Venkataraman Srinivasan

Radioprotection by vitamin E: injectable vitamin E administered alone or with WR-3689 enhances survival of irradiated mice.

Radioprotection by injectable vitamin E (alpha-tocopherol) was investigated in mice exposed to 60Co radiation (0.2 Gy/min). Vitamin E injected subcutaneously either 1 hr before or within 15 min after irradiation significantly increased 30-day postirradiation survival in CD2F1 male mice. A dose reduction factor (DRF) of 1.11 (95% confidence interval [1.08, 1.14]) was observed for vitamin E at a dose of 100 IU/kg body weight administered within 15 min after irradiation. Combination studies with the phosphorothioate WR-3689 (S-2([3-methylaminopropyl]amino)ethylphosphorothioic acid) were undertaken to determine whether radioprotection by WR-3689 could be enhanced by vitamin E. Mice were given WR-3689 (150-225 mg/kg, intraperitoneally) 30 min before irradiation and were given vitamin E (100 IU/kg) either 1 hr before or within 15 min after irradiation. Survival was significantly increased in mice given vitamin E and WR-3689 before irradiation as compared to mice given WR-3689 alone: the DRF for WR-3689 (150 mg) was 1.35 [1.32, 1.38]; for WR-3689 combined with vitamin E (100 IU), the DRF was 1.49 [1.45, 1.53].

δ-tocotrienol protects mouse and human hematopoietic progenitors from γ-irradiation through extracellular signal-regulated kinase/mammalian target of rapamycin signaling

Background Exposure to γ-radiation causes rapid hematopoietic cell apoptosis and bone marrow suppression. However, there are no approved radiation countermeasures for the acute radiation syndrome. In this study, we demonstrated that natural δ-tocotrienol, one of the isomers of vitamin E, significantly enhanced survival in total body lethally irradiated mice. We explored the effects and mechanisms of δ-tocotrienol on hematopoietic progenitor cell survival after γ-irradiation in both in vivo and in vitro experiments. Design and Methods CD2F1 mice and human hematopoietic progenitor CD34+ cells were treated with δ-tocotrienol or vehicle control 24 h before or 6 h after γ-irradiation. Effects of δ-tocotrienol on hematopoietic progenitor cell survival and regeneration were evaluated by clonogenicity studies, flow cytometry, and bone marrow histochemical staining. δ-tocotrienol and γ-irradiation-induced signal regulatory activities were assessed by immunofluorescence staining, immunoblotting and short-interfering RNA assay. Results δ-tocotrienol displayed significant radioprotective effects. A single injection of δ-tocotrienol protected 100% of CD2F1 mice from total body irradiation-induced death as measured by 30-day post-irradiation survival. δ-tocotrienol increased cell survival, and regeneration of hematopoietic microfoci and lineage−/Sca-1+/ckit+ stem and progenitor cells in irradiated mouse bone marrow, and protected human CD34+ cells from radiation-induced damage. δ-tocotrienol activated extracellular signal-related kinase 1/2 phosphorylation and significantly inhibited formation of DNA-damage marker γ-H2AX foci. In addition, δ-tocotrienol up-regulated mammalian target of rapamycin and phosphorylation of its downstream effector 4EBP-1. These alterations were associated with activation of mRNA translation regulator eIF4E and ribosomal protein S6, which is responsible for cell survival and growth. Inhibition of extracellular signal-related kinase 1/2 expression by short interfering RNA abrogated δ-tocotrienol-induced mammalian target of rapamycin phosphorylation and clonogenicity, and increased γ-H2AX foci formation in irradiated CD34+ cells. Conclusions Our data indicate that δ-tocotrienol protects mouse bone marrow and human CD34+ cells from radiation-induced damage through extracellular signal-related kinase activation-associated mammalian target of rapamycin survival pathways.

Radioprotection, pharmacokinetic and behavioural studies in mouse implanted with biodegradable drug (amifostine) pellets.

Purpose : We evaluated the use of a subcutaneously (s.c.) implantable, biodegradable pellet as a drug delivery system for the radioprotector amifostine. Materials and methods : Mice were implanted s.c. with either the custom-made biodegradable amifostine drug pellet or the placebo pellet without amifostine, exposed to cobalt-60 γ-radiation (bilateral, 1 Gy min -1, 7-16 Gy), and the 30-day survival rate was monitored. The non-irradiated mouse was used for pharmacokinetic and behavioural tests. Results : Significant radioprotection (85-95% survival) at 10 Gy was observed in the three-amifostine pellet implanted group 3-5 h after implantation. LD 50/30 was 7.97, 8.74 and 16.64 Gy for the control, three-placebo pellet (dose reduction factor, DRF=1.10, p <0.01), and three-amifostine pellet (DRF=1.79, p <0.01) groups respectively in mouse exposed to radiation 2h after implantation. Radioprotection at 12 Gy was observed up to 4h after s.c. amifostine administration and up to 3h after implantation. Pharmacokinetic data revealed that the three-amifostine pellet group had sustained blood WR-1065 levels at 2 h after implantation, in contrast to the reported sharp peak at 30 min for s.c. administration. Although locomotor activity was significantly reduced (p <0.01) in the amifostine pellet group, the onset of the locomotor decrement was delayed as compared with groups that received 400 and 750 mg kg -1 s.c. amifostine. Conclusions : Amifostine in biodegradable implant was effective. The radioprotection observed was comparable between conventional s.c. administration of the drug and implantation. Pharmacokinetic data and locomotor activity suggest that the implantation was beneficial though radioprotection data warrants formulation improvements in implants.

Radioprotection by metals: Selenium

The need exists for compounds that will protect individuals from high-dose acute radiation exposure in space and the agents that might be less protective but less toxic and longer acting. Metals and metal derivatives provide a small degree of radioprotection (dose reduction factor < or = 1.2 for animal survival after whole-body irradiation). Emphasis is placed here on the radioprotective potential of selenium (Se). Both the inorganic salt, sodium selenite, and the organic Se compound, selenomethionine, enhance the survival of irradiated mice (60Co, 0.2 Gy/min) when injected IP either before (-24 hr and -1 hr) or shortly after (+15 min) radiation exposure. When administered at equitoxic doses (one-fourth LD10; selenomethionine = 4.0 mg/kg Se, sodium selenite = 0.8 mg/kg Se), both drugs enhanced the 30-day survival of mice irradiated at 9 Gy. Survival after 10-Gy exposure was significantly increased only after selenomethionine treatment. An advantage of selenomethionine is lower lethal and behavioral toxicity (locomotor activity depression) compared to sodium selenite, when they are administered at equivalent doses of Se. Sodium selenite administered in combination with WR-2721, S-2-(3-aminopropylamino)ethylphosphorothioic acid, enhances the radioprotective effect and reduces the lethal toxicity, but not the behavioral toxicity, of WR-2721. Other studies on radioprotection and protection against chemical carcinogens by different forms of Se are reviewed. As additional animal data and results from human chemoprevention trials become available, consideration also can be given to prolonged administration of Se compounds for protection against long-term radiation effects in space.

Oral delivery of spray dried PLGA/amifostine nanoparticles

Amifostine (Ethyol, WR‐2721) is a cytoprotective drug approved by the US Food & Drug Administration for intravenous administration in cancer patients receiving radiation therapy and certain forms of chemotherapy. The primary objective of this project was to develop orally active amifostine nanoparticles using spray drying technique. Two different nanoparticle formulations (Amifostine‐PLGA (0.4:1.0 and 1.0:1.0)) were prepared using a Buchi B191 Mini Spray Dryer. A water‐in‐oil emulsion of amifostine and PLGA (RG 502) was spray dried using an airflow of 600 Lh−1 and input temperature of 55°C. A tissue distribution study in mice was conducted following oral administration of the formulation containing drug‐polymer (0.4:1.0). The efficiency of encapsulation was 90% and 100%, respectively, for the two formulations while the median particle sizes were 257 and 240 nm, with 90% confidence between 182 and 417 nm. Since amifostine is metabolized to its active form, WR‐1065, by intracellular alkaline phosphatase, the tissue levels of WR‐1065 were measured, instead of WR‐2721. WR‐1065 was detected in significant amounts in all tissues, including bone marrow, jejunum and the kidneys, and there was some degree of selectivity in its distribution in various tissues. This work demonstrates the feasibility of developing an orally effective formulation of amifostine that can be used clinically.

A TPO receptor agonist, ALXN4100TPO, mitigates radiation-induced lethality and stimulates hematopoiesis in CD2F1 mice.

Abstract Thrombopoietin (TPO) receptor agonists lacking sequence homology to TPO were designed by grafting a known peptide sequence into the hinge and/or kappa constant regions of a human anti-anthrax antibody. Some of these proteins were equipotent to TPO in stimulating cMpl-r activity in vitro and in increasing platelet levels in vivo. ALXN4100TPO (4100TPO), the best agonist in this series with a Kd of 30 nM for cMpl-r, exhibited potent activity as a radiation countermeasure in CD2F1 mice exposed to lethal total-body radiation from a cobalt-60 γ-ray source. 4100TPO (2 mg/kg, s.c.) administered once either 24 h before or 6 h after TBI showed superior protection to five daily doses given before or after TBI. Prophylactic administration (69 to 94% survival) was superior to therapeutic schedules (60% survival). 4100TPO conferred a significant survival benefit (P < 0.01) when administered 4 days before or even 12 h after exposure and across a dose range of 0.1 to 8 mg/kg. The dose reduction factors (DRFs) with a single dose of 1 mg/kg 4100TPO 24 h before or 12 h after TBI were 1.32 and 1.11, respectively (P < 0.0001). Furthermore, 4100TPO increased bone marrow cellularity and megakaryocytic development and accelerated multi-lineage hematopoietic recovery in irradiated mice, demonstrating the potential of 4100TPO as both a protector and a mitigator in the event of a radiological incident.

Evaluation of EUK-189, a synthetic superoxide dismutase/catalase mimetic as a radiation countermeasure.

EUK-189, a salen-manganese complex and superoxide dismutase/catalase mimetic, was administered subcutaneously (sc; 30 or 70 mg/kg) to mice at −   24, −   1, +1, or +6 h relative to whole-body cobalt-60 gamma irradiation (LD90/30 dose), and survival was monitored for 30 days. Cell counts and cytokines in circulation were measured in sublethally irradiated mice treated with EUK-189. EUK-189 (70 mg/kg, −   24 h) enhanced 30-day survival with a dose reduction factor (DRF) of 1.15 (p = 0.047, 95% confidence limits: 1.053, 1.244). LD50/30s were 7.96 and 9.13 Gy for saline- and EUK-189–treated groups, respectively. Drug treatment was associated with elevations in numbers of total white blood cells, eosinophils, lymphocytes, and platelets in irradiated mice, compared to vehicle-injected, irradiated controls. EUK-189 did not stimulate production of any cytokine or chemokine tested.

Radioprotection by N-palmitoylated nonapeptide of human interleukin-1β

Interleukin-1beta (IL-1beta) is a cytokine involved in homeostatic processes of the immune system and specifically in inflammatory reactions. The nonapeptide of human IL-1beta (VQGEESNDK, position 163-171) has been shown to retain adjuvant and immunostimulatory activities of the native molecule without any inflammatory and pyrogenic properties. A lipophilic derivative of IL-1beta nonapeptide having a palmitoyl residue at the amino terminus was synthesized in order to determine the effects of such structural modification on its bioactivities. The structurally modified peptide derivative, palmitoylated peptide, significantly protected C3H/HeN mice against potentially lethal doses of ionizing radiation. The dose reduction factor was found to be 1.07. Hematological studies show improved recovery of red blood cells and platelets in irradiated and palmitoylated peptide treated mice as compared with the untreated and irradiated group. These results suggest the importance of the derivatization of small peptides of radioprotective, but toxic cytokines in order to enhance radioprotective activity while reducing unwanted toxic side effects.

Suppression of delayed-type hypersensitivity to oxazolone in whole-body-irradiated mice and protection by WR-2721

The effect of whole-body irradiation on cellular immunity, as measured in vivo by delayed-type hypersensitivity (DTH) to oxazolone (4- ethoxymethylene -2-phenyl- oxazol -5-one), was determined in CD2F1 mice. DTH, determined by changes in ear swelling after challenge with oxazolone, was significantly depressed in irradiated mice (500-900 rad of 60Co) in a dose-dependent fashion when animals were irradiated after sensitization and before challenge with oxazolone. Administration of WR-2721 [S-2-(3-aminopropylamino)ethylphosphorothioic acid] 30 min before irradiation (2 days after sensitization) resulted in protection against suppression of DTH, which was dependent on drug and radiation dose. An effective dose of WR-2721 (200 mg/kg body wt) provided an approximate dose-modifying factor of 1.3. The data suggest that WR-2721 interacts with cells involved in that DTH response (lymphocytes and/or macrophages) and that WR-2721 may be useful in protecting against radiation-induced decrements in cell-mediated immunity.