchymal inflammatory and fibrotic lesions. Fibrosis was confirmed by elevated levels of collagen when compared to lungs of age-matched naïve mice. These findings were validated by findings of elevated levels of pro-fibrotic biomarkers and reduction in anti-inflammatory proteins. In conclusion, a long-term model for radiation-induced pulmonary fibrosis was established, and countermeasures could be screened in this model for survival and protection/mitigation or recovery from radiation-induced pulmonary damage.