Liquid Chromatography-Mass Spectrometry-Based Metabolomics of Nonhuman Primates after 4 Gy Total Body Radiation Exposure: Global Effects and Targeted Panels.

Rapid assessment of radiation signatures in noninvasive biofluids may aid in assigning proper medical treatments for acute radiation syndrome (ARS) and delegating limited resources after a nuclear disaster. Metabolomic platforms allow for rapid screening of biofluid signatures and show promise in differentiating radiation quality and time postexposure. Here, we use global metabolomics to differentiate temporal effects (1-60 d) found in nonhuman primate (NHP) urine and serum small molecule signatures after a 4 Gy total body irradiation. Random Forests analysis differentially classifies biofluid signatures according to days post 4 Gy exposure. Eight compounds involved in protein metabolism, fatty acid ß oxidation, DNA base deamination, and general energy metabolism were identified in each urine and serum sample and validated through tandem MS. The greatest perturbations were seen at 1 d in urine and 1-21 d in serum. Furthermore, we developed a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) method to quantify a six compound panel (hypoxanthine, carnitine, acetylcarnitine, proline, taurine, and citrulline) identified in a previous training cohort at 7 d after a 4 Gy exposure. The highest sensitivity and specificity for classifying exposure at 7 d after a 4 Gy exposure included carnitine and acetylcarnitine in urine and taurine, carnitine, and hypoxanthine in serum. Receiver operator characteristic (ROC) curve analysis using combined compounds show excellent sensitivity and specificity in urine (area under the curve [AUC] = 0.99) and serum (AUC = 0.95). These results highlight the utility of MS platforms to differentiate time postexposure and acquire reliable quantitative biomarker panels for classifying exposed individuals.

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.

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.

Pharmacodynamics of romiplostim alone and in combination with pegfilgrastim on acute radiation-induced thrombocytopenia and neutropenia in non-human primates.

Purpose: Evaluation of the pharmacodynamics (PD) and pharmacokinetics (PK) of romiplostim alone and in combination with pegfilgrastim in a non-human primate (NHP) model of acute radiation syndrome (ARS).Materials and methods: Male and female rhesus macaques were subjected to Cobalt-60 γ irradiation, at a dose of 550 cGy 24 h prior to subcutaneous administration of either romiplostim alone as a single (2.5 or 5.0 mg/kg on Day 1) or repeat dose (5.0 mg/kg on Days 1 and 8), pegfilgrastim alone as a repeat dose (0.3 µg/kg on Day 1 and 8), or a combination of both agents (romiplostim 5.0 mg/kg on Day 1; pegfilgrastim 0.3 µg/kg on Days 1 and 8). Clinical outcome, hematological parameters and PK were assessed throughout the 45 d study period post-irradiation.Results: Administration of romiplostim, pegfilgrastim or the combination of both resulted in significant improvements in hematological parameters, notably prevention of severe thrombocytopenia, compared with irradiated, vehicle control-treated NHPs. The largest hematologic benefit was observed when romiplostim and pegfilgrastim were administered as a combination therapy with much greater effects on both platelet and neutrophil recovery following irradiation compared to single agents alone.Conclusions: These results indicate that romiplostim alone or in combination with pegfilgrastim is effective at improving hematological parameters in an NHP model of ARS. This study supports further study of romiplostim as a medical countermeasure to improve primary hemostasis and survival in ARS.

Salivary Metabolomics of Total Body Irradiated Nonhuman Primates Reveals Long-Term Normal Tissue Responses to Radiation.

PURPOSE: To identify metabolomic biomarkers of acute radiation exposure in saliva that show time-dependent changes. METHODS AND MATERIALS: Nonhuman primates were exposed to 4 Gy of total body irradiation with γ-rays. Saliva was collected from 7 animals twice before and at days 1, 3, 5, 7, 15, 21, 28, and 60 after irradiation. Profiling was conducted with liquid chromatography time-of-flight mass spectrometry. Multivariate data analysis and potential biomarker identification was conducted through random Forests and the software MetaboAnalyst. Candidate biomarkers were validated through tandem mass spectrometry, and receiver operating characteristic curves were constructed to show the diagnostic ability of the signature over time. RESULTS: Untargeted metabolomic analysis revealed significant and persistent effects up to the 60 days evaluated in this study. Biomarkers spanning primarily amino acids and nucleotides were identified, with a significant number showing long-term responses. Fifteen biomarkers showed high statistical significance in the first week after irradiation and 16 at >7 days after irradiation (false discovery rate-adjusted P < .05). The combination of the biomarkers in a single biosignature was able to accurately show the diagnostic ability of the signature in a binary classifier system with receiver operating characteristic curves. CONCLUSIONS: Radiation can alter the metabolome in saliva, and metabolomics could effectively be used to monitor radiation responses, as a biodosimetry method, in the event of a radiological incident. Saliva metabolomics also has potential relevance in a clinical setting.

Temporal Effects on Radiation Responses in Nonhuman Primates: Identification of Biofluid Small Molecule Signatures by Gas Chromatography⁻Mass Spectrometry Metabolomics.

Whole body exposure to ionizing radiation damages tissues leading to physical symptoms which contribute to acute radiation syndrome. Radiation biodosimetry aims to determine characteristic early biomarkers indicative of radiation exposure and is necessary for effective triage after an unanticipated radiological incident. Radiation metabolomics can address this aim by assessing metabolic perturbations following exposure. Gas chromatography-mass spectrometry (GC-MS) is a standardized platform ideal for compound identification. We performed GC time-of-flight MS for the global profiling of nonhuman primate urine and serum samples up to 60 d after a single 4 Gy γ-ray total body exposure. Multivariate statistical analysis showed higher group separation in urine vs. serum. We identified biofluid markers involved in amino acid, lipid, purine, and serotonin metabolism, some of which may indicate host microbiome dysbiosis. Sex differences were observed for amino acid fold changes in serum samples. Additionally, we explored mitochondrial dysfunction by tricarboxylic acid intermediate analysis in the first week with a GC tandem quadrupole MS platform. By adding this temporal component to our previous work exploring dose effects at 7 d, we observed the highest fold changes occurring at 3 d, returning closer to basal levels by 7 d. These results emphasize the utility of both MS-based metabolomics for biodosimetry and complementary analytical platforms for increased metabolome coverage.

In Vivo Activity of Amodiaquine against Ebola Virus Infection.

During the Ebola virus disease (EVD) epidemic in Western Africa (2013‒2016), antimalarial treatment was administered to EVD patients due to the high coexisting malaria burden in accordance with World Health Organization guidelines. In an Ebola treatment center in Liberia, EVD patients receiving the combination antimalarial artesunate-amodiaquine had a lower risk of death compared to those treated with artemether-lumefantrine. As artemether and artesunate are derivatives of artemisinin, the beneficial anti-Ebola virus (EBOV) effect observed could possibly be attributed to the change from lumefantrine to amodiaquine. Amodiaquine is a widely used antimalarial in the countries that experience outbreaks of EVD and, therefore, holds promise as an approved drug that could be repurposed for treating EBOV infections. We investigated the potential anti-EBOV effect of amodiaquine in a well-characterized nonhuman primate model of EVD. Using a similar 3-day antimalarial dosing strategy as for human patients, plasma concentrations of amodiaquine in healthy animals were similar to those found in humans. However, the treatment regimen did not result in a survival benefit or decrease of disease signs in EBOV-infected animals. While amodiaquine on its own failed to demonstrate efficacy, we cannot exclude potential therapeutic value of amodiaquine when used in combination with artesunate or another antiviral.

A long-duration dihydroorotate dehydrogenase inhibitor (DSM265) for prevention and treatment of malaria.

Malaria is one of the most significant causes of childhood mortality, but disease control efforts are threatened by resistance of the Plasmodium parasite to current therapies. Continued progress in combating malaria requires development of new, easy to administer drug combinations with broad-ranging activity against all manifestations of the disease. DSM265, a triazolopyrimidine-based inhibitor of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH), is the first DHODH inhibitor to reach clinical development for treatment of malaria. We describe studies profiling the biological activity, pharmacological and pharmacokinetic properties, and safety of DSM265, which supported its advancement to human trials. DSM265 is highly selective toward DHODH of the malaria parasite Plasmodium, efficacious against both blood and liver stages of P. falciparum, and active against drug-resistant parasite isolates. Favorable pharmacokinetic properties of DSM265 are predicted to provide therapeutic concentrations for more than 8 days after a single oral dose in the range of 200 to 400 mg. DSM265 was well tolerated in repeat-dose and cardiovascular safety studies in mice and dogs, was not mutagenic, and was inactive against panels of human enzymes/receptors. The excellent safety profile, blood- and liver-stage activity, and predicted long half-life in humans position DSM265 as a new potential drug combination partner for either single-dose treatment or once-weekly chemoprevention. DSM265 has advantages over current treatment options that are dosed daily or are inactive against the parasite liver stage.