Integrated testing strategy for the study of the effects of the human pharmaceutical dutasteride on fish

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.In recent years, a growing number of human pharmaceuticals have been detected in the aquatic environment, generally at low concentrations (sub-ng/L to low μg/L). These compounds are characterised by highly specific mechanisms of action, high potency and prolonged activity in order to minimise dosing requirements and potential toxicity in patients. Among the various classes of pharmaceuticals, steroids and anti-steroids are widely used, as shown by the analysis of their clinical use carried out at the beginning of this Ph.D. project. Although the amounts used are much lower than the amounts of some other pharmaceuticals (e.g. analgesics), their ability to affect important physiological processes in fish (e.g. reproduction) at very low concentrations (ng/L) suggest that this class of compounds should represent a high priority for ecotoxicological research. In particular, this Ph.D. project addressed the question of whether or not dutasteride, a human pharmaceutical mainly used to treat benign prostatic hyperplasia, may cause adverse effects in the teleost fathead minnow (Pimephales promelas) by inhibiting the activity of both isoforms of 5α-reductase (5αR), the enzyme which convert testosterone into dihydrotestosterone (DHT). The theoretical framework used to guide the design of the experimental studies was based on the combination of several conceptual approaches, including the study of the evolutionary degree of conservation and functionality of the drug target in non-target species, and the cross-species extrapolation of pharmacological and toxicological information generated during pre-clinical and clinical studies in mammals during drug development. The results obtained during the first phase of this Ph.D. project strongly suggested that DHT has a physiological role in the fathead minnow. In fact, 5αRs are evolutionary conserved in this species, 5αRs genes are expressed in tissues such as the testis, and DHT circulates in fathead minnow plasma at concentrations similar to those detected in humans. These findings represented the rationale for testing the effects of dutasteride in the fathead minnow. Dutasteride caused significant adverse effects in all the in vivo studies performed in order to evaluate its potential toxicity on fish, including early life stage and short term reproduction studies, and all the tested life stages were sensitive to the inhibition of 5αRs activity; however, none of the observed adverse effects occurred at concentrations of exposure lower than 32 μg/L (measured concentration). The results also showed that female fish are highly sensitive to disruption of the androgenic pathways, highlighting their utility for the evaluation of potential adverse effects caused by anti-androgens on fish. In conclusion, the results presented in this Thesis suggest that, at present, the potential presence of dutasteride in the environment does not represent a risk to wild fish populations, due to the high concentrations required to elict significant adverse effect (LOEC = 32 μg/L) and the low volume of drug prescribed every year (5.07 kg in UK in 2006). However, the high bioaccumulation factor of dutasteride suggest that further studies should be conducted to elucidate the role played by the bioaccumulation process in the toxicity responses observed in fish.This work is funded by GlaxoSmithKlin

Integrated testing strategy for the study of the effects of the human pharmaceutical dutasteride on fish

In recent years, a growing number of human pharmaceuticals have been detected in the aquatic environment, generally at low concentrations (sub-ng/L to low μg/L). These compounds are characterised by highly specific mechanisms of action, high potency and prolonged activity in order to minimise dosing requirements and potential toxicity in patients. Among the various classes of pharmaceuticals, steroids and anti-steroids are widely used, as shown by the analysis of their clinical use carried out at the beginning of this Ph.D. project. Although the amounts used are much lower than the amounts of some other pharmaceuticals (e.g. analgesics), their ability to affect important physiological processes in fish (e.g. reproduction) at very low concentrations (ng/L) suggest that this class of compounds should represent a high priority for ecotoxicological research. In particular, this Ph.D. project addressed the question of whether or not dutasteride, a human pharmaceutical mainly used to treat benign prostatic hyperplasia, may cause adverse effects in the teleost fathead minnow (Pimephales promelas) by inhibiting the activity of both isoforms of 5α-reductase (5αR), the enzyme which convert testosterone into dihydrotestosterone (DHT). The theoretical framework used to guide the design of the experimental studies was based on the combination of several conceptual approaches, including the study of the evolutionary degree of conservation and functionality of the drug target in non-target species, and the cross-species extrapolation of pharmacological and toxicological information generated during pre-clinical and clinical studies in mammals during drug development. The results obtained during the first phase of this Ph.D. project strongly suggested that DHT has a physiological role in the fathead minnow. In fact, 5αRs are evolutionary conserved in this species, 5αRs genes are expressed in tissues such as the testis, and DHT circulates in fathead minnow plasma at concentrations similar to those detected in humans. These findings represented the rationale for testing the effects of dutasteride in the fathead minnow. Dutasteride caused significant adverse effects in all the in vivo studies performed in order to evaluate its potential toxicity on fish, including early life stage and short term reproduction studies, and all the tested life stages were sensitive to the inhibition of 5αRs activity; however, none of the observed adverse effects occurred at concentrations of exposure lower than 32 μg/L (measured concentration). The results also showed that female fish are highly sensitive to disruption of the androgenic pathways, highlighting their utility for the evaluation of potential adverse effects caused by anti-androgens on fish. In conclusion, the results presented in this Thesis suggest that, at present, the potential presence of dutasteride in the environment does not represent a risk to wild fish populations, due to the high concentrations required to elict significant adverse effect (LOEC = 32 μg/L) and the low volume of drug prescribed every year (5.07 kg in UK in 2006). However, the high bioaccumulation factor of dutasteride suggest that further studies should be conducted to elucidate the role played by the bioaccumulation process in the toxicity responses observed in fish.EThOS - Electronic Theses Online ServiceGlaxoSmithKlineGBUnited Kingdo

Big Question To Developing Solutions : A Decade of Progress in the Development of Aquatic New Approach Methodologies from 2012 to 2022

In 2012, 20 key questions related to hazard and exposure assessment and environmental and health risks of pharmaceuticals and personal care products in the natural environment were identified. A decade later, this article examines the current level of knowledge around one of the lowest-ranking questions at that time, number 19: "Can nonanimal testing methods be developed that will provide equivalent or better hazard data compared with current in vivo methods?" The inclusion of alternative methods that replace, reduce, or refine animal testing within the regulatory context of risk and hazard assessment of chemicals generally faces many hurdles, although this varies both by organism (human-centric vs. other), sector, and geographical region or country. Focusing on the past 10 years, only works that might reasonably be considered to contribute to advancements in the field of aquatic environmental risk assessment are highlighted. Particular attention is paid to methods of contemporary interest and importance, representing progress in (1) the development of methods which provide equivalent or better data compared with current in vivo methods such as bioaccumulation, (2) weight of evidence, or (3) -omic-based applications. Evolution and convergence of these risk assessment areas offer the basis for fundamental frameshifts in how data are collated and used for the protection of taxa across the breadth of the aquatic environment. Looking to the future, we are at a tipping point, with a need for a global and inclusive approach to establish consensus. Bringing together these methods (both new and old) for regulatory assessment and decision-making will require a concerted effort and orchestration. Environ Toxicol Chem 2023;00:1-15. (c) 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.Peer reviewe

Uptake and Metabolism of Human Pharmaceuticals by Fish - A Case Study with the Opioid Analgesic Tramadol.

This document is the unedited Author’s version of a Submitted Work that was subsequently accepted for publication in Environmental science & technology, copyright © American Chemical Society after peer review. To access the final edited and published work see https://doi.org/10.1021/acs.est.7b03441Recent species-extrapolation approaches to predict the potential effects of pharmaceuticals present in the environment on wild fish are based on the assumption that pharmacokinetics and metabolism in humans and fish are comparable. To test this hypothesis, we exposed fathead minnows to the opiate pro-drug tramadol and examined uptake from the water into the blood and brain, and metabolism of the drug into its main metabolites. We found that plasma concentrations could be predicted reasonably accurately based on the lipophilicity of the drug, once the pH of the water was taken into account. The concentrations of the drug and its main metabolites were higher in the brain than in the plasma, and the observed brain/plasma concentration ratios were within the range of values reported in mammalian species. This fish species was able to metabolise the pro-drug tramadol into the highly active metabolite O-desmethyl tramadol and the inactive metabolite N-desmethyl tramadol in a similar manner to mammals. However, we found that concentration ratios of O-desmethyl tramadol to tramadol were lower in the fish than values in most humans administered the drug. Our pharmacokinetic data of tramadol in fish help bridge the gap between widely available mammalian pharmacological data and potential effects on aquatic organisms, and highlight the importance of understanding drug uptake and metabolism in fish to enable the full implementation of predictive toxicology approaches.We would like to thank members of the Ecotoxicology Research Group, Brunel University London, particularly J. Walker, N. Brodigan, and A. Ferreira for fish husbandry, and T. Thrupp, E. Lawton, and A. Baynes for fish sampling. The research at Brunel University London was internally funded by the university. This study was also supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT) to a project on Joint Usage/Research Center– Leading Academia in Marine and Environment Pollution Research (LaMer), and Research Fellowships from the Japan Society for the Promotion of Science (JSPS) for Young Scientists in Japan (PD) provided to R. Tanoue (26·2800), Grants in Bid (KAKENHI) for Scientific Research (A) (25257403), Scientific Research (A) (16H01784). This study was also funded by the Sasakawa Scientific Research Grant from The Japan Science Society

Quantitative cross-species extrapolation between humans and fish: The case of the anti-depressant fluoxetine

This article has been made available through the Brunel Open Access Publishing Fund.Fish are an important model for the pharmacological and toxicological characterization of human pharmaceuticals in drug discovery, drug safety assessment and environmental toxicology. However, do fish respond to pharmaceuticals as humans do? To address this question, we provide a novel quantitative cross-species extrapolation approach (qCSE) based on the hypothesis that similar plasma concentrations of pharmaceuticals cause comparable target-mediated effects in both humans and fish at similar level of biological organization (Read-Across Hypothesis). To validate this hypothesis, the behavioural effects of the anti-depressant drug fluoxetine on the fish model fathead minnow (Pimephales promelas) were used as test case. Fish were exposed for 28 days to a range of measured water concentrations of fluoxetine (0.1, 1.0, 8.0, 16, 32, 64 μg/L) to produce plasma concentrations below, equal and above the range of Human Therapeutic Plasma Concentrations (HTPCs). Fluoxetine and its metabolite, norfluoxetine, were quantified in the plasma of individual fish and linked to behavioural anxiety-related endpoints. The minimum drug plasma concentrations that elicited anxiolytic responses in fish were above the upper value of the HTPC range, whereas no effects were observed at plasma concentrations below the HTPCs. In vivo metabolism of fluoxetine in humans and fish was similar, and displayed bi-phasic concentration-dependent kinetics driven by the auto-inhibitory dynamics and saturation of the enzymes that convert fluoxetine into norfluoxetine. The sensitivity of fish to fluoxetine was not so dissimilar from that of patients affected by general anxiety disorders. These results represent the first direct evidence of measured internal dose response effect of a pharmaceutical in fish, hence validating the Read-Across hypothesis applied to fluoxetine. Overall, this study demonstrates that the qCSE approach, anchored to internal drug concentrations, is a powerful tool to guide the assessment of the sensitivity of fish to pharmaceuticals, and strengthens the translational power of the cross-species extrapolation

The read-across hypothesis and environmental risk assessment of pharmaceuticals

This article is made available through the Brunel Open Access Publishing Fund. Copyright © 2013 American Chemical Society.Pharmaceuticals in the environment have received increased attention over the past decade, as they are ubiquitous in rivers and waterways. Concentrations are in sub-ng to low μg/L, well below acute toxic levels, but there are uncertainties regarding the effects of chronic exposures and there is a need to prioritise which pharmaceuticals may be of concern. The read-across hypothesis stipulates that a drug will have an effect in non-target organisms only if the molecular targets such as receptors and enzymes have been conserved, resulting in a (specific) pharmacological effect only if plasma concentrations are similar to human therapeutic concentrations. If this holds true for different classes of pharmaceuticals, it should be possible to predict the potential environmental impact from information obtained during the drug development process. This paper critically reviews the evidence for read-across, and finds that few studies include plasma concentrations and mode of action based effects. Thus, despite a large number of apparently relevant papers and a general acceptance of the hypothesis, there is an absence of documented evidence. There is a need for large-scale studies to generate robust data for testing the read-across hypothesis and developing predictive models, the only feasible approach to protecting the environment.BBSRC Industrial Partnership Award BB/ I00646X/1 and BBSRC Industrial CASE Partnership Studentship BB/I53257X/1 with AstraZeneca Safety Health and Environment Research Programme

Response-Adapted Postinduction Strategy in Patients With Advanced-Stage Follicular Lymphoma: The FOLL12 Study

Purpose: We compared 2 years of rituximab maintenance (RM) with a response-adapted postinduction approach in patients with follicular lymphoma who responded to induction immunochemotherapy. Methods: We randomly assigned treatment-naïve, advanced-stage, high-tumor burden follicular lymphoma patients to receive standard RM or a response-adapted postinduction approach on the basis of metabolic response and molecular assessment of minimal residual disease (MRD). The experimental arm used three types of postinduction therapies: for complete metabolic response (CMR) and MRD-negative patients, observation; for CMR and MRD-positive (end of induction or follow-up) patients, four doses of rituximab (one per week, maximum three courses) until MRD-negative; and for non-CMR patients, one dose of ibritumomab tiuxetan followed by standard RM. The study was designed as noninferiority trial with progression-free survival (PFS) as the primary end point. Results: Overall, 807 patients were randomly assigned. After a median follow-up of 53 months (range 1-92 months), patients in the standard arm had a significantly better PFS than those in the experimental arm (3-year PFS 86% v 72%; P < .001). The better PFS of the standard vs experimental arm was confirmed in all the study subgroups except non-CMR patients (n = 65; P = .274). The 3-year overall survival was 98% (95% CI, 96 to 99) and 97% (95% CI, 95 to 99) in the reference and experimental arms, respectively (P = .238). Conclusion: A metabolic and molecular response-adapted therapy as assessed in the FOLL12 study was associated with significantly inferior PFS compared with 2-year RM. The better efficacy of standard RM was confirmed in the subgroup analysis and particularly for patients achieving both CMR and MRD-negative

The goliath project: Towards an internationally harmonised approach for testing metabolism disrupting compounds

Copyright © 2020 by the authors. The purpose of this project report is to introduce the European “GOLIATH” project, a new research project which addresses one of the most urgent regulatory needs in the testing of endocrine-disrupting chemicals (EDCs), namely the lack of methods for testing EDCs that disrupt metabolism and metabolic functions. These chemicals collectively referred to as “metabolism disrupting compounds” (MDCs) are natural and anthropogenic chemicals that can promote metabolic changes that can ultimately result in obesity, diabetes, and/or fatty liver in humans. This project report introduces the main approaches of the project and provides a focused review of the evidence of metabolic disruption for selected EDCs. GOLIATH will generate the world’s first integrated approach to testing and assessment (IATA) specifically tailored to MDCs. GOLIATH will focus on the main cellular targets of metabolic disruption—hepatocytes, pancreatic endocrine cells, myocytes and adipocytes—and using an adverse outcome pathway (AOP) framework will provide key information on MDC-related mode of action by incorporating multi-omic analyses and translating results from in silico, in vitro, and in vivo models and assays to adverse metabolic health outcomes in humans at real-life exposures. Given the importance of international acceptance of the developed test methods for regulatory use, GOLIATH will link with ongoing initiatives of the Organisation for Economic Development (OECD) for test method (pre-)validation, IATA, and AOP development