Removing Critical Gaps in Chemical Test Methods by Developing New Assays for the Identification of Thyroid Hormone System-Disrupting Chemicals-The ATHENA Project.

The test methods that currently exist for the identification of thyroid hormone system-disrupting chemicals are woefully inadequate. There are currently no internationally validated in vitro assays, and test methods that can capture the consequences of diminished or enhanced thyroid hormone action on the developing brain are missing entirely. These gaps put the public at risk and risk assessors in a difficult position. Decisions about the status of chemicals as thyroid hormone system disruptors currently are based on inadequate toxicity data. The ATHENA project (Assays for the identification of Thyroid Hormone axis-disrupting chemicals: Elaborating Novel Assessment strategies) has been conceived to address these gaps. The project will develop new test methods for the disruption of thyroid hormone transport across biological barriers such as the blood-brain and blood-placenta barriers. It will also devise methods for the disruption of the downstream effects on the brain. ATHENA will deliver a testing strategy based on those elements of the thyroid hormone system that, when disrupted, could have the greatest impact on diminished or enhanced thyroid hormone action and therefore should be targeted through effective testing. To further enhance the impact of the ATHENA test method developments, the project will develop concepts for better international collaboration and development in the area of thyroid hormone system disruptor identification and regulation.

MTH1 as a target to alleviate T cell driven diseases by selective suppression of activated T cells.

T cell-driven diseases account for considerable morbidity and disability globally and there is an urgent need for new targeted therapies. Both cancer cells and activated T cells have an altered redox balance, and up-regulate the DNA repair protein MTH1 that sanitizes the oxidized nucleotide pool to avoid DNA damage and cell death. Herein we suggest that the up-regulation of MTH1 in activated T cells correlates with their redox status, but occurs before the ROS levels increase, challenging the established conception of MTH1 increasing as a direct response to an increased ROS status. We also propose a heterogeneity in MTH1 levels among activated T cells, where a smaller subset of activated T cells does not up-regulate MTH1 despite activation and proliferation. The study suggests that the vast majority of activated T cells have high MTH1 levels and are sensitive to the MTH1 inhibitor TH1579 (Karonudib) via induction of DNA damage and cell cycle arrest. TH1579 further drives the surviving cells to the MTH1low phenotype with altered redox status. TH1579 does not affect resting T cells, as opposed to the established immunosuppressor Azathioprine, and no sensitivity among other major immune cell types regarding their function can be observed. Finally, we demonstrate a therapeutic effect in a murine model of experimental autoimmune encephalomyelitis. In conclusion, we show proof of concept of the existence of MTH1high and MTH1low activated T cells, and that MTH1 inhibition by TH1579 selectively suppresses pro-inflammatory activated T cells. Thus, MTH1 inhibition by TH1579 may serve as a novel treatment option against autoreactive T cells in autoimmune diseases, such as multiple sclerosis.

Associations between clinical signs and pathological findings in toxicity testing

Animal testing for toxicity assessment of chemicals and pharmaceuticals must take the 3R principles into consideration. During toxicity testing in vivo, clinical signs are used to monitor animal welfare and to inform about potential toxicity. This study investigated possible associations between clinical signs, body weight change and histopathological findings observed after necropsy. We hypothesized that clinical signs and body weight loss observed during experiments could be used as early markers of organ toxicity. This represents a potential for refinement in terms of improved study man­agement and decreasing of pain and distress experienced during animal experiments. Data from three sequential toxicity studies of an anti-cancer drug candidate in rats were analyzed using the multivariate partial least squares (PLS) regression method. Associations with a predictive value over 80% were found between the occurrence of mild to severe clinical signs and histopathological findings in the thymus, testes, epididymides and bone marrow. Piloerection, eyes half shut and slightly decreased motor activity were most strongly associated with the pathological findings. A 5% body weight loss was found to be a strong empirical predictor of pathological findings but could also be predicted accurately by clinical signs. Thus, we suggest using mild clinical signs and a 5% body weight loss as toxicity markers and as a non-invasive surveillance tool to monitor research animal welfare in toxicity testing. These clinical signs may also enable reduction of animal use due to their informative potential to support scientific decisions regarding drug candidate selection, dose setting, study design, and toxicity assessment.

Efficacy of EBL-1003 (apramycin) against Acinetobacter baumannii lung infections in mice.

OBJECTIVES: Novel therapeutics are urgently required for the treatment of carbapenem-resistant Acinetobacter baumannii (CRAB) causing critical infections with high mortality. Here we assessed the therapeutic potential of the clinical-stage drug candidate EBL-1003 (crystalline free base of apramycin) in the treatment of CRAB lung infections. METHODS: The genotypic and phenotypic susceptibility of CRAB clinical isolates to aminoglycosides and colistin was assessed by database mining and broth microdilution. The therapeutic potential was assessed by target attainment simulations on the basis of time-kill kinetics, a murine lung infection model, comparative pharmacokinetic analysis in plasma, epithelial lining fluid (ELF) and lung tissue, and pharmacokinetic/pharmacodynamic (PKPD) modelling. RESULTS: Resistance gene annotations of 5451 CRAB genomes deposited in the National Database of Antibiotic Resistant Organisms (NDARO) suggested >99.9% of genotypic susceptibility to apramycin. Low susceptibility to standard-of-care aminoglycosides and high susceptibility to EBL-1003 were confirmed by antimicrobial susceptibility testing of 100 A. baumannii isolates. Time-kill experiments and a mouse lung infection model with the extremely drug-resistant CRAB strain AR Bank #0282 resulted in rapid 4-log CFU reduction both in vitro and in vivo. A single dose of 125 mg/kg EBL-1003 in CRAB-infected mice resulted in an AUC of 339 h × µg/mL in plasma and 299 h × µg/mL in ELF, suggesting a lung penetration of 88%. PKPD simulations suggested a previously predicted dose of 30 mg/kg in patients (creatinine clearance (CLCr) = 80 mL/min) to result in >99% probability of -2 log target attainment for MICs up to 16 µg/mL. CONCLUSIONS: This study provides proof of concept for the efficacy of EBL-1003 in the treatment of CRAB lung infections. Broad in vitro coverage, rapid killing, potent in vivo efficacy, and a high probability of target attainment render EBL-1003 a strong therapeutic candidate for a priority pathogen for which treatment options are very limited.

Antibacterial activity of apramycin at acidic pH warrants wide therapeutic window in the treatment of complicated urinary tract infections and acute pyelonephritis.

BACKGROUND: The clinical-stage drug candidate EBL-1003 (apramycin) represents a distinct new subclass of aminoglycoside antibiotics for the treatment of drug-resistant infections. It has demonstrated best-in-class coverage of resistant isolates, and preclinical efficacy in lung infection models. However, preclinical evidence for its utility in other disease indications has yet to be provided. Here we studied the therapeutic potential of EBL-1003 in the treatment of complicated urinary tract infection and acute pyelonephritis (cUTI/AP). METHODS: A combination of data-base mining, antimicrobial susceptibility testing, time-kill experiments, and four murine infection models was used in a comprehensive assessment of the microbiological coverage and efficacy of EBL-1003 against Gram-negative uropathogens. The pharmacokinetics and renal toxicology of EBL-1003 in rats was studied to assess the therapeutic window of EBL-1003 in the treatment of cUTI/AP. FINDINGS: EBL-1003 demonstrated broad-spectrum activity and rapid multi-log CFU reduction against a phenotypic variety of bacterial uropathogens including aminoglycoside-resistant clinical isolates. The basicity of amines in the apramycin molecule suggested a higher increase in positive charge at urinary pH when compared to gentamicin or amikacin, resulting in sustained drug uptake and bactericidal activity, and consequently in potent efficacy in mouse infection models. Renal pharmacokinetics, biomarkers for toxicity, and kidney histopathology in adult rats all indicated a significantly lower nephrotoxicity of EBL-1003 than of gentamicin. INTERPRETATION: This study provides preclinical proof-of-concept for the efficacy of EBL-1003 in cUTI/AP. Similar efficacy but lower nephrotoxicity of EBL-1003 in comparison to gentamicin may thus translate into a higher safety margin and a wider therapeutic window in the treatment of cUTI/API. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Acute and chronic temperature effects on cardiovascular regulation in the red-eared slider (Trachemys scripta).

Acute and chronic changes in ambient temperature alter several aspects of reptilian physiology. We investigated the effects of each type of temperature change on reptilian cardiovascular regulation in red-eared slider turtles (Trachemys scripta), a species known to experience marked seasonal changes in ambient temperature. Turtles were instrumented with occlusive catheters in the femoral artery and vein. Following an acclimation period of 10 days at 13 °C (13(1)), cardiovascular responses to adrenaline, and the cardiac limb of the baroreflex were quantified. Ambient temperature was then reduced 1 °C day(-1) until 3 °C was reached (3(1)). Turtles were maintained at this temperature for 1-week before cardiovascular responses were reassessed. Turtles were then gradually (1 °C day(-1)) returned to an ambient temperature of 13 °C, (13(2)). After a 1-week re-acclimation period, cardiovascular responses were again determined. Finally, 1-week post-pharmacological manipulation of turtles in the 13(2) treatment, ambient temperature was reduced to 3 °C over 24 h (3(2)), and cardiovascular responses were again assessed. Temperature reduction from 13(1) to 3(1) decreased mean arterial blood pressure (P(m)) and heart rate (f(H)) by ~38 and ~63%, respectively. Acute temperature reduction, from 13(2) to 3(2), decreased f(H) similarly, ~66%; however, while P(m) decreased ~28%, this was not significantly different than P(m) at 13(2). The adrenaline injections increased f(H) ranging from 90 to 170% at 13 °C which was a greater change than that observed at 3 °C ranging from a 40 to 70% increase. The increase in P m at the lowest dose of adrenaline did not differ across the temperature treatment groups. The operational point (set-point) P(m) of the baroreflex was decreased similarly by both methods of temperature reduction (3(1) or 3(2)). Further, a hypertensive cardiac baroreflex was absent in the majority of the animals studied independent of temperature. Baroreflex gain and normalized gain based on individual estimates of the relationship were decreased by temperature reduction similarly. Collectively, the data suggest that red-eared slider turtles modulate (down-regulate) some cardiovascular control mechanisms during reduced ambient temperature.

Characterization and cardiovascular actions of endothelin-1 and endothelin-3 from the American alligator.

The structures and biological activities of the isoforms of endothelin (ET) in a reptile are unknown. ET-3, whose primary structure is identical to human ET-3 except for the substitution Phe4 --> Tyr, and a peptide identical to human ET-1 were isolated from an extract of the lung of the alligator, Alligator mississipiensis. Bolus intravenous injections of alligator ET-3 (10, 30, and 100 pmol/kg) into anesthetized alligators produced dose-dependent decreases in systemic blood pressure (P(sys)) and systemic vascular resistance (R(sys)) without change in heart rate (HR), systemic blood flow (Q(sys)), pulmonary pressure (P(pul)), pulmonary vascular resistance (R(pul)), or pulmonary blood flow (Q(pul)). At a dose of 300 pmol/kg, the initial vasodilatation was followed by an increase in R(sys) and decreases in Q(sys) and P(pul). The response to intravenous human/alligator ET-1 (10, 30, 100, and 300 pmol/kg) was biphasic at all doses with initial decreases in P(sys) and R(sys) being followed by sustained increases in these parameters. In the pulmonary circulation, ET-1 produced a dose-dependent decrease in Q(pul) and an increase in R(pul) during the first phase of the response but no significant change during the second phase. There was no change in HR in response to ET-1. The vasodilatator action of arginine, but not ET-1, was attenuated by N(omega)-nitro-L-arginine methyl ester, indicating that the effect of the peptide is probably not mediated through increased synthesis of nitric oxide. The data demonstrate that the structure of the ET isoforms has been strongly conserved during the evolution of vertebrates but that cardiovascular actions differ significantly between the alligator and mammals, especially in the magnitude and duration of the hypotensive response.