Delayed effects of radiation exposure in a C57L/J mouse model of partial body irradiation with ~2.5% bone marrow shielding.

Introduction: Mouse models of radiation injury are critical to the development of medical countermeasures (MCMs) against radiation. Now that MCMs against hematopoietic acute radiation syndrome (H-ARS) have achieved regulatory approval, attention is shifting to develop MCMs against the adverse effects of gastrointestinal acute radiation syndrome (GI-ARS) and delayed effects of acute radiation exposure (DEARE). The C57L/J mouse model of partial body irradiation (PBI) with 2.5% bone marrow shielding (BM2.5) is being leveraged to examine both GI-ARS and DEARE effects. Within days of PBI, mice may develop H- and GI-ARS followed several months later by DEARE as a multi-organ injury, which typically involves the lung and kidney (L- and K-DEARE, respectively). The objective of this manuscript is to describe the dose response relationship and progression of radiation injury in the C57L/J mouse and to evaluate its suitability for use in DEARE MCM testing. Materials and methods: In two separate studies conducted over 2 years, male and female C57L/J mice were exposed to PBI BM2.5 with one hindlimb shielded from radiation, representing ~2.5% bone marrow shielding/sparing. Mice were X-ray irradiated at doses ranging from 9 to 13 Gy at 10 to 12 weeks of age for the purposes of assessing ARS survival at 30 days and DEARE survival at 182 days post-irradiation. Clinical indicators of ARS and DEARE were determined by clinical observations, body weights, hematology, clinical chemistry, magnetic resonance imaging (MRI) of lung, and histopathology of selected tissues. Results: C57L/J mice developed canonical ARS responses of hematopoietic atrophy and gastrointestinal injury resulting in dose dependent mortality at doses ≥11 Gy between 1- and 15-days post-irradiation. In animals that survived ARS, DEARE associated mortality occurred in dose dependent fashion at ≥9 Gy for both sexes between 60- and 159-days post-irradiation with histopathology examinations indicating lung injury as the primary cause of death in moribund animals. Conclusion: The PBI BM2.5 C57L/J mouse model reliably produced known H- and GI-ARS effects at doses greater than those resulting in DEARE effects. Because of this, the C57L/J mouse can be used to test MCMs against L-DEARE injury, while avoiding ARS associated mortality.

Novel progestogenic androgens for male contraception: design, synthesis, and activity of C7 α-substituted testosterone†.

Male contraceptive development has included use of testosterone (T) with or without a progestin or the use of a single molecule such as progestogenic androgens (PA) for suppression of testicular T production. Expanding upon the vast amount of data accumulated from nortestosterone (NT), NT analogs, and their prodrugs, a new series of PA, the C7 methyl, and ethyl α-substituted T analogs 7α-Methyltestosterone (7α-MT) and 7α-Ethyltestosterone (7α-ET), respectively, were hypothesized and designed to have superior androgenic and progestogenic activities when compared with parent T. Results from androgen receptor and progesterone receptor competitive binding and transcriptional activation assays showed favorable activities for these T analogs. Additionally, 7α-MT and 7α-ET were shown to be active substrates for aromatase in vitro, mitigating a potential negative impact on bone mineral density with long-term use. In conjunction with this observation, the diminished metabolism of these T analogs by 5α-reductase may reduce potential concerns for prostatic growth. In the Hershberger in vivo rat bioassay, 7α-MT and 7α-ET showed superior androgenic and anabolic activities as compared with T. These C7 α-substituted T analogs also showed clear progestogenic activity in the McPhail bioassay which evaluated endometrial glandular arborization in a rabbit model. The discovery of aromatizable molecules with reduced metabolism by 5α-reductase that have androgenic, anabolic, and progestogenic properties indicates that the core and/or prodrugs of 7α-MT and 7α-ET are promising molecules for further development as male contraceptive PAs.

Survival and Hematologic Benefits of Romiplostim After Acute Radiation Exposure Supported FDA Approval Under the Animal Rule.

PURPOSE: Patients exposed to acute high doses of ionizing radiation are susceptible to dose-dependent bone marrow depression with resultant pancytopenia. Romiplostim (RP; Nplate) is a recombinant thrombopoietin receptor agonist protein that promotes progenitor megakaryocyte proliferation and platelet production and is an approved treatment for patients with chronic immune thrombocytopenia. The goal of our study was to evaluate the postirradiation survival and hematologic benefits of a single dose of RP with or without pegfilgrastim (PF; Neulasta, granulocyte colony stimulating factor) by conducting a well-controlled, treatment-concealed, good laboratory practice-compliant study in rhesus macaques that was compliant with the United States Food and Drug Administration Animal Rule regulatory approval pathway. METHODS AND MATERIALS: Irradiated male and female rhesus macaques (20/sex in each of 3 groups: control, RP, and RP + PF) were subcutaneously administered vehicle or RP (5 mg/kg, 10 mL/kg) on day 1 in the presence or absence of 2 doses of PF (0.3 mg/kg, 0.03 mL/kg, days 1 and 8). Total body radiation (680 cGy, 50 cGy/min from cobalt-60 gamma ray source) occurred 24 ± 2 hours previously at a dose targeting 70% lethality for the control cohort over 60 days. The study examined 60-day survival postirradiation as the primary endpoint. Secondary endpoints included incidence, severity, and duration of thrombocytopenia and neutropenia, other hematology parameters, coagulation parameters, and body weight change to provide insights into potential mechanisms of action. RESULTS: Compared with sham-treated controls, treated animals demonstrated a 40% to 55% survival benefit compared with controls, less severe clinical signs, reduced incidence of thrombocytopenia and/or neutropenia, earlier hematologic recovery, and reduced morbidity from bacterial infection. CONCLUSIONS: These results were pivotal in obtaining Food and Drug Administration approval in January 2021 for RP's new indication as a single administration therapy to increase survival in adults and pediatric patients acutely exposed to myelosuppressive doses of radiation.

The progression of radiation injury in a Wistar rat model of partial body irradiation with ∼5% bone marrow shielding.

PURPOSE: To describe the dose response relationship and natural history of radiation injury in the Wistar rat and its suitability for use in medical countermeasures (MCM) testing. MATERIALS & METHODS: In two separate studies, male and female rats were exposed to partial body irradiation (PBI) with 5% bone marrow sparing. Animals were X-ray irradiated from 7 to 12 Gy at 7-10 weeks of age. Acute radiation syndrome (ARS) survival at 30 days and delayed effects of acute radiation exposure (DEARE) survival at 182 days were assessed. Radiation effects were determined by clinical observations, body weights, hematology, clinical chemistry, magnetic resonance imaging of lung, whole-body plethysmography, and histopathology. RESULTS: Rats developed canonical ARS responses of hematopoietic atrophy and gastrointestinal injury resulting in mortality at doses ≥8Gy in males and ≥8.5 Gy in females. DEARE mortality occurred at doses ≥8Gy for both sexes. Findings indicate lung, kidney, and/or liver injury, and persistent hematological dysregulation, revealing multi-organ injury as a DEARE. CONCLUSION: The Wistar rat PBI model is suitable for testing MCMs against hematopoietic and gastrointestinal ARS. DEARE multi-organ injury occurred in both sexes irradiated with 8-9Gy, also suggesting suitability for polypharmacy studies addressing the combination of ARS and DEARE injury.

A tandem liquid chromatography and tandem mass spectrometry (LC/LC-MS/MS) technique to separate and quantify steroid isomers 11ß-methyl-19-nortestosterone and testosterone.

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has become a mainstay analytical technique in pharmaceutical research and development and clinical diagnosis due to several advantages including excellent selectivity, specificity, and high sensitivity. LC-MS/MS has become the method of choice for steroids analysis due to its fast analytical time and improved specificity yet has a challenge in the separation and measurement of isomers with the same product ions. Here we describe a high-sensitivity LC/LC-MS/MS method that combines chiral chromatography and reverse-phase chromatography (LC/LC) along with MS/MS to rapidly separate and quantify steroid isomers of 11ß-methyl-19-nortestosterone (11ß-MNT) and endogenous testosterone in serum.

Total body irradiation models in NHPs - consideration of animal sex and provision of supportive care to advance model development.

PURPOSE: Harmonized animal models are an indispensable tool for the development of safe and effective medical countermeasures (MCMs) against radiation injury, and rhesus macaques (referred herein as NHPs) play a critical role in FDA approval of radiation medical countermeasures for acute and delayed radiation syndromes. Reliance on such models requires that they be well characterized, which consists, in part, of a reproducible dose to mortality response relationship (DRR). However, data describing the DRR for both male and female NHPs from the same study are scarce. Furthermore, the level of supportive care and the use of blood transfusions may shift the DRR, yet such information can be difficult to compare across publications. To address these knowledge gaps, the DRRs of two different NHP total body irradiation (TBI) models are compared in this paper, one which is reliant on the use of male animals provided blood transfusions, and the other which incorporates both sexes wherein animals are not provided transfusions. MATERIALS AND METHODS: Studies were conducted using NHPs (Macacca mulatta) receiving TBI, with survival reported over a 60 days. Two primary studies, incorporating both male and female animals not receiving blood transfusions as a provision of supportive care, were compared to two previously published studies, which incorporated only male animals provided blood transfusions as a part of the supportive care regimen. Criterion for euthanasia, and all other provisions of supportive care were comparable. Linear probit plots estimating the lethal dose (LD) and upper and lower limits of the 95% confidence interval (CI) for 10, 30, 50, 70 and 90% mortality, were compared between individual studies and the two models presented. RESULTS: Comparison of probit estimates reveals two important findings. (1) Females have higher mortality than males at identical radiation doses, and (2) blood transfusions increased survival of male animals at lower doses but not at high doses of radiation exposure. CONCLUSIONS: The use of single sex animal models may lead to an incomplete understanding of potential sex differences in the dose to mortality response of the TBI model. Consistent use of both sexes and type of supportive care will improve the transferability and reliability of NHP-TBI models currently in use, assist in the selection of radiation doses for single dose lethality studies, and allow investigators to determine the effectiveness of a particular MCM.

Romiplostim (Nplate®) as an effective radiation countermeasure to improve survival and platelet recovery in mice.

Purpose: Rapid depletion of white blood cells, platelets, and reticulocytes are hallmarks of hematopoietic injury of acute radiation syndrome (H-ARS) and, if left untreated, can lead to severe health consequences including death. While the granulocyte colony stimulating factors (G-CSF) filgrastim (Neupogen®), pegfilgrastim (Neulasta®), and sargramostim (Leukine®) are approved to increase survival in patients exposed to a myelosuppressive dose of radiation, no medical countermeasure is currently available for treatment of the thrombocytopenia that also results following radiation exposure. Romiplostim (Nplate®), a thrombopoietin receptor agonist, is the first FDA-approved thrombopoiesis-stimulating protein for the treatment of low platelet (PLT) counts in adults with chronic immune thrombocytopenia. Herein, we present the results of an analysis in mice of romiplostim as a medical countermeasure to improve survival and PLT recovery following acute radiation.Materials and methods: Male and female C57BL/6J mice (11 - 12 weeks of age, n = 21/sex/group) were total body irradiated (TBI) with 6.8 Gy X-rays that reduces 30-day survival to 30% (LD70/30). Vehicle, romiplostim, and/or pegfilgrastim were administered subcutaneously beginning 24 h after TBI for 1-5 days. Evaluation parameters included 30-day survival, pharmacokinetics, and hematology.Results: Full or maximal efficacy with an ∼40% increase in survival was achieved after a single 30 µg/kg dose of romiplostim. No further survival benefit was seen with higher (100 µg/kg) or more frequent dosing (3 or 5 once daily doses at 30 µg/kg) of romiplostim or combined treatment with pegfilgrastim. Pharmacodynamic analysis revealed that the platelet nadir was not as low and recovery was faster in the irradiated mice treated with romiplostim when compared with irradiated control animals (Day 8 versus 10 nadir; Day 22 versus 29 recovery to near baseline). Platelet volume also increased more rapidly after romiplostim injection. Kinetic profiles of other hematology parameters were similar between TBI romiplostim-treated and control mice. Peak serum levels of romiplostim in TBI mice occurred 4 - 24 h (Tmax) after injection with a t1/2 of ∼24 h. Cmax values were at ∼6 ng/ml after 30 µg/kg ± TBI and ∼200 ng/ml after 300 µg/kg. A 10-fold higher romiplostim dose increased the AUClast values by ∼35-fold.Conclusion: A single injection of romiplostim administered 24 h after TBI is a promising radiation medical countermeasure that dramatically increased survival, with or without pegfilgrastim, and hastened PLT recovery in mice.

Biodistribution of the multidentate hydroxypyridinonate ligand [(14) C]-3,4,3-LI(1,2-HOPO), a potent actinide decorporation agent.

The pharmacokinetics and biodistribution of the (14) C-labeled actinide decorporation agent 3,4,3-LI(1,2-HOPO) were investigated in young adult Swiss Webster mice and Sprague Dawley rats, after intravenous, intraperitoneal, and oral dose administration. In all routes investigated, the radiolabeled compound was rapidly distributed to various tissues and organs of the body. In mice, the 24 h fecal elimination profiles suggested that the biliary route is the predominant route of elimination. In contrast, lower fecal excretion levels were observed in rats. Tissue uptake and retention of the compound did not differ significantly between sexes although some differences were observed in the excretion patterns over time. The male mice eliminated a greater percentage of (14) C through the renal pathway than the female mice after receiving an intravenous or intraperitoneal dose, while the opposite trend was seen in rats that received an intravenous dose. Metabolite profiling performed on selected rat samples demonstrated that a putative major metabolite of [(14) C]-3,4,3-LI(1,2-HOPO) is formed, accounting for approximately 10% of an administered oral dose. Finally, to improve its oral bioavailability, 3,4,3-LI(1,2-HOPO) was coformulated with a proprietary permeability enhancer, leading to a notable increase in oral bioavailability of the compound.

In vitro metabolism and stability of the actinide chelating agent 3,4,3-LI(1,2-HOPO).

The hydroxypyridinonate ligand 3,4,3-LI(1,2-HOPO) is currently under development for radionuclide chelation therapy. The preclinical characterization of this highly promising ligand comprised the evaluation of its in vitro properties, including microsomal, plasma, and gastrointestinal fluid stability, cytochrome P450 inhibition, plasma protein binding, and intestinal absorption using the Caco-2 cell line. When mixed with active human liver microsomes, no loss of parent compound was observed after 60 min, indicating compound stability in the presence of liver microsomal P450. At the tested concentrations, 3,4,3-LI(1,2-HOPO) did not significantly influence the activities of any of the cytochromal isoforms screened. Thus, 3,4,3-LI(1,2-HOPO) is unlikely to cause drug-drug interactions by inhibiting the metabolic clearance of coadministered drugs metabolized by these enzymes. Plasma protein-binding assays revealed that the compound is protein-bound in dogs and less extensively in rats and humans. In the plasma stability study, the compound was stable after 1 h at 37°C in mouse, rat, dog, and human plasma samples. Finally, a bidirectional permeability assay demonstrated that 3,4,3-LI(1,2-HOPO) is not permeable across the Caco-2 monolayer, highlighting the need to further evaluate the effects of various compounds with known permeability enhancement properties on the permeability of the ligand in future studies.

Characterization of Batracylin-induced Renal and Bladder Toxicity in Rats.

Batracylin (NSC-320846) is a dual inhibitor of DNA topoisomerases I and II. Batracylin advanced as an anticancer agent to Phase I clinical trials where dose limiting hemorrhagic cystitis (bladder inflammation and bleeding) was observed. To further investigate batracylin's mechanism of toxicity, studies were conducted in Fischer 344 rats. Once daily oral administration of 16 or 32 mg/kg batracylin to rats for 4 days caused overt toxicity. Abnormal clinical observations and adverse effects on clinical pathology, urinalysis, and histology indicated acute renal damage and urothelial damage and bone marrow dysfunction. Scanning electron microscopy revealed sloughing of the superficial and intermediate urothelial layers. DNA damage was evident in kidney and bone marrow as indicated by histone γ-H2AX immunofluorescence. After a single oral administration of 16 or 32 mg/kg, the majority of batracylin was converted to N-acetylbatracylin (NAB) with a half-life of 4 hr to 11 hr. Mesna (Mesnex™), a drug known to reduce the incidence of hemorrhagic cystitis induced by ifosfamide or cyclophosphamide, was administered to rats prior to batracylin, but did not alleviate batracylin-induced bladder and renal toxicity. These findings suggest that batracylin results in DNA damage-based mechanisms of toxicity and not an acrolein-based mechanism of toxicity as occurs after ifosfamide or cyclophosphamide administration.

Dose-dependent efficacy and safety toxicology of hydroxypyridinonate actinide decorporation agents in rodents: towards a safe and effective human dosing regimen.

Two hydroxypyridinone-containing actinide decorporation agents, 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO), are being developed for the treatment of internal actinide contamination by chelation therapy. Dose-response efficacy profiles in mice were established for the removal of intravenously injected (238)Pu and (241)Am after parenteral and oral treatment with these chelators. In both cases, presumed efficacious doses promoted substantially greater actinide elimination rates than the currently approved agent, diethylenetriamine-pentaacetic acid, considering two different interspecies scaling methods for the conversion of human doses to equivalent rodent dose levels. In addition, genotoxicity of both ligands was assessed using the Salmonella/ Escherichia coli /microsome plate incorporation test and the Chinese hamster ovary cell chromosome aberration assay, showing that neither ligand is genotoxic, in the presence and absence of metabolic activation. Finally, maximum tolerated dose studies were performed in rats for seven consecutive daily oral administrations with the chelators, confirming the safety of the presumed efficacious doses for 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO). The results of these studies add to the growing body of evidence that both decorporation agents have remarkable decorporation efficacy properties and promising safety toxicology profiles. These results are necessary components of the regulatory approval process and will help determine the optimal human dosing regimens for the treatment of internal radionuclide contamination.

Toxicogenomics and metabolomics of pentamethylchromanol (PMCol)-induced hepatotoxicity.

Pentamethyl-6-chromanol (PMCol), a chromanol-type compound related to vitamin E, was proposed as an anticancer agent with activity against androgen-dependent cancers. In repeat dose-toxicity studies in rats and dogs, PMCol caused hepatotoxicity, nephrotoxicity, and hematological effects. The objectives of this study were to determine the mechanisms of the observed toxicity and identify sensitive early markers of target organ injury by integrating classical toxicology, toxicogenomics, and metabolomic approaches. PMCol was administered orally to male Sprague-Dawley rats at 200 and 2000 mg/kg daily for 7 or 28 days. Changes in clinical chemistry included elevated alanine aminotransferase, total bilirubin, cholesterol and triglycerides-indicative of liver toxicity that was confirmed by microscopic findings (periportal hepatocellular hydropic degeneration and cytomegaly) in treated rats. Metabolomic evaluations of liver revealed time- and dose-dependent changes, including depletion of total glutathione and glutathione conjugates, decreased methionine, and increased S-adenosylhomocysteine, cysteine, and cystine. PMCol treatment also decreased cofactor levels, namely, FAD and increased NAD(P)+. Microarray analysis of liver found that differentially expressed genes were enriched in the glutathione and cytochrome P450 pathways by PMCol treatment. Reverse transcription-polymerase chain reaction of six upregulated genes and one downregulated gene confirmed the microarray results. In conclusion, the use of metabolomics and toxicogenomics demonstrates that chronic exposure to high doses of PMCol induces liver damage and dysfunction, probably due to both direct inhibition of glutathione synthesis and modification of drug metabolism pathways. Depletion of glutathione due to PMCol exposure ultimately results in a maladaptive response, increasing the consumption of hepatic dietary antioxidants and resulting in elevated reactive oxygen species levels associated with hepatocellular damage and deficits in liver function.

Biomimetic actinide chelators: an update on the preclinical development of the orally active hydroxypyridonate decorporation agents 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO).

The threat of a dirty bomb or other major radiological contamination presents a danger of large-scale radiation exposure of the population. Because major components of such contamination are likely to be actinides, actinide decorporation treatments that will reduce radiation exposure must be a priority. Current therapies for the treatment of radionuclide contamination are limited and extensive efforts must be dedicated to the development of therapeutic, orally bioavailable, actinide chelators for emergency medical use. Using a biomimetic approach based on the similar biochemical properties of plutonium(IV) and iron(III), siderophore-inspired multidentate hydroxypyridonate ligands have been designed and are unrivaled in terms of actinide-affinity, selectivity, and efficiency. A perspective on the preclinical development of two hydroxypyridonate actinide decorporation agents, 3,4,3-LI(1,2-HOPO) and 5-LIO(Me-3,2-HOPO), is presented. The chemical syntheses of both candidate compounds have been optimized for scale-up. Baseline preparation and analytical methods suitable for manufacturing large amounts have been established. Both ligands show much higher actinide-removal efficacy than the currently approved agent, diethylenetriaminepentaacetic acid (DTPA), with different selectivity for the tested isotopes of plutonium, americium, uranium and neptunium. No toxicity is observed in cells derived from three different human tissue sources treated in vitro up to ligand concentrations of 1 mM, and both ligands were well tolerated in rats when orally administered daily at high doses (>100 micromol kg d) over 28 d under good laboratory practice guidelines. Both compounds are on an accelerated development pathway towards clinical use.