Serum RNA biomarkers for predicting survival in non-human primates following thoracic radiation.

In a mass radiation exposure, the healthcare system may rely on differential expression of miRNA to determine exposure and effectively allocate resources. To this end, miRNome analysis was performed on non-human primate serum after whole thorax photon beam irradiation of 9.8 or 10.7 Gy with dose rate 600 cGy/min. Serum was collected up to 270 days after irradiation and sequenced to determine immediate and delayed effects on miRNA expression. Elastic net based GLM methods were used to develop models that predicted the dose vs. controls at 81% accuracy at Day 15. A three-group model at Day 9 achieved 71% accuracy in determining if an animal would die in less than 90 days, between 90 and 269 days, or survive the length of the study. At Day 21, we achieved 100% accuracy in determining whether an animal would later develop pleural effusion. These results demonstrate the potential ability of miRNAs to determine thorax partial-body irradiation dose and forecast survival or complications early following whole thorax irradiation in large animal models. Future experiments incorporating additional doses and independent animal cohorts are warranted to validate these results. Development of a serum miRNA assay will facilitate the administration of medical countermeasures to increase survival and limit normal tissue damage following a mass exposure.

Acute Radiation Effects, the H-ARS in the Non-human Primate: A Review and New Data for the Cynomolgus Macaque with Reference to the Rhesus Macaque.

ABSTRACT: Medical countermeasure development under the US Food and Drug Administration animal rule requires validated animal models of acute radiation effects. The key large animal model is the non-human primate, rhesus macaque. To date, only the rhesus macaque has been used for both critical supportive data and pivotal efficacy trials seeking US Food and Drug Administration approval. The potential for use of the rhesus for other high priority studies such as vaccine development underscores the need to identify another non-human primate model to account for the current lack of rhesus for medical countermeasure development. The cynomolgus macaque, Macaca fascicularis, has an existing database of medical countermeasure development against the hematopoietic acute radiation syndrome, as well as the use of radiation exposure protocols that mimic the likely nonuniform and heterogenous exposure consequent to a nuclear terrorist event. The review herein describes published studies of adult male cynomolgus macaques that used two exposure protocols-unilateral, nonuniform total-body irradiation and partial-body irradiation with bone marrow sparing-with the administration of subject-based medical management to assess mitigation against the hematopoietic acute radiation syndrome. These studies assessed the efficacy of cytokine combinations and cell-based therapy to mitigate acute radiation-induced myelosuppression. Both therapeutics were shown to mitigate the myelosuppression of the hematopoietic acute radiation syndrome. Additional studies being presented herein further defined the dose-dependent hematopoietic acute radiation syndrome of cynomolgus and rhesus macaques and a differential dose-dependent effect with young male and female cynomolgus macaques. The database supports the investigation of the cynomolgus macaque as a comparable non-human primate for efficacy testing under the US Food and Drug Administration animal rule. Critical gaps in knowledge required to validate the models and exposure protocols are also identified.

Plasma Metabolomics in a Nonhuman Primate Model of Abdominal Radiation Exposure.

The acute radiation syndrome is defined in large part by radiation injury in the hematopoietic and gastrointestinal (GI) systems. To identify new pathways involved in radiation-induced GI injury, this study assessed dose- and time-dependent changes in plasma metabolites in a nonhuman primate model of whole abdominal irradiation. Male and female adult Rhesus monkeys were exposed to 6 MV photons to the abdomen at doses ranging between 8 and 14 Gy. At time points from 1 to 60 days after irradiation, plasma samples were collected and subjected to untargeted metabolomics. With the limited sample size of females, different discovery times after irradiation between males and females were observed in metabolomics pattern. Detailed analyses are restricted to only males for the discovery power. Radiation caused an increase in fatty acid oxidation and circulating levels of corticosteroids which may be an indication of physiological stress, and amino acids, indicative of a cellular repair response. The largest changes were observed at days 9 and 10 post-irradiation, with most returning to baseline at day 30. In addition, dysregulated metabolites involved in amino acid pathways, which might indicate changes in the microbiome, were detected. In conclusion, abdominal irradiation in a nonhuman primate model caused a plasma metabolome profile indicative of GI injury. These results point to pathways that may be targeted for intervention or used as early indicators of GI radiation injury. Moreover, our results suggest that effects are sex-specific and that interventions may need to be tailored accordingly.

Differential Recovery of Small Intestinal Segments after Partial-Body Irradiation in Non-Human Primates.

In the event of a radiological attack or accident, it is more likely that the absorbed radiation dose will be heterogeneous, rather than uniformly distributed throughout the body. This type of uneven dose distribution is known as partial-body irradiation (PBI). Partial exposure of the vital organs, specifically the highly radiosensitive intestines, may cause death, if the injury is significant and the post-exposure recovery is considerably compromised. Here we investigated the recovery rate and extent of recovery from PBI-induced intestinal damage in large animals. Rhesus macaques (Macaca mulatta) were randomly divided into four groups: sham-irradiated (0 Gy), 8 Gy PBI, 11 Gy PBI and 14 Gy PBI. A single dose of ionizing radiation was delivered in the abdominal region using a uniform bilateral anteroposterior and posteroanterior technique. Irradiated animals were scheduled for euthanasia on days 10, 28 or 60 postirradiation, and sham-irradiated animals on day 60. Intestinal structural injuries were assessed via crypt depth, villus height, and mucosal surface length in the four different intestinal regions (duodenum, proximal jejunum, distal jejunum and ileum) using H&E staining. Higher radiation doses corresponded with more injury at 10 days post-PBI and a faster recovery rate. However, at 60 days post-PBI, damage was still evident in all regions of the intestine. The proximal and distal ends (duodenum and ileum, respectively) sustained less damage and recovered more fully than the jejunum.

Identification of miRNA Associated with Reduced Survival after Whole-Thorax Lung Irradiation in Non-Human Primates.

Thoracic exposure to ionizing radiation can lead to delayed injuries to the heart and lung that are serious and even life-threatening. These injuries are difficult to predict since they manifest over many weeks and months. To identify noninvasive, tissue-specific biomarkers for the early detection of late radiation injury, circulating microRNA (miRNA) levels were measured in non-human primates (NHP, Macaca mulatta) that received a single exposure of whole-thorax lung irradiation (WTLI) at a dose likely to result in 20% or 75% mortality within 180 days (9.8 or 10.7 Gy). Animals were observed for 270 days after WTLI. Approximately 58% of 9.8 Gy WTLI animals (7 of 12) and 94% of 10.7 Gy WTLI animals (15 out of 16) did not survive to the primary end point. Evidence of pulmonary fibrosis/pneumonitis was observed in all animals. Animals that received 10.7 Gy WTLI experienced more severe and early-onset pneumonitis, as indicated by reduced aerated lung volume, high non-sedated respiratory rate, earlier and more frequent dexamethasone treatments, and evidence of onset of heart disease. Radiation-induced changes in the circulating miRNA profile were most prominent within the first 30 days postirradiation, before the manifestation of symptoms, and included miRNA sequences known to regulate pathways associated with pulmonary fibrosis (TGF-ß/SMAD signaling) and pneumonitis/inflammation (p53 signaling). The abundance of several circulating miRNA differentially expressed at day 6 or 15, such as miR-199a-3p and miR-25-3p, correlated with statistically significant differences in survival. This study supports the hypothesis that it is feasible to use plasma miRNA profiles to identify individuals at high risk of organ-specific late radiation injury. These miRNA profiles could improve radiation oncology clinical practice and serve as biomarkers to predict who might develop late complications in the aftermath of a radiological or nuclear (RAD-NUC) incident.

The impact of supportive care on survival in large animal models of total body irradiation.

PURPOSE: Well-characterized animal models that mimic the human response to potentially lethal doses of radiation are necessary in order to assess the efficacy of candidate medical countermeasures under the criteria of the U.S. Food and Drug Administration 'Animal Rule'. Development of a model requires the determination of the radiation dose response relationship and time course of mortality and morbidity under scenarios likely to be present in the human population during mass casualty situations. These scenarios include understanding the impact of medical management on survival of the hematopoietic acute radiation syndrome (H-ARS). Little information is available to compare the impact of medical management under identical study conditions. The work presented here provides a comparison of the impact of different levels of medical management (supportive care) on the survival outcome in two large animal models: the male Gottingen minipig and the male rhesus macaque (NHP). MATERIALS AND METHODS: In the context of this comparison, limited supportive care consisted of administration of analgesics only, standard supportive care consisted of prophylactic administration of analgesics, antibiotics and fluids (minipigs) or analgesics, antibiotics, antidiarrheals, nutritional and fluid support (NHP) on a set schedule regardless of indication, and full supportive care (NHP only) consisted of analgesics, antibiotics, antidiarrheals, nutritional and fluid support, antiemetics and blood transfusions on an individual animal, trigger-to-treat regimen. Regardless of level of supportive care, minipigs were exposed to total body irradiation using a Co60 source and NHPs were exposed to total body irradiation using 6 MV photon energy. RESULTS: Based on estimated LD50 values, the inclusion of antimicrobial or broad-spectrum antibiotics provided a dose modifying factor (DMF) of 1.09 in the minipig, and by 1.15 in the NHP (standard supportive care to limited supportive care ratio. For the NHP, the administration of supportive care based on symptomology rather than a set schedule, and inclusion of blood transfusions yielded a DMF of 1.05 (full supportive care to standard supportive care ratio). Conversely, comparison of the estimated LD50 values between full supportive care and limited supportive care in the NHP provided a DMF of 1.21. CONCLUSION: The study reported here provides a comparison of the impact of antibiotic administration on radiation-induced lethality.

A gottingen minipig model of radiation-induced coagulopathy.

PURPOSE: Total body irradiation of the Gottingen minipig results in a characteristic hematopoietic response, including anemia, neutropenia, lymphocytopenia, and thrombocytopenia. Currently, there are no well-characterized large or small animal models for radiation-induced thrombocytopenia. The study described here using the Gottingen minipig was focused on understanding which aspects of the coagulation cascade leads to radiation-induced coagulopathy. In this study, multiple clinical pathology parameters were determined prior to and for 45-days following total body irradiation using a 6 MV photon linear accelerator. MATERIALS AND METHODS: Following irradiation, frequent analyses of conventional hematology and coagulation parameters provided time-course information on the onset and recovery of thrombocytopenia. In addition, thromboelastography (TEG) was utilized to monitor coagulation dysfunction, namely clotting time, clot formation time, clot strength, and fibrinolysis. Coagulation factor activity levels were measured for factors II, V, VII, VIII, IX, X, XI, XII, XIII, Protein C, fibrin monomers, antiplasmin and D-dimer using a Siemen's coagulation analyzer to provide time course information of changes in activity post irradiation exposure. RESULTS: These analyses revealed that in total body irradiated minipigs, TEG tracings demonstrate long R (time to initial clot formation) and K (time to achieve a certain clot strength) times, and low alpha-angle (rate of clot formation) and MA (overall stability of the clot) during onset of thrombocytopenia (typically post irradiation day 10-15). Low alpha-angle and MA directly correlated with decreased platelet counts. A long R time is suggestive of a deficiency in clotting factors and was compared to measured activity levels of individual coagulation factors. The data indicates that coagulation factors are significantly changed early after irradiation exposure prior to thrombocytopenia and factors VIII, XI, XII and XIII are markedly altered during the critical point of thrombocytopenia. CONCLUSION: These data support the continued use of multiple approaches to evaluate the coagulation cascade in order to provide the most meaningful interpretation of the hematopoietic changes that occur post irradiation.

Use of computed tomography to assess subcutaneous drug dispersion with recombinant human hyaluronidase PH20 in a swine model.

INTRODUCTION: Subcutaneous (SC) formulations of therapeutics with recombinant human hyaluronidase PH20 (rHuPH20) are currently approved across various disease indications. The rHuPH20-mediated enzymatic degradation of SC hyaluronan (HA) facilitates bulk fluid flow and dispersion of co-administered therapeutics. However, current methods of quantifying dispersion in the SC space are limited. Here, a novel method is outlined to quantify and follow rapid SC volumetric dispersion of a representative therapeutic fluid in the presence of rHuPH20 using computed tomography (CT). METHODS: Ten Yucatan miniature swine were randomized to three groups. Animals received simultaneous infusions of contrast agent (CA) alone (left side of the animal) or in combination with rHuPH20 (right side) at infusion rates of 2.5, 5, or 10 mL/min. Spiral CT scans (1.5 mm thickness) were conducted before and after the infusion and at regular time intervals throughout. Scans were used to create three-dimensional (3D) reconstructions of the fluid pockets and analyze surface area, volume, and sphericity. RESULTS: 3D reconstruction showed increased dispersion of CA with rHuPH20 compared with CA alone, with fenestration and increased dispersion in the craniocaudal and lateromedial directions. The CA with rHuPH20 fluid pockets showed an average increase of 46% in surface area (p = 0.001), a 35% increase in volume (p = 0.001) and a 17% decrease in sphericity post-infusion compared with CA alone at 30 min post-infusion. DISCUSSION: This exploratory study confirms the value of CT imaging as a non-invasive method of assessing real-time spatial and temporal behavior of SC-administered fluids. This technique could help to assess the dispersion pattern of novel rHuPH20 SC co-formulations.

A Comparative Dose-response Relationship Between Sexes for Mortality and Morbidity of Radiation-induced Lung Injury in the Rhesus Macaque.

Radiation-induced lung injury is a characteristic, dose- and time-dependent sequela of potentially lethal, delayed effects of acute radiation exposure. Understanding of these delayed effects to include development of medical countermeasures requires well-characterized and validated animal models that mimic the human response to acute radiation and adhere to the criteria of the US Food and Drug Administration Animal Rule. The objective herein was to establish a nonhuman primate model of whole-thorax lung irradiation in female rhesus macaques. Definition of the dose-response relationship to include key signs of morbidity and mortality in the female macaque served to independently validate the recent model performed with male macaques and importantly, to establish the lack of sex and institutional bias across the dose-response relationship for radiation-induced lung injury. The study design was similar to that described previously, with the exception that female rhesus macaques were utilized. In brief, a computed tomography scan was conducted prior to irradiation and used for treatment planning. Animals in 5 cohorts (n = 8 per cohort) were exposed to a single 6-MV photon exposure focused on the lung as determined by the computed tomography scan and treatment planning at a dose of 9.5, 10, 10.5, 11, or 11.5 Gy. Subject-based supportive care, including administration of dexamethasone, was based on trigger-to-treat criteria. Clearly defined euthanasia criteria were used to determine a moribund condition over the 180-day study duration post-whole-thorax lung irradiation. Percent mortality per radiation dose was 12.5% at 9.5 Gy, 25% at 10 Gy, 62.5% at 10.5 Gy, 87.5% at 11 Gy, and 100% at 11.5 Gy. The resulting probit plot for the whole-thorax lung irradiation model estimated an LD50/180 of 10.28 Gy, which was not significantly different from the published estimate of 10.27 Gy for the male rhesus. The key parameters of morbidity and mortality support the conclusion that there is an absence of a sex influence on the radiation dose-response relationship for whole-thorax lung irradiation in the rhesus macaque. This work also provides a significant interlaboratory validation of the previously published model.

An Interlaboratory Validation of the Radiation Dose Response Relationship (DRR) for H-ARS in the Rhesus Macaque.

The Medical Countermeasures against Radiological Threats (MCART) consortium has established a dose response relationship for the hematopoietic acute radiation syndrome (HARS) in the rhesus macaque conducted under an individualized supportive care protocol, including blood transfusions. Application of this animal model as a platform for demonstrating efficacy of candidate medical countermeasures is significantly strengthened when the model is independently validated at multiple institutions. The study reported here describes implementation of standard operating procedures at an institute outside the consortium in order to evaluate the ability to establish an equivalent radiation dose response relationship in a selected species. Validation of the animal model is a significant component for consideration of the model protocol as an FDA-recommended drug development tool in the context of the "Animal Rule." In the current study, 48 male rhesus macaques (4-8 kg) were exposed to total-body irradiation (TBI) using 6 MV photon energy at a dose rate of approximately 0.8 Gy min. Results show that onset and duration of the hematological response, including anemia, neutropenia, and thrombocytopenia, following TBI ranging from 6.25 to 8.75 Gy correlate well with previously reported findings. The lethality values at 60 d following TBI were estimated to be 6.88 Gy (LD30/60), 7.43 Gy (LD50/60), and 7.98 Gy (LD70/60). These values are equivalent to those published previously of 7.06 Gy (LD30/60), 7.52 Gy (LD50/60), and 7.99 Gy (LD70/60); the DRR slope (p = 0.68) and y-intercepts show agreement along the complete dose range for HARS. The ability to replicate the previously established institutional lethality profile (PROBIT) and model outcomes through careful implementation of defined procedures is a testament to the robustness of the model and highlights the need for consistency in procedures.

Breath biomarkers of whole-body gamma irradiation in the Göttingen minipig.

There is widespread interest in the development of tools to estimate radiation exposures. Exhaled breath provides a novel matrix for assessing biomarkers that could be correlated with exposures. The use of exhaled breath for estimating radiation exposure is warranted, as studies have shown that external exposure to ionizing radiation causes oxidative stress that accelerates lipid peroxidation of polyunsaturated fatty acids, liberating alkanes and alkane metabolites that are excreted in the breath as volatile organic compounds (VOCs). As a proof of principle study, small groups (n = 4) of Göttingen minipigs were whole-body irradiated with gamma rays delivered by a 60Co source at absorbed doses of 0, 0.25, 0.5, 0.75, 1, 1.25, 2, and 4 Gy. Additional groups (n = 4) were treated with lipopolysaccharide (LPS) or granulocyte colony stimulating factor (G-CSF), with and without concurrent 60Co exposure, at an absorbed dose of 1 Gy. Breath and background air VOC samples were collected on days -3, -2, -1, 0 pre-irradiation, then at 0.25, 24, 48, 72, and 168 h post-irradiation. VOCs were analyzed by automated thermal desorption with two-dimensional gas chromatography and time-of-flight mass spectrometry (ATD GCxGC TOF MS). The results show significant changes in 58 breath VOCs post-irradiation, mainly consisting of methylated and other derivatives of alkanes, alkenes, and benzene. Using a multivariate combination of these VOCs, a radiation response function was constructed, which was significantly elevated at 15 min post irradiation and remained elevated throughout the study (to 168 h post irradiation). As a binary test of radiation absorbed doses ≥ 0.25 Gy, the radiation response function distinguished irradiated animals from shams (0 Gy) with 83-84% accuracy. A randomly derived radiation response function was robust: When half of the biomarkers were removed, accuracy was 75%. An optimally derived function with two biomarkers was 82% accurate. As a binary test of radiation absorbed doses ≥ 0.5 Gy, the radiation response function identified irradiated animals with an accuracy of 87% at 15 min post irradiation and 75.5% at 168 h post irradiation. Treatment with LPS and G-CSF did not affect the radiation response function. This proof-of-principle study supports the hypothesis that breath VOCs may be used for estimating radiation exposures. Further studies will be required to validate the sensitivity and specificity of these potential biomarkers.

A Dose-Dependent Hematological Evaluation of Whole-Body Gamma-Irradiation in the Göttingen Minipig.

There is a great deal of interest in the establishment of a standardized animal model for the acute radiation syndrome to allow development of diagnostic approaches and countermeasure treatments following radiological terrorist events. Due to physiological, anatomical, and biochemical similarities to humans, the minipig is an attractive large animal model for evaluating countermeasure efficacy. This study was conducted in order to aid in the establishment of the minipig, and the Göttingen minipig in particular, as an animal model for the hematopoietic acute radiation syndrome. Animals were exposed whole-body to Co at doses of 0 (sham control), 0.25, 0.5, 0.75, 1.0, and 2.0 Gy, and hematological parameters followed in time from pre-irradiation to post-irradiation Day 7. Following irradiation, a dose-dependent decrease in total white blood cells was observed, which was determined to be statistically different as compared to control animals at all dose levels above 0.25 Gy at 24 h post-irradiation. Similarly, a dose-dependent reduction in both absolute lymphocyte count and absolute neutrophil count occurred by the earliest time point measured for all exposed animals. A significant decrease in platelets was observed at post-irradiation Day 7 in animals exposed only at the highest (2.0 Gy) level. The platelet-to-lymphocyte ratio generated for exposures ranging from 0.25-2.0 Gy was able to differentiate response between high and low exposure levels even at 7 d post exposure. In conclusion, the present study supports the development of the Göttingen minipig as a suitable large animal model to study radiation-induced hematopoietic syndrome.

Evaluation of Cuprimine® and Syprine® for decorporation of radioisotopes of cesium, cobalt, iridium and strontium.

Cuprimine® and Syprine® are therapeutics approved by the USFDA to treat copper overload in Wilson Disease (a genetic defect in copper transport) by chelation and accelerated excretion of internally-deposited copper. These oral therapeutics are based on the respective active ingredients D-penicillamine (DPA) and N,N'-bis (2-aminoethyl) -1,2-ethanediamine dihydrochloride (Trien). Cuprimine is considered the primary treatment, although physicians are increasingly turning to Syprine as a first-line therapy. Both drugs exhibit oral systemic activity and low toxicity; their biological effects and safety are established. Previous in vivo studies using a rodent animal model established the decorporation potential of Cuprimine and Syprine for (60)Co and (210)Po. Currently these studies are being expanded to evaluate the in vivo decorporation efficacy of these drugs for several additional radionuclides. In this report, results of this investigation are discussed using the radionuclides (137)Cs, (60)Co, (192)Ir and (85)Sr. Short-term 48-h pilot studies were undertaken to evaluate DPA and Trien for their in vivo decorporation potential using male Wistar-Han rats. In these studies, a radionuclide solution was administered to the animals by intravenous (IV) injection, followed by a single IV dose of either DPA or Trien. Control animals received the radionuclide alone. Results show effective decorporation of (60)Co by DPA within the time frame evaluated. DPA and Trien were also modestly effective in decorporation of (137)Cs and (85)Sr, respectively. The study did not find DPA or Trien effective for decorporation of (192)Ir. Based on these encouraging findings, further studies to evaluate the dose-response profiles and timing of the chelator administration post exposure to radionuclides are warranted.

Biomaterials for the decorporation of (85)Sr in the rat.

Although four stable isotopes of strontium occur naturally, Sr is produced by nuclear fission and is present in surface soil around the world as a result of fallout from atmospheric nuclear weapons tests. It can easily transfer to humans in the event of a nuclear/radiological emergency or through the plant-animal-human food chain causing long-term exposures. Strontium is chemically and biologically similar to calcium, and is incorporated primarily into bone following internal deposition. Alginic acid (alginate) obtained from seaweed (kelp) extract selectively binds ingested strontium in the gastrointestinal tract blocking its systemic uptake and reducing distribution to bone in rats, while other natural polysaccharides including chitosan and hyaluronic acid had little in vivo affinity for strontium. Alginate exhibits the unique ability to discriminate between strontium and calcium and has been previously shown to reduce intestinal absorption and skeletal retention of strontium without changing calcium metabolism. In our studies, the effect of commercially available alginate on intestinal absorption of strontium was examined. One problem associated with alginate treatment is its limited solubility and gel formation in water. The aqueous solubility of sodium alginate was improved in a sodium chloride/sodium bicarbonate electrolyte solution containing low molecular weight polyethylene glycol (PEG). Furthermore, oral administration of the combined alginate/electrolyte/PEG solution accelerated removal of internal strontium in rats when compared to treatment with individual sodium alginate/electrolyte or electrolyte/PEG solutions. Importantly, both alginate and PEG are nontoxic, readily available materials that can be easily administered orally in case of a national emergency when potentially large numbers of the population may require medical treatment for internal depositions. Our results suggest further studies to optimize in vivo decorporation performance of engineered alginate material via modification of its chemical and physicochemical properties are warranted.

Evaluation of Cuprimine and Syprine for decorporation of (60)Co and (210)Po.

The acknowledged risk of deliberate release of radionuclides into local environments by terrorist activities has prompted a drive to improve novel materials and methods for removing internally deposited radionuclides. These decorporation treatments will also benefit workers in the nuclear industry, should an exposure occur. Cuprimine and Syprine are oral therapeutics based on the active ingredients D-penicillamine and N,N'-bis-(2-aminoethyl)-1,2-ethanediamine dihydrochloride, respectively. These therapeutic drugs have been used for several decades to treat Wilson's disease, a genetic defect leading to copper overload, by chelation and accelerated excretion of internally deposited copper. Studies were undertaken to evaluate these FDA-approved drugs for the in vivo decorporation of radioactive cobalt (Co) and polonium (Po) using male Wistar-Han rats. In these studies, Co or Po was administered to animals by IV injection, followed by oral gavage doses of either Cuprimine or Syprine. Control animals received the radionuclide alone. For Co studies, animals received a single dose of Cuprimine or Syprine, while for Po studies animals were repeatedly dosed at 24-h intervals for a total of 5 doses. Results show that Syprine significantly increased urinary elimination and skeletal concentrations of Co compared to controls. While Cuprimine had little effect on total excretion of Co, the skeletal, kidney, liver, muscle, and stomach tissues had significantly lower radioactivity compared to control animals. The low overall excretion of Po made it difficult to reliably measure urinary or fecal radioactivity and draw a definitive conclusion on the effect of Cuprimine or Syprine treatment on excretion. However, Cuprimine treatment was effective at reducing spleen levels of Po compared to controls. Similarly, Syprine treatment produced statistically significant reductions of Po in the spleen and skeletal tissues compared to control animals. Based on these promising findings, further studies to evaluate the dose-response pharmacokinetic profiles for decorporation are warranted.

Aminothiol receptors for decorporation of intravenously administered (60)Co in the rat.

This report provides a comparison of the oral decorporation efficacy of L-glutathione (GSH), L-cysteine (Cys), and a liposomal GSH formulation (ReadiSorb) toward systemic (60)Co to that observed following intravenous administration of GSH and Cys in F344 rats. Aminoacid L-histidine (His) containing no thiol functionality was tested intravenously to compare in vivo efficacy of the aminothiol (GSH, Cys) chelators with that of the aminoimidazole (His) chelator. In these studies, (60)Co was administered to animals by intravenous injection, followed by intravenous or oral gavage doses of a chelator repeated at 24-h intervals for a total of 5 doses. The results suggest that GSH and Cys are potent decorporation agents for (60)Co in the rat model, although the efficacy of treatment depends largely on the systemic availability of the chelator. The intravenous route of administration of GSH or Cys was most effective in reducing tissue (60)Co levels and in increasing excretion of radioactivity compared to control animals. Liposomal encapsulation was found to markedly enhance the oral bioavailability of GSH compared to non-formulated GSH. The oral administration of liposomal GSH reduced (60)Co levels in nearly all tissues by 12-43% compared to that observed for non-formulated GSH. Efficacy of oral Cys was only slightly reduced in comparison with intravenous Cys. Further studies to optimize the dosing regimen in order to maximize decorporation efficiency are warranted.

Investigation of chitosan for decorporation of 60Co in the rat.

With the increased threat of terrorist release of radioactive materials, there is a need for non-toxic decorporation agents to treat internal contamination with radionuclides. In this study, low molecular weight chitosan was evaluated for decorporation of radioactive cobalt (60Co). The affinity of chitosan for Co(II) was tested in vitro using spectrophotometric and potentiometric titration techniques. For in vivo studies, the effect of chitosan on ingested 60Co was evaluated using F344 rats administered a single dose followed by oral chitosan. Chitosan was also evaluated for systemic decorporation of 60Co following intravenous injection with repeated chitosan administration over 5 d. Control animals received 60Co without chelation treatment. Excreta and tissues were collected for analysis using gamma-counting techniques. Results from in vitro experiments confirmed the binding of Co(II) to chitosan, with the postulated formation of a mixed cobalt-chitosan-hydroxide complex species; a stability constant was calculated for this complex. For in vivo studies, oral administration of chitosan significantly reduced systemic absorption of orally administered 60Co as evidenced by an increase in fecal elimination and decrease in urinary elimination. However, oral administration of chitosan lactate slightly decreased fecal excretion of 60Co. Further, oral administration of chitosan significantly reduced 60Co levels in kidney, liver, and skeleton compared to control animals receiving 60Co alone. By the i.v. route, chitosan slightly reduced levels of 60Co in tissues compared to controls, although statistically significant reductions were only observed for blood and kidney. Overall, this commercially available chitosan oligosaccharide exhibited promising potential; further studies are warranted to evaluate the optimal dosing regimen and chemical modifications to increase effectiveness.

In vitro glutathione conjugation of methyl iodide in rat, rabbit, and human blood and tissues.

Methyl iodide (MeI) is an intermediate in the manufacture of some pesticides and pharmaceuticals, and is under review for US registration as a non-ozone depleting alternative for methyl bromide for pre-plant soil fumigation. MeI is primarily metabolized via conjugation with glutathione (GSH), with further metabolism to S-methyl cysteine and methanethiol. To facilitate extrapolations of animal pharmacokinetic data to humans, rate constants for the GSH metabolism of MeI were determined in cytosols prepared from the liver and kidneys of rats, human donors, female rabbits, and rabbit fetuses, from rabbit olfactory and respiratory epithelium, and from rabbit and rat blood using a headspace vial equilibration technique and two-compartment mathematical model. MeI was metabolized in liver and kidney from adults of all three species, but metabolism was not detectable in fetal rabbit kidney. Maximal metabolic rates (V(max)) were similar in liver from rat and human donors (approximately 40 and 47 nmol/min/mg, respectively) whereas the V(max) rates in kidney cytosols varied approximately three-fold between the three species. No difference was observed in the loss of MeI from active and inactive whole blood from either rats or rabbits. The metabolism in olfactory and respiratory epithelial cytosol had Michaelis-Menten constant (K(m)) values that were several times higher than for any other tissue, suggesting essentially first-order metabolism in the nose. The metabolism of MeI in human liver cytosol prepared from five individual donors indicated two potential populations, one high affinity/low capacity and one with a lower affinity but higher capacity.