Obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial

Background Non-alcoholic steatohepatitis (NASH) is a common type of chronic liver disease that can lead to cirrhosis. Obeticholic acid, a farnesoid X receptor agonist, has been shown to improve the histological features of NASH. Here we report results from a planned interim analysis of an ongoing, phase 3 study of obeticholic acid for NASH. Methods In this multicentre, randomised, double-blind, placebo-controlled study, adult patients with definite NASH,non-alcoholic fatty liver disease (NAFLD) activity score of at least 4, and fibrosis stages F2–F3, or F1 with at least oneaccompanying comorbidity, were randomly assigned using an interactive web response system in a 1:1:1 ratio to receive oral placebo, obeticholic acid 10 mg, or obeticholic acid 25 mg daily. Patients were excluded if cirrhosis, other chronic liver disease, elevated alcohol consumption, or confounding conditions were present. The primary endpointsfor the month-18 interim analysis were fibrosis improvement (≥1 stage) with no worsening of NASH, or NASH resolution with no worsening of fibrosis, with the study considered successful if either primary endpoint was met. Primary analyses were done by intention to treat, in patients with fibrosis stage F2–F3 who received at least one dose of treatment and reached, or would have reached, the month 18 visit by the prespecified interim analysis cutoff date. The study also evaluated other histological and biochemical markers of NASH and fibrosis, and safety. This study is ongoing, and registered with ClinicalTrials.gov, NCT02548351, and EudraCT, 20150-025601-6. Findings Between Dec 9, 2015, and Oct 26, 2018, 1968 patients with stage F1–F3 fibrosis were enrolled and received at least one dose of study treatment; 931 patients with stage F2–F3 fibrosis were included in the primary analysis (311 in the placebo group, 312 in the obeticholic acid 10 mg group, and 308 in the obeticholic acid 25 mg group). The fibrosis improvement endpoint was achieved by 37 (12%) patients in the placebo group, 55 (18%) in the obeticholic acid 10 mg group (p=0·045), and 71 (23%) in the obeticholic acid 25 mg group (p=0·0002). The NASH resolution endpoint was not met (25 [8%] patients in the placebo group, 35 [11%] in the obeticholic acid 10 mg group [p=0·18], and 36 [12%] in the obeticholic acid 25 mg group [p=0·13]). In the safety population (1968 patients with fibrosis stages F1–F3), the most common adverse event was pruritus (123 [19%] in the placebo group, 183 [28%] in the obeticholic acid 10 mg group, and 336 [51%] in the obeticholic acid 25 mg group); incidence was generally mild to moderate in severity. The overall safety profile was similar to that in previous studies, and incidence of serious adverse events was similar across treatment groups (75 [11%] patients in the placebo group, 72 [11%] in the obeticholic acid 10 mg group, and 93 [14%] in the obeticholic acid 25 mg group). Interpretation Obeticholic acid 25 mg significantly improved fibrosis and key components of NASH disease activity among patients with NASH. The results from this planned interim analysis show clinically significant histological improvement that is reasonably likely to predict clinical benefit. This study is ongoing to assess clinical outcomes

Molecular and metabolic mechanisms of transgenerational effects in Daphnia exposed to radionuclides

International audienceUnderstanding how radioactive contaminants affect wildlife species at every level of biological organization is a major challenge in radioecology. Mechanistic links among observed effects are necessary to predict consequences for survival, growth and reproduction which are critical for population dynamics. With its short life cycle, the cladoceran Daphnia magna is particularly suitable to address toxicity over several generations, on a much more relevant time scale to natural biota. Multigenerational toxicity tests were conducted with depleted uranium (U), americium-241 (Am-241) and cesium-137 (Cs-137), inducing respectively a chemical toxicity, an internal alpha radiotoxicity and an external gamma radiotoxicity. Experimental results showed in all cases that toxic effects on survival, body size, fecundity increased in severity across generations, demonstrating that measured effects in one generation might not be representative of toxicity in offspring generations, and ultimately of population response.Reduction in body size and fecundity induced by depleted U, Cs-137 and Am-241 were analyzed using DEBtox models with respectively internal concentration, external gamma dose rate and internal alpha dose rate as dose metrics. For each radionuclide, a combination of several modes of action was necessary to explain observed effects. A damage compartment with hereditary damage level was introduced in order to explore possible modes of action associated with the increase in toxicity across generations. Modelling was performed using a Bayesian framework, in order to quantify uncertainty in parameter estimations and model predictions. Studies of DNA alterations, using a qPCR technique, confirmed that molecular damage was accumulated in daphnids exposed to depleted U and Cs-137 and transmitted to their progeny, as a possible underlying mechanism causing the increase in effect severity over generations. Further studies of DNA methylation recently investigated the role of epigenetic processes in the transmission of effects from parents exposed to Cs-137 to their unexposed progeny

Molecular and metabolic mechanisms of transgenerational effects in Daphnia exposed to radionuclides

International audienceUnderstanding how radioactive contaminants affect wildlife species at every level of biological organization is a major challenge in radioecology. Mechanistic links among observed effects are necessary to predict consequences for survival, growth and reproduction which are critical for population dynamics. With its short life cycle, the cladoceran Daphnia magna is particularly suitable to address toxicity over several generations, on a much more relevant time scale to natural biota. Multigenerational toxicity tests were conducted with depleted uranium (U), americium-241 (Am-241) and cesium-137 (Cs-137), inducing respectively a chemical toxicity, an internal alpha radiotoxicity and an external gamma radiotoxicity. Experimental results showed in all cases that toxic effects on survival, body size, fecundity increased in severity across generations, demonstrating that measured effects in one generation might not be representative of toxicity in offspring generations, and ultimately of population response.Reduction in body size and fecundity induced by depleted U, Cs-137 and Am-241 were analyzed using DEBtox models with respectively internal concentration, external gamma dose rate and internal alpha dose rate as dose metrics. For each radionuclide, a combination of several modes of action was necessary to explain observed effects. A damage compartment with hereditary damage level was introduced in order to explore possible modes of action associated with the increase in toxicity across generations. Modelling was performed using a Bayesian framework, in order to quantify uncertainty in parameter estimations and model predictions. Studies of DNA alterations, using a qPCR technique, confirmed that molecular damage was accumulated in daphnids exposed to depleted U and Cs-137 and transmitted to their progeny, as a possible underlying mechanism causing the increase in effect severity over generations. Further studies of DNA methylation recently investigated the role of epigenetic processes in the transmission of effects from parents exposed to Cs-137 to their unexposed progeny

Mechanistic and ecophysiological study of carbon 14 transfer in fish

International audienceCarbon-14 (14C) is one of the two radionuclides which are released in greatest amounts into rivers by the nuclear power plants in routine operation. In France, 14C contributes to a major fraction of the estimated annual dose absorbed by local populations living in the vicinity of nuclearized rivers, mainly by ingestion of radiocontaminated fish. However, mathematical models used to assess the transfer of 14C to the aquatic ecosystems are unable to explain 14C values observed in sampled fish. One of the reason for this is that these models are very simplistic, neglecting the potential influence of changes in 14C speciation, biochemical transformations occurring along trophic chains and the potential influence of fish physiology as a function of environmental factors, such as temperature or food. In this context, the present PhD project aims to adapt the conceptual approach known as the “Dynamic Isotope Budget” (DIB), an extension of the Dynamic Energetic Budget (DEB) used to describe stable isotope fluxes in organisms, to the case of 14C transfers among water, sediment, phytoplankton, zooplankton, and fish. In this aim, Cyprinus carpio is selected as a robust laboratory species, commonly found during radioecological monitoring in French rivers and available in the Add-My-Pet collection. First, a literature search is conducted and size and reproduction data are used to improve the parameterization of the DEB model for C. carpio. Second, sensitivity analyses (MORRIS, SOBOL’) are carried out to identify most influent parameters. Finally, on-going experiments are conducted to quantify transfer efficiencies of 14C in different processes of the DIB model using different forms of 14C (glycerol, arginine, glucose, as precursors in lipid, protein and glucid metabolims)

Mechanistic and ecophysiological study of carbon 14 transfer in fish

International audienceCarbon-14 (14C) is one of the two radionuclides which are released in greatest amounts into rivers by the nuclear power plants in routine operation. In France, 14C contributes to a major fraction of the estimated annual dose absorbed by local populations living in the vicinity of nuclearized rivers, mainly by ingestion of radiocontaminated fish. However, mathematical models used to assess the transfer of 14C to the aquatic ecosystems are unable to explain 14C values observed in sampled fish. One of the reason for this is that these models are very simplistic, neglecting the potential influence of changes in 14C speciation, biochemical transformations occurring along trophic chains and the potential influence of fish physiology as a function of environmental factors, such as temperature or food. In this context, the present PhD project aims to adapt the conceptual approach known as the “Dynamic Isotope Budget” (DIB), an extension of the Dynamic Energetic Budget (DEB) used to describe stable isotope fluxes in organisms, to the case of 14C transfers among water, sediment, phytoplankton, zooplankton, and fish. In this aim, Cyprinus carpio is selected as a robust laboratory species, commonly found during radioecological monitoring in French rivers and available in the Add-My-Pet collection. First, a literature search is conducted and size and reproduction data are used to improve the parameterization of the DEB model for C. carpio. Second, sensitivity analyses (MORRIS, SOBOL’) are carried out to identify most influent parameters. Finally, on-going experiments are conducted to quantify transfer efficiencies of 14C in different processes of the DIB model using different forms of 14C (glycerol, arginine, glucose, as precursors in lipid, protein and glucid metabolims)

Short-term accumulation and elimination of carbon-14 in the common carp Cyprinus carpio under laboratory conditions

International audienceThis study examined the short term transfer of carbon-14 (14C) in the common carp Cyprinus carpio under laboratory conditions. Various experiments were achieved in order to investigate direct or trophic transfer for 4 days, using waterborne 14C-labelled arginine or 14C-labelled food pellets respectively. Radiolabelled food was prepared with 14C-labelled arginine or glucose in order to test how transfer kinetics might vary with the biochemical form of 14C. Elimination experiments were achieved using fish fed for 5 days on radiolabelled food and then placed under starvation for 4 days. In all experiments, water, food and fish activities were monitored every day. Different fish fractions (whole body, muscle) were sampled in order to elucidate the role of muscle as a potential storage. Results suggested that direct water-to-fish absorption rate was 20% d-1 per fish. Carps incorporated 14.3% of the absorbed 14C. Fish activity did not increase over days, due to a strong decrease in 14C concentration in the water (resulting from aquarium sorption). During trophic transfer experiments, food was entirely ingested and 14C sources rapidly assimilated. For either arginine or glucose, results suggested that 19-20% of ingested 14C was incorporated, yielding a significant increase in fish activity over days. No difference in mass-specific activity was observed among muscle and whole body. Total activity in the muscle represented 29%-32% of whole body activity, this proportion reflecting the contribution of muscle to whole body weight. During elimination experiments, results showed a significant decrease in whole body total activity and mass-specific activity with arginine. The decrease was not significant with glucose due to a great variability among fish. Results suggested that an essential amino-acid like arginine can be used as an energy source under starvation and that muscles can act as a storage for essential amino-acids

Epigenetic modifications as a molecular mechanism for transgenerational effects in Daphnia exposed to ionising radiation

International audienceIn order to predict long term consequences of pollution on wildlife, toxic effects have to be assessed on a multigenerational scale. DEBtox models were recently developped to analyze effects of alpha and gamma radiation over several generations of Daphnia magna. A transgenerational damage compartement which level is transmitted from parent to offspring, was used to account for changes in effect severity observed among generations. However, the nature of molecular mechanisms involved in the transmission of radio-induced damage accross generations are poorly understood. In this context, the role of epigenetic processes was recently investigated in D. magna, as part of the transgenerational response to external Cs-137 gamma irradiation (at dose rates of 6.5 µGy/h and 41.3 mGy/h). In this aim, we studied DNA methylation changes after a 25-day chronic exposure of a parental generation (F0) and their potential inheritance by subsequent recovering generations, including F2 (exposed as germ cells in F1 embryos) and F3 (the first truly unexposed generation). Effects on survival, growth and reproduction were examined in all generations and DNA was extracted for whole genome bisulfite sequencing. Results showed that effects on reproduction were significant in generation F0 at the high dose rate and absent in subsequent generations (F1, F2 and F3). In contrast, we detected significant DNA methylation changes in every generation independent of dose rate. Some of these changes were shared between dose rates and among generations. Involved genes included gene families and gene functions which were previously shown to play roles during exposure to ionising radiation. Common DNA methylation changes detected between generations F2 and F3 clearly showed that epigenetic modifications were transmitted to unexposed generations, most likely through the germline

Transgenerational effects and epigenetic inheritance following a chronic external gamma irradiation in Daphnia magna

Aquatic and terrestrial ecosystems are exposed to radionuclides through planned discharges or accidental releases linked with the nuclear industry activities. Over the past decade, ecosystems protection against ionizing radiation has become a growing public, regulatory and scientific concern. Nowadays, ecological risk assessment for ionizing radiation is based on a majority of radiotoxocity tests under acute doses and short exposure time, far from realistic environmental conditions in which natural biota are actually exposed. Recently, studies in daphnids at chronic low doses have been conducted over several generations in order to understand the long-term effects of low doses of gamma radiation and explore the contribution of underlying molecular mechanisms, such as DNA alterations. In this context, radio-induced epigenetic modifications have never been investigated in invertebrate species although ionizing radiation were shown to induce changes in DNA methylation patterns and gene expression. This suggests that epigenetic modifications might be inherited across generations. In this context, the present study examines the potential transmission of epigenetic alterations caused by a life-long gamma irradiation to their non-irradiated progeny up to the third generation, using the microcrustacean Daphnia magna as a model organism. The aim of our study is to test whether effects on survival, growth and reproduction observed in daphnids exposed to gamma radiation (in generation referred as F0) might persist in their non-exposed offspring generations (referred as F1, F2 and F3) and whether it might be linked to modifications of DNA methylation patterns inherited across generations. Results showed significant effects of gamma radiation on fecundity only for the highest dose rate and no effect at organism level seemed to last across non-exposed generations. However, effects on DNA methylation at low dose rate and across generations are not excluded. Our study will give a better insight in the effect of ionizing radiation on epigenetic processes, their heritability across generation and their link to organism level effects

Combined exposures to stable and radioactive substances for ecological risk assessment

International audienc

Issues and practices in the use of effects data from FREDERICA in the ERICA Integrated Approach

The ERICA Integrated Approach requires that a risk assessment screening dose rate is defined for the risk characterisation within Tiers 1 and 2. At Tier 3, no numerical screening dose rate is used, and the risk characterisation is driven by methods that can evaluate the possible effects of ionising radiation on reproduction, mortality and morbidity. Species sensitivity distribution has been used to derive the ERICA risk assessment predicted no-effect dose rate (PNEDR). The method used was based on the mathematical processing of data from FRED (FASSET radiation effects database merged with the EPIC database to form FREDERICA) and resulted in a PNEDR of 10 μGy/h. This rate was assumed to ascribe sufficient protection of all ecosystems from detrimental effects on structure and function under chronic exposure. The value was weighed against a number of points of comparison: (i) PNEDR values obtained by application of the safety factor method, (ii) background levels, (iii) dose rates triggering effects on radioactively contaminated sites and (iv) former guidelines from literature reviews. In Tier 3, the effects analysis must be driven by the problem formulation and is thus highly case specific. Instead of specific recommendations on numeric values, guidance on the sorts of methods that may be applied for refined effect analysis is provided and illustrated