Safety, Pharmacokinetics, and Biomarkers of an Amorphous Solid Dispersion of Genistein, a Radioprotectant, in Healthy Volunteers.

A pharmaceutical formulation of genistein, produced as an amorphous solid dispersion by hot melt extrusion (genistein HME), has been developed that can be administered prophylactically to improve outcomes and survival following radiation exposure. Here, genistein HME was evaluated in a phase 1, open-label, single ascending dose (SAD) and multiple single dose (MSD) study enrolling 34 healthy volunteers. In the SAD study, participants were administered a single dose (500, 1000, 2000, or 3000 mg) and in the MSD study, participants were administered a single daily dose for six consecutive days (3000 mg/day). The overall adverse event profile and pharmacokinetics of genistein HME were determined. Additionally, biomarkers of genistein HME were evaluated by profiling whole blood for changes in gene expression by RNA sequencing. Genistein HME was found to be safe at doses up to 3000 mg. Most toxicities were mild to moderate gastrointestinal events, and no dose-limiting toxicities were reported. The maximum tolerated dose was not determined and the no observable adverse effect level was 500 mg. Genistein HME bioavailability greatly increased between the 2000 mg and 3000 mg doses. RNA sequencing analysis revealed that the majority of drug-related changes in gene expression occurred 8-12 hours after the sixth dose in the MSD study. Based on these results, the putative effective dose in humans is 3000 mg.

Preclinical Evaluation of Safety, Pharmacokinetics, Efficacy, and Mechanism of Radioprotective Agent HL-003.

Amifostine is a radioprotector with high efficacy but poor safety, short half-life, no oral formulation, and poor compliance, which limits its application. With the increasing risk of exposure to radiation, the development of new radioprotective agents is critical. We previously synthesized a new amifostine derivative, the small molecule compound HL-003. In this study, we focused on evaluating the radioprotective properties of HL-003. Using the in vitro 2,2-diphenyl-1-picrylhydrazyl assay, we initially confirmed HL-003 as a strong antioxidant and demonstrated that its free radical scavenging activity was stronger than that of amifostine. Then, we performed an acute toxicity test, a 28-day toxicity test, a 30-day survival rate test, and a pharmacokinetic study, all of which provided aggregate evidence that HL-003 functioned as a small molecule radioprotector with high efficacy, a favorable safety profile, a long half-life, and oral administration. The intestinal radioprotective mechanism of HL-003 was explored in male C57 mice after abdominal irradiation by analyzing intestinal tissue samples with hematoxylin-eosin staining, immunohistochemistry, TUNEL staining, and immunofluorescence detection. The results showed that HL-003 protected intestinal DNA from radiation damage and suppressed the expression of phosphorylated histone H2AX, phosphorylated p53, and the apoptosis-related proteins caspase-8 and caspase-9, which contributed to maintaining the normal morphology of the small intestine and provided insights into the mechanism of radioprotection. Thus, HL-003 is a small molecule radioprotector with a potential application in radiation medicine.

γ-Tocotrienol-Loaded Liposomes for Radioprotection from Hematopoietic Side Effects Caused by Radiotherapeutic Drugs.

With the successful development and increased use of targeted radionuclide therapy for treating cancer comes the increased risk of radiation injury to bone marrow-both direct suppression and stochastic effects, leading to neoplasia. Herein, we report a novel radioprotector drug, a liposomal formulation of γ-tocotrienol (GT3), or GT3-Nano for short, to mitigate bone marrow radiation damage during targeted radionuclide therapy. Methods: GT3 was loaded into liposomes using passive loading. 64Cu-GT3-Nano and 3H-GT3-Nano were synthesized to study the in vivo biodistribution profile of the liposome and GT3 individually. The radioprotection efficacy of GT3-Nano was assessed after acute 137Cs whole-body irradiation at a sublethal (4 Gy), a lethal (9 Gy), or a single high-dose administration of 153Sm-ethylenediamine-N,N,N',N'-tetrakis(methylene phosphonic acid) (EDTMP). Flow cytometry and fluorescence microscopy were used to analyze hematopoietic cell population dynamics and the cellular site of GT3-Nano localization in the spleen and bone marrow, respectively. Results: Bone marrow uptake and retention (percentage injected dose per gram of tissue) at 24 h was 6.98 ± 2.34 for 64Cu-GT3-Nano and 7.44 ± 2.52 for 3H-GT3-Nano. GT3-Nano administered 24 h before or after 4 Gy of total-body irradiation (TBI) promoted rapid and complete hematopoietic recovery, whereas recovery of controls stalled at 60%. GT3-Nano demonstrated dose-dependent radioprotection, achieving 90% survival at 50 mg/kg against lethal 9-Gy TBI. Flow cytometry of the bone marrow indicated that progenitor bone marrow cells MPP2 and CMP were upregulated in GT3-Nano-treated mice. Immunohistochemistry showed that GT3-Nano accumulates in CD105-positive sinusoid epithelial cells. Conclusion: GT3-Nano is highly effective in mitigating the marrow-suppressive effects of sublethal and lethal TBI in mice. GT3-Nano can facilitate rapid recovery of hematopoietic components in mice treated with the endoradiotherapeutic agent 153Sm-EDTMP.

Role of Hypotaurine in Protection against UVA-Induced Damage in Keratinocytes.

Photoageing and skin cancer are major causes of morbidity and are a high cost to society. Interest in the development of photoprotective agents for inclusion in topical cosmetic and sunscreen products is profound. Recently, amino acids with a sulfinic group, notably hypotaurine, have been included as ingredients in cosmetic preparations. However, the mechanism of action of hypotaurine as a possible anti-aging agent is unknown, despite its use as a free radical scavenger. To address this issue, we investigated hypotaurine uptake in a human keratinocyte model and examined its effect on UVR-induced cytotoxicity. Hypotaurine was taken up by keratinocytes in a time- and concentration-dependent manner, with levels remaining significantly above baseline 48 h after washout. A cytoprotective effect of pre-incubation with 2.5-5 mMhypotaurine was shown as indicated by increased cell viability when keratinocytes were irradiated with UVA at 5 or 10 Jcm-2 , with the level of hypotaurine also significantly reduced. These findings indicate a potential cytoprotective effect of hypotaurine against the deleterious effects of UVA irradiation. This provides support for further studies to evaluate the potential photoprotective benefits of hypotaurine supplementation of topical cosmetic and sunscreen products.

Pre-clinical evaluation of an innovative oral nano-formulation of baicalein for modulation of radiation responses.

There is an unmet medical need for non-toxic and effective radiation countermeasures for prevention of radiation toxicity during planned exposures. We have earlier shown that intraperitoneal administration of baicalein (BCL) offers significant survival benefit in animal model. Safety, tolerability, pharmacokinetics (PK) and pharmacodynamics of baicalein has been reported in pre-clinical model systems and also in healthy human volunteers. However, clinical translation of baicalein is hindered owing to poor bioavailability due to lipophilicity. In view of this, we fabricated and characterized in-situ solid lipid nanoparticles of baicalein (SLNB) with effective drug entrapment and release kinetics. SLNB offered significant protection to murine splenic lymphocytes against 4 Gy ionizing radiation (IR) induced apoptosis. Oral administration of SLNB exhibited ~70% protection to mice against whole body irradiation (WBI 7.5 Gy) induced mortality. Oral relative bioavailability of BCL was enhanced by over ~300% after entrapment in the SLNB as compared to BCL. Oral dosing of SLNB resulted in transient increase in neutrophil abundance in peripheral blood. Interestingly, we observed that treatment of human lung cancer cells (A549) with radioprotective dose of SLNB exhibited radio-sensitization as evinced by decrease in survival and clonogenic potential. Contrary to antioxidant nature of baicalein in normal cells, SLNB treatment induced significant increase in cellular ROS levels in A549 cells probably due to higher uptake and inhibition of TrxR. Thus, a pharmaceutically acceptable SLNB exhibited improved bioavailability, better radioprotection to normal cells and sensitized cancer cells to radiation induced killing as compared to BCL suggesting its possible utility as an adjuvant during cancer radiotherapy.

3,3'-Diselenodipropionic acid (DSePA): A redox active multifunctional molecule of biological relevance.

BACKGROUND: Extensive research is being carried out globally to design and develop new selenium compounds for various biological applications such as antioxidants, radio-protectors, anti-carcinogenic agents, biocides, etc. In this pursuit, 3,3'-diselenodipropionic acid (DSePA), a synthetic organoselenium compound, has received considerable attention for its biological activities. SCOPE OF REVIEW: This review intends to give a comprehensive account of research on DSePA so as to facilitate further research activities on this organoselenium compound and to realize its full potential in different areas of biological and pharmacological sciences. MAJOR CONCLUSIONS: It is an interesting diselenide structurally related to selenocystine. It shows moderate glutathione peroxidase (GPx)-like activity and is an excellent scavenger of reactive oxygen species (ROS). Exposure to radiation, as envisaged during radiation therapy, has been associated with normal tissue side effects and also with the decrease in selenium levels in the body. In vitro and in vivo evaluation of DSePA has confirmed its ability to reduce radiation induced side effects into normal tissues. Administration of DSePA through intraperitoneal (IP) or oral route to mice in a dose range of 2 to 2.5 mg/kg body weight has shown survival advantage against whole body irradiation and a significant protection to lung tissue against thoracic irradiation. Pharmacokinetic profiling of DSePA suggests its maximum absorption in the lung. GENERAL SIGNIFICANCE: Research work on DSePA reported in fifteen years or so indicates that it is a promising multifunctional organoselenium compound exhibiting many important activities of biological relevance apart from radioprotection.

Radiomodifying action, Pharmacokinetic and Biodistribution of Ethyl 3, 4, 5-trihydroxybenzoate-Implication in development of radiomitigator.

Ethyl 3, 4, 5-trihydroxybenzoate (GAE) is a major bioactive constituent of Hippophae Rhamnoides L. leaves and extract prepared from H. rhamnoides leaves exhibited radioprotective and pharmacological activity. Radiomodifying properties of polyphenol compounds through free radical neutralizing have been reported earlier. However, to date pharmacokinetic (PK) and biodistribution of polyphenol compounds post 60Co-γ-irradiation (5 Gy) exposure have not been studied yet. The study aims to investigate the radio modifying and inflammatory action, PK and biodistribution of GAE at a radioprotective dose and changes, if any, induced after irradiation. Male C 57 BL/6 mice (28-30 g) were administered GAE (200 mg/kg b.wt) orally 15 minutes post to irradiation. Mice were sacrificed at 15, 30 min, 1,2,4,8 and 24 h. PK and biodistribution of GAE in plasma and tissues were studied. The radiomodifying potential was assessed in terms of mitigating NF-kB activity and SGOT, SGPT, urea and creatinine levels in liver and kidney post irradiation. Our study suggested the potential use of GAE as radiomodifying agent inhibits NF-kB expression and maintains the SGOT 24.10 ± 2.4, SGPT 36.01 ± 6.1 U/l, urea18.16 ± 0.003, and creatinine 1.05 ± 0.04 mg/dL upto 8 h in comparison to irradiated mice. Moreover, in biodistribution studies, showed that GAE crosses the blood-brain barrier and is found in brain tissue. Plasma level of GAE peaked at about 15 min, with Cmax 4390.85 ± 285.20 in GAE and in 3391.78 ± 78.13 ng/mL in radiation + GAE-treated animals, Biodistribution resulted in the highest concentration to be found in liver and kidney. These radiomodifying and pharmacokinetic result may be useful for study of the bioactive mechanism associated with radiation injury and to develop a potent formulation of GAE for clinical application.

Highly Catalytic Niobium Carbide (MXene) Promotes Hematopoietic Recovery after Radiation by Free Radical Scavenging.

Ionizing radiation (IR) has been extensively used in industry and radiotherapy, but IR exposure from nuclear or radiological accidents often causes serious health effects in an exposed individual, and its application in radiotherapy inevitably brings undesirable damage to normal tissues. In this work, we have developed ultrathin two-dimensional (2D) niobium carbide (Nb2C) MXene as a radioprotectant and explored its application in scavenging free radicals against IR. The 2D Nb2C MXene features intriguing antioxidant properties in effectively eliminating hydrogen peroxide (H2O2), hydroxyl radicals (•OH), and superoxide radicals (O2•-). Pretreatment with biocompatible polyvinylpyrrolidone (PVP)-functionalized Nb2C nanosheets (Nb2C-PVP NSs) significantly reduces IR-induced production of reactive oxygen species (ROS), resulting in enhanced cell viability in vitro. A single intravenous injection of Nb2C-PVP significantly enhances the survival rate of 5 and 6.5 Gy irradiated mice to 100% and 81.25%, respectively, and significantly increases bone marrow mononuclear cells after IR. Critically, Nb2C-PVP reverses the damage of the hematopoietic system in irradiated mice. Single administration of Nb2C-PVP significantly increases superoxide dismutase (SOD) activities, decreases malondialdehyde levels, and thereby reduces IR-induced pathological damage in the testis, small intestine, lung, and liver of 5 Gy irradiated mice. Importantly, Nb2C-PVP is almost completely eliminated from the mouse body on day 14 post treatment, and no obvious toxicities are observed during the 30-day post treatment period. Our study pioneers the application of 2D MXenes with intrinsic radioprotective nature in vivo.

Simultaneous quantitation of E0703, a novel radioprotective agent and its oxidative metabolite M1 in human plasma by UPLC-MS/MS, and application to clinical pharmacokinetics.

A rapid, sensitive and reliable bioanalytical method was firstly developed and validated based on ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), for simultaneous quantitation of the novel radioprotective compound E0703 and its oxidative metabolite M1 in human plasma. Plasma samples were deproteinated with acetonitrile containing the internal standard IS1229 as precipitant and separated on a short CAPCELL PAK C18 IF2 column (2.0 mm × 20 mm, 2 µm) by gradient elution using acetonitrile (containing 0.1% formic acid) and water (containing 0.1% formic acid) with a run time of 2.5 min per sample. MS detection was carried out on a triple quadrupole mass spectrometer (Xevo TQ-S) coupled with electrospray ionization in positive multiple reaction monitoring (MRM) mode. The method was linear over the concentration ranges of 0.100-50.0 ng/mL for E0703 and 0.200-100 ng/mL for M1, with correlation coefficient (r2) values ≥0.993. A full validation of this method was performed, and all results were within acceptable limits. The novel assay was sensitive enough to monitor E0703 and M1 levels in human plasma, and was successfully applied to a clinical pharmacokinetic study of healthy Chinese subjects after a single oral administration of 30 mg E0703 tablets. In conclusion, the validated method is accurate, sensitive and high-throughput, and can be successfully implemented for subsequent clinical pharmacokinetic studies of E0703 and M1.

PK-PD based optimal dose and time for orally administered supra-pharmacological dose of melatonin to prevent radiation induced mortality in mice.

AIMS: The study reports preclinical pharmacokinetics (PK) and correlation with pharmacological effect at suprapharmacological dose of orally administered melatonin along with time and dose optimization, which have been lacking in earlier reports of radioprotection using melatonin. METHODS: PK of melatonin in C57BL/6 mice was evaluated after dose of 250 mg/kg using HPLC. Tissue distribution study was conducted in vital organs following oral administration. Plasma total antioxidant capacity (TAC) was determined by ABTS+ radical assay and was correlated to plasma concentrations of melatonin. Using the outcomes of PK and Pharmacodynamics (PD), survival study was conducted for optimization of 'drug radiation gap period' (DRGP). Optimal oral dose for radioprotection was determined using survival as an end point. KEY FINDINGS: PK analysis of melatonin revealed Tmax at 5 min with closely spaced another distinct concentration peak at 20 min. Plasma TAC of melatonin showed similar peaks at 5 min and 45 min, with the highest TAC at 45 min. Survival following a lethal (9 Gy) radiation dose was 20% and 40% after 5 and 45 min of melatonin administration, respectively. DRGP for melatonin was thus 45 min, while optimal oral dose ranged from 125 to 250 mg/kg. PK parameters at 250 mg/kg dose were qualitatively similar to low dose of melatonin, thus preventing chances of unexpected toxicity. SIGNIFICANCE: Survival enhancement at 45 min suggested as probable interval required as 'DRGP'. The optimum oral therapeutic window appears large with no substantial toxicity. The outcomes will be useful in development of radioprotectors as well as other therapeutic applications.

Repeated injection of KMRC011, a medical countermeasure for radiation, can cause adverse health effects in cynomolgus monkeys.

High-dose radiation-induced tissue damage is a major limiting factor in the medical application of nuclear technology. Herein, we tested 28-day repeated-dose toxicity of KMRC011, an agonist of toll-like receptor (TLR) 5, which is being developed as a medical countermeasure for radiation, using cynomolgus monkeys. KMRC011 (0.01, 0.02 or 0.04 mg/kg/day) was intramuscularly injected once daily for 4 weeks, and each two monkeys in both control and 0.04 mg/kg/day group were observed for an additional 2-week recovery period. There were no dose-related toxicological changes in mortality, clinical observations, body weight, food consumption, ophthalmological findings, electrocardiographs, coagulation, serum chemistry, organ weights, or urinalysis and urine chemistry. Although treatment-related changes, such as increased white blood cells, increased absolute and relative neutrophils, decreased relative lymphocytes and inflammatory lesions, were noted in the maximum dose group, these findings were not observed after the 2-week recovery period. Further, we considered that the kidneys and heart may be target organs of TLR5 agonists, as well as the spleen, and that autophagic signals can be triggered in tissue damage and the repair process. Importantly, accumulation of p62 protein, an indicator of autophagy, and a decrease of caveolin-1 protein, a regulator of TLR5 protein half-life, were found in both tissues from the highest dose group. Therefore, we conclude that the no-observed-adverse-effect level for KMRC011 may be greater than 0.04 mg/kg/day in male and female monkeys. Additionally, we propose that further studies are needed to identify the molecular signals, which are related to KMRC011-induced adverse effects.

Evaluation of Different Formulations and Routes for the Delivery of the Ionizing Radiation Mitigator GS-Nitroxide (JP4-039).

BACKGROUND/AIM: The mitochondrial targeted GS-nitroxide, JP4-039, is an effective total body irradiation (TBI) mitigator when delivered intravenously (IV) up to 72 h after exposure. Effective systemic and localized administration to oral cavity/oropharynx and esophagus has been demonstrated. The objective of the study was to establish alternatives to IV administration suitable for JP4-039 delivery to mass casualties. MATERIALS AND METHODS: JP4-039 was administered to C57BL/6 mice by topically applied carboxy-methyl-cellulose microneedle arrays (MNAs) or by intramuscular (IM) injection. Three different formulations that have passed Food and Drug Administration review, namely Captisol, 2-hydroxypropyl-ß-cyclodextrin (cyclodextrin), and Miglyol-812-N, were used for drug delivery. Intraoral (IO) administration with each formulation was also evaluated. RESULTS: All tested formulations and MNAs successfully delivered JP4-039. However, IM delivery of the Miglyol-812-N displayed very efficient and highly reproducible radiation mitigation. CONCLUSION: Effective IM delivery of JP4-039 in animal models after TBI or partial-body irradiation suggested the use of the Miglyol-812-N formulation in both medical indications and radiation countermeasures.

Novel Formulation Strategy to Improve the Feasibility of Amifostine Administration.

PURPOSE: Amifostine (AMF), a radioprotectant, is FDA-approved for intravenous administration in cancer patients receiving radiation therapy (XRT). Unfortunately, it remains clinically underutilized due to adverse side effects. The purpose of this study is to define the pharmacokinetic profile of an oral AMF formulation potentially capable of reducing side effects and increasing clinical feasibility. METHODS: Calvarial osteoblasts were radiated under three conditions: no drug, AMF, and WR-1065 (active metabolite). Osteogenic potential of cells was measured using alkaline phosphatase staining. Next, rats were given AMF intravenously or directly into the jejunum, and pharmacokinetic profiles were evaluated. Finally, rats were given AMF orally or subcutaneously, and blood samples were analyzed for pharmacokinetics. RESULTS: WR-1065 preserved osteogenic potential of calvarial osteoblasts after XRT to a greater degree than AMF. Direct jejunal AMF administration incurred a systemic bioavailability of 61.5%. Subcutaneously administrated AMF yielded higher systemic levels, a more rapid peak exposure (0.438 vs. 0.875 h), and greater total systemic exposure of WR-1065 (116,756 vs. 16,874 ng*hr/ml) compared to orally administered AMF. CONCLUSIONS: Orally administered AMF achieves a similar systemic bioavailability and decreased peak plasma level of WR-1065 compared to intravenously administered AMF, suggesting oral AMF formulations maintain radioprotective efficacy without causing onerous side effects, and are clinically feasible.

Evaluation of a method for measuring the radioprotective metabolite WR-1065 in plasma using chemical derivatization combined with UHPLC-MS/MS.

Hypotension is the dose-limiting side effect of the radio-protective drug Amifostine and results from relaxation of the vascular smooth muscle, which is directly mediated by the active metabolite, WR-1065, of Amifostine. The route of administration (currently FDA-approved only for intravenous administration) and the rapid metabolic conversion of Amifostine combine to yield high systemic levels of WR-1065 and facilitate the onset of hypotension. Research efforts aiming to optimize the delivery of WR-1065 to maintain efficacy while reducing its peak, systemic concentration below levels that induce hypotension are underway. To fully characterize the effect of reduced dose levels and alternative routes of administration of Amifostine on systemic WR-1065 concentrations, improved analytical techniques are needed. We have developed and evaluated a highly sensitive method for measuring WR-1065 in rat plasma that employs chemical derivatization, protein precipitation and UPLC-MS/MS analysis. The method exhibits a limit of quantification (LOQ) of 7.4 nM in plasma, which is a significant improvement over conventional approaches that utilize LC-electrochemical detection (ECD) (LOQ 150 nM or higher). The method was assessed in a pharmacokinetics study in rats administered Amifostine intravenously and via direct jejunal injection (10 mg/kg each route). The bioavailability of WR-1065 was 61.5% after direct jejunal injection indicating rapid conversion and absorption of the metabolite in the intestinal tract. This demonstrates that an oral formulation of Amifostine designed for site-specific release of the drug in the upper GI tract can deliver systemic absorption/conversion to WR-1065, provided that the formulation protects the therapeutic from gastric decomposition in the stomach.

Liquid chromatography-tandem mass spectrometric assay for the quantitation of the novel radiation protective agent and radiation mitigator JP4-039 in murine plasma.

JP4-039 radio-protects prior to, and radio-mitigates after ionizing radiation by neutralizing reactive oxygen species. We developed and validated an LC-MS/MS assay for the quantitation of JP4-039 in murine plasma. Methanol protein precipitation of 50µL plasma was followed by isocratic reverse phase chromatography for a 6min run time, and electrospray positive mode ionization mass spectrometric detection. The plasma assay was linear from 1 to 1000ng/mL with appropriate accuracy (97.1-107.6%) and precision (3.7-12.5%CV), and fulfilled FDA guidance criteria. Recovery was 77.2-136.1% with moderate ionization enhancement (10.9-39.5%). Plasma freeze-thaw stability (98.8-104.2%), stability for 13.5 months at -80°C (93.1-105.6%), and stability for 4h at room temperature (94.2-97.6%) were all acceptable. Limited cross-validation to tissue homogenates suggested that these could also be analyzed for JP4-039 accurately. This assay has been directly applied to determine the pharmacokinetics of JP4-039 in C57BL/6 male mice after IV administration of 20mg/kg JP4-039 and will be extended to other studies of this agent.

A Metabolomic and Lipidomic Serum Signature from Nonhuman Primates Administered with a Promising Radiation Countermeasure, Gamma-Tocotrienol.

The development of radiation countermeasures for acute radiation syndrome (ARS) has been underway for the past six decades, leading to the identification of multiple classes of radiation countermeasures. However, to date, only two growth factors (Neupogen and Neulasta) have been approved by the United States Food and Drug Administration (US FDA) for the mitigation of hematopoietic acute radiation syndrome (H-ARS). No radioprotector for ARS has been approved by the FDA yet. Gamma-tocotrienol (GT3) has been demonstrated to have radioprotective efficacy in murine as well as nonhuman primate (NHP) models. Currently, GT3 is under advanced development as a radioprotector that can be administered prior to radiation exposure. We are studying this agent for its safety profile and efficacy using the NHP model. In this study, we analyzed global metabolomic and lipidomic changes using ultra-performance liquid chromatography (UPLC) quadrupole time-of-flight mass spectrometry (QTOF-MS) in serum samples of NHPs administered GT3. Our study, using 12 NHPs, demonstrates that alterations in metabolites manifest only 24 h after GT3 administration. Furthermore, metabolic changes are associated with transient increase in the bioavailability of antioxidants, including lactic acid and cholic acid and anti-inflammatory metabolites 3 deoxyvitamin D3, and docosahexaenoic acid. Taken together, our results show that the administration of GT3 to NHPs causes metabolic shifts that would provide an overall advantage to combat radiation injury. This initial assessment also highlights the utility of metabolomics and lipidomics to determine the underlying physiological mechanisms involved in the radioprotective efficacy of GT3.

A DPP-IV-resistant glucagon-like peptide-2 dimer with enhanced activity against radiation-induced intestinal injury.

Although radiotherapy is a highly effective treatment for abdominal or pelvic cancer patients, it can increase the incidence of severe gastrointestinal (GI) toxicity. As an intestinal growth factor, glucagon-like peptide 2 (GLP-2) has been shown to improve the preclinical models of both short bowel syndrome and inflammatory bowel disease by stimulating intestinal growth. Teduglutide ([Gly2]GLP-2), a recombinant human GLP-2 variant, has a prolonged half-life and stability as compared to the native GLP-2 peptide, but still requires daily application in the clinic. Here, we designed and prepared a new degradation-resistant GLP-2 analogue dimer, designated GLP-2②, with biotechnological techniques. The purity of GLP-2②reached 97% after ammonium sulphate precipitation and anion exchange chromatography purification, and the purification process was simple and cost-effective. We next confirmed that the GLP-2② exhibited enhanced activities compared with [Gly2]GLP-2, the long-acting, degradation-resistant analogue. Notably, GLP-2② offers a pharmacokinetic and therapeutic advantage in the treatment of radiation-induced intestinal injury over [Gly2]GLP-2. We further demonstrated that GLP-2② rapidly activates divergent intracellular signaling pathways involved in cell survival and apoptosis. Taken together, our data revealed a potential novel and safe peptide drug for limiting the adverse effect of radiotherapy on the gastrointestinal system.

CNS bioavailability and radiation protection of normal hippocampal neurogenesis by a lipophilic Mn porphyrin-based superoxide dismutase mimic, MnTnBuOE-2-PyP5.

Although radiation therapy can be effective against cancer, potential damage to normal tissues limits the amount that can be safely administered. In central nervous system (CNS), radiation damage to normal tissues is presented, in part, as suppressed hippocampal neurogenesis and impaired cognitive functions. Mn porphyrin (MnP)-based redox active drugs have demonstrated differential effects on cancer and normal tissues in experimental animals that lead to protection of normal tissues and radio- and chemo-sensitization of cancers. To test the efficacy of MnPs in CNS radioprotection, we first examined the tissue levels of three different MnPs - MnTE-2-PyP5+(MnE), MnTnHex-2-PyP5+(MnHex), and MnTnBuOE-2-PyP5+(MnBuOE). Nanomolar concentrations of MnHex and MnBuOE were detected in various brain regions after daily subcutaneous administration, and MnBuOE was well tolerated at a daily dose of 3mg/kg. Administration of MnBuOE for one week before cranial irradiation and continued for one week afterwards supported production and long-term survival of newborn neurons in the hippocampal dentate gyrus. MnP-driven S-glutathionylation in cortex and hippocampus showed differential responses to MnP administration and radiation in these two brain regions. A better understanding of how preserved hippocampal neurogenesis correlates with cognitive functions following cranial irradiation will be helpful in designing better MnP-based radioprotection strategies.

A sharp, robust, and quantitative method by liquid chromatography tandem mass spectrometry for the measurement of EAD for acute radiation syndrome and its application.

17-Ethinyl-3,17-dihydroxyandrost-5-ene (EAD) is an agent designed for the treatment of acute radiation syndrome (ARS). Given its vital role played in the prevention and mitigation of ARS, the development of a sharp, sensitive and robust liquid chromatography tandem mass spectrometry (LC-MS/MS) method to monitor the metabolism of EAD in vivo was crucial. A new method was constructed and validated for the determination of EAD with the internal standard of androst-5-ene-3ß,17ß-diol (5-AED). The blood samples were precipitated with methanol, centrifuged, from which the supernatant was separated on UPLC with C18 column and eluted in gradient with acetonitrile and Milli-Q water both containing 0.1% formic acid (FA). Quantification was performed by a triple quadrupole mass spectrometer with electro spray ionization (ESI) in multiple reactive monitoring (MRM) positive mode. A good linearity was obtained with R>0.99 for EAD within its calibration range from 5 to 1000ngmL-1 with a lowest limit of quantification (LLOQ) of 5ngmL-1. Inter- and intra-day accuracy and precision of three levels of quality control (QC) samples were within the range of 15%, while the LLOQ was within 20%. Samples were stable under the circumstances of the experiments. The method was simple, accurate and robust applied to determine the concentrations of EAD in Wistar rat after a single administration of EAD orally at the dose of 100mgkg-1.

Supercritical anti-solvent technique assisted synthesis of thymoquinone liposomes for radioprotection: Formulation optimization, in-vitro and in-vivo studies.

The aim of this study was to develop Thymoquinone (TQ) loaded PEGylated liposomes using supercritical anti-solvent (SAS) process for enhanced blood circulation, and greater radioprotection. The SAS process of PEGylated liposomes synthesis was optimized by Box-Behnken design. Spherical liposomes with a particle size of 195.6±5.56nm and entrapment efficiency (%EE) of 89.4±3.69% were obtained. Optimized SAS process parameters; temperature, pressure and solution flow rate were 35°C, 140bar and 0.18mL/min, respectively, while 7.5mmol phospholipid, 0.75mmol of cholesterol, and 1mmol TQ were optimized formulation ingredients. Incorporation of MPEG-2000-DSPE (5% w/w) provided the PEGylated liposomes (FV-17B; particle size=231.3±6.74nm, %EE=91.9±3.45%, maximum TQ release >70% in 24h). Pharmacokinetics of FV-17B in mice demonstrated distinctly superior systemic circulation time for TQ in plasma. Effectiveness of radioprotection by FV-17B in mice model was demonstrated by non-significant body weight change, normal vital blood components (WBCs, RBCs, and Platelets), micronuclei and spleen index and increased survival probability in post irradiation animal group as compared to controls (plain TQ and marketed formulation). Altogether, the results anticipated that the SAS process could serve as a single step environmental friendly technique for the development of stable long circulating TQ loaded liposomes for effective radioprotection.