Generation of a drug-induced renal injury list to facilitate the development of new approach methodologies for nephrotoxicity.

Drug-induced renal injury (DIRI) causes >1.5 million adverse events annually in the USA alone. Although standard biomarkers exist for DIRI, they lack the sensitivity or specificity to detect nephrotoxicity before the significant loss of renal function. In this study, we describe the creation of DIRIL - a list of drugs associated with DIRI and nephrotoxicity - from two literature datasets with DIRI annotation, confirmed using FDA drug labeling. DIRIL comprises 317 orally administered drugs covering all 14 anatomical, therapeutic and chemical (ATC) classification categories. Of the 317 drugs, 171 were DIRI-positive and 146 were DIRI-negative. DIRIL will be a relevant and invaluable resource for discovery of new approach methods (NAMs) to predict the occurrence and possible severity of DIRI earlier in drug development.

Academic drug discovery: Challenges and opportunities.

There are many different approaches to drug discovery in academia, some of which are based broadly on the industrial model of discovering novel targets and then conducting screening within academic drug discovery centres to identify hit molecules. Here we describe our approach to drug discovery, which makes more efficient use of the capabilities and resources of the different stakeholders. Specifically, we have created a large portfolio of drug projects and conducted small amounts of derisking work to ensure projects are investment ready. In this feature we will describe this model, including its limitations and advantages, since we believe the ideas and concepts will be of interest to other academic institutions and consortia.

Data science in drug discovery safety: Challenges and opportunities.

Early de-risking of drug targets and chemistry is essential to provide drug projects with the best chance of success. Target safety assessments (TSAs) use target biology, gene and protein expression data, genetic information from humans and animals, and competitor compound intelligence to understand the potential safety risks associated with modulating a drug target. However, there is a vast amount of information, updated daily that must be considered for each TSA. We have developed a data science-based approach that allows acquisition of relevant evidence for an optimal TSA. This is built on expert-led conventional and artificial intelligence-based mining of literature and other bioinformatics databases. Potential safety risks are identified according to an evidence framework, adjusted to the degree of target novelty. Expert knowledge is necessary to interpret the evidence and to take account of the nuances of drug safety, the modality, and the intended patient population for each TSA within each project. Overall, TSAs take full advantage of the most recent developments in data science and can be used within drug projects to identify and mitigate risks, helping with informed decision-making and resource management. These approaches should be used in the earliest stages of a drug project to guide decisions such as target selection, discovery chemistry options, in vitro assay choice, and end points for investigative in vivo studies.

AI-based language models powering drug discovery and development.

The discovery and development of new medicines is expensive, time-consuming, and often inefficient, with many failures along the way. Powered by artificial intelligence (AI), language models (LMs) have changed the landscape of natural language processing (NLP), offering possibilities to transform treatment development more effectively. Here, we summarize advances in AI-powered LMs and their potential to aid drug discovery and development. We highlight opportunities for AI-powered LMs in target identification, clinical design, regulatory decision-making, and pharmacovigilance. We specifically emphasize the potential role of AI-powered LMs for developing new treatments for Coronavirus 2019 (COVID-19) strategies, including drug repurposing, which can be extrapolated to other infectious diseases that have the potential to cause pandemics. Finally, we set out the remaining challenges and propose possible solutions for improvement.

Species-Specific Urothelial Toxicity With an Anti-HIV Noncatalytic Site Integrase Inhibitor (NCINI) Is Related to Unusual pH-Dependent Physicochemical Changes.

GS-9695 and GS-9822 are next-generation noncatalytic site integrase inhibitors (NCINIs) with significantly improved potency against human immunodeficiency virus compared with previous drugs such as BI-224436. Development stopped due to vacuolation of the bladder urothelium seen in cynomolgus monkey but not in rat; this lesion was absent in equivalent preclinical studies with BI-224436 (tested in dog and rat). Lesions were unlikely to be attributable to target because NCINIs specifically target viral integrase protein and no mammalian homologue is known. Secondary pharmacology studies, mitochondrial toxicity studies, immunophenotyping, and analysis of proteins implicated in cell-cell interactions and/or bladder integrity (E-cadherin, pan-cytokeratin, uroplakins) failed to offer any plausible explanation for the species specificity of the lesion. Because it was characterized by inflammation and disruption of urothelial morphology, we investigated physicochemical changes in the bladder of cynomolgus monkey (urinary pH 5.5-7.4) that might not occur in the bladder of rats (urinary pH 7.3-8.5). In measurements of surface activity, GS-9822 showed an unusual transition from a monolayer to a bilayer at the air/water interface with decreasing pH, attributed to the strong association between drug molecules in adjacent bilayer leaflets and expected to be highly disruptive to the urothelium. Structural analysis of GS-9822 and GS-9695 showed zwitterionic characteristics over the range of pH expected in cynomolgus monkey but not rat urine. This exotic surface behavior is unlikely with BI-224436 since it would transition from neutral to cationic (never zwitterionic) with decreasing pH. These data provide useful insights to guide discovery and development of NCINIs, related compounds, and zwitterions.

Key Characteristics of Human Hepatotoxicants as a Basis for Identification and Characterization of the Causes of Liver Toxicity.

Hazard identification regarding adverse effects on the liver is a critical step in safety evaluations of drugs and other chemicals. Current testing paradigms for hepatotoxicity rely heavily on preclinical studies in animals and human data (epidemiology and clinical trials). Mechanistic understanding of the molecular and cellular pathways that may cause or exacerbate hepatotoxicity is well advanced and holds promise for identification of hepatotoxicants. One of the challenges in translating mechanistic evidence into robust decisions about potential hepatotoxicity is the lack of a systematic approach to integrate these data to help identify liver toxicity hazards. Recently, marked improvements were achieved in the practice of hazard identification of carcinogens, female and male reproductive toxicants, and endocrine disrupting chemicals using the key characteristics approach. Here, we describe the methods by which key characteristics of human hepatotoxicants were identified and provide examples for how they could be used to systematically identify, organize, and use mechanistic data when identifying hepatotoxicants.

A decade of toxicological trends: what the papers say.

Here we look at popular trends and concepts in toxicology over the decade 2009-2019. The top 10 concepts included methodological approaches such as zebrafish and genomics as well as broader concepts such as personalized medicine and adverse outcome pathways. The total number and rank order for each of the top 10 were tracked year by year via PubMed with >9500 papers contributing to the analysis. The data revealed a slow upward trend in the number of papers across all the concepts from 260 in 2009 to >1700 in 2019. Zebrafish, genomics and personalized medicine remained in the top four slots since 2009 with zebrafish dominating the rankings over the entire decade. Genomics was a strong second until 2013 when it was displaced first by the microbiome in 2014 and secondly by personalized medicine in 2015. Other notable trends were the ascendancy of the microbiome and adverse outcome pathways and the descendancy of hormesis and the 3Rs (replacement, reduction and refinement of animals in testing). The observation that the top four slots have been static over the past 4 years suggests that new ideas are introduced and increase in popularity until they find their place in scientific culture. This may suggest that relatively new concepts such as artificial intelligence and microphysiological systems have yet to find their steady state in the rankings. Similarly, as a relatively new player in toxicology, the full impact of the human microbiome on drug efficacy and safety remains to be seen.

New ideas for non-animal approaches to predict repeated-dose systemic toxicity: Report from an EPAA Blue Sky Workshop.

The European Partnership for Alternative Approaches to Animal Testing (EPAA) convened a 'Blue Sky Workshop' on new ideas for non-animal approaches to predict repeated-dose systemic toxicity. The aim of the Workshop was to formulate strategic ideas to improve and increase the applicability, implementation and acceptance of modern non-animal methods to determine systemic toxicity. The Workshop concluded that good progress is being made to assess repeated dose toxicity without animals taking advantage of existing knowledge in toxicology, thresholds of toxicological concern, adverse outcome pathways and read-across workflows. These approaches can be supported by New Approach Methodologies (NAMs) utilising modern molecular technologies and computational methods. Recommendations from the Workshop were based around the needs for better chemical safety assessment: how to strengthen the evidence base for decision making; to develop, standardise and harmonise NAMs for human toxicity; and the improvement in the applicability and acceptance of novel techniques. "Disruptive thinking" is required to reconsider chemical legislation, validation of NAMs and the opportunities to move away from reliance on animal tests. Case study practices and data sharing, ensuring reproducibility of NAMs, were viewed as crucial to the improvement of non-animal test approaches for systemic toxicity.

Collaboration, competition and publication in toxicology: views of British Toxicology Society members.

To ascertain attitudes to resourcing, collaboration and publication in toxicology, a survey was developed and distributed to British Toxicology Society (BTS) members. The survey comprised 14 questions with 5 response options (strongly agree; agree; conflicted; disagree; strongly disagree) and a free text box. One hundred completed surveys were received by the cut-off date for data analysis. Unsurprisingly, 60% of participants disagreed or strongly disagreed that toxicology research is adequately funded in the UK; only 12% agreed with this statement. A similar proportion of participants (53%) disagreed with the statement that funding councils give equal opportunity to toxicology whereas 31% were conflicted on this point. An overwhelming 97% of respondents agreed that collaboration is important in driving toxicology research whereas only 38% agreed that competition is important. When this question was broadened out beyond the discipline of toxicology, a similar profile was seen suggesting that participants held similar views on toxicology versus other types of research. Many respondents were conflicted regarding the role of competition both in toxicology and in other research disciplines. Free text comments suggested that some competition is good to drive quality but can be counterproductive when competing for limited resources. Most participants were in favour of making toxicology research data openly available (86%) and in favour of open access publication (89%) although there were reservations about the cost of open access. Many (60%) thought the current system of peer review is fair but 65% also supported the idea of double-blind peer review (where both reviewer and author are anonymized). Others suggested a step in the opposite direction towards increased transparency (revealing and holding reviewers to account) would be preferable. Overall, there was a broad theme in free text responses that the need for experienced toxicologists has increased at a time when training and investment in the discipline has declined. However, not all respondents held that view with some noting that toxicology both as a research and as an applied discipline is strong within the UK scientific community.

Innovative models for in vitro detection of seizure.

Data show that toxicity to the central nervous system (CNS) is the most frequent cause of safety failures during the clinical phase of drug development. CNS endpoints such as seizure pose a safety risk to patients and volunteers and can lead to a loss of competitiveness, delays, and increased costs. Current methods rely on detection in the nonclinical rodent and non-rodent studies required to support clinical trials. There are two main issues with this approach; seizure may be missed in the animal studies and, even if seizure is detected, significant resource has already been invested in the project by this stage. Thus, there is a need to develop improved screening methods that can be used earlier in drug discovery to predict seizure. Advances in stem cell biology coupled with an increased understanding of the role of ion channels in seizure offer an opportunity for a new paradigm in screening. Human derived induced pluripotent stem cells (hiPSCs) representative of almost all cellular subtypes present in the brain can be incorporated into physiologically relevant in vitro models that can be used to determine seizure risk using high-throughput methods. Akin to the success of screening against a panel of ion channels such as hERG to reduce cardiovascular safety liability, the involvement of ion channels in seizure suggests that a similar approach to early seizure detection is valid. Profiling of the ion channels expressed in hiPSC models showing the seizurogenic phenotype coupled with electrophysiological assessment of ion channel function could translate into an ion channel seizure panel for rapid and reliable in vitro detection of seizure. The mechanistic information gathered would support optimal drug design early in development before resources, animals and time have been wasted.

Drug discovery and development: Biomarkers of neurotoxicity and neurodegeneration.

IMPACT STATEMENT: Attrition in drug discovery and development remains a major challenge. Safety/toxicity is the most prevalent reason for failure with cardiovascular and CNS toxicities predominating. Non-invasive biomarkers of neurotoxicity would provide significant advantage by allowing earlier prediction of likely neurotoxicity in preclinical studies as well as facilitating clinical trials of new therapies for neurodegenerative conditions such as Parkinson's disease (PD) and multiple sclerosis (MS).

Collaboration and competition: ethics in toxicology.

From animal research through adverse events in clinical trials to health scares around food contamination, toxicology has frequently been a focus of scientific and societal concern. As these concerns shift with each new drug, new technology or public health scare, how can toxicology stay current, relevant and ethical? Two of the biggest ethical challenges in pharmaceutical toxicology are the use of animals in testing and the high safety-related attrition rates in new drug development. Both of these require progress in the discipline that will only be driven by research funding. Yet, very little is invested in these two fields compared with investment in new efficacy models, new disease targets and new technologies. How can this be addressed? Here, we explore current paradigms in toxicology that may have the potential for perceived or actual unethical ramifications. We discuss the underpinnings of such practices and make recommendations for change around peer review, resourcing, transparency and data sharing. These ideas build on the analysis presented in the 2004 Paton Prize lecture (Purchase, 2004) where issues around conflict of interest (COI), collaboration and competition in the context of ethical behaviours were highlighted. These areas are clearly relevant to many aspects of scientific research but here we focus on toxicology and specifically toxicology in the pharmaceutical industry.

Understanding drug targets: no such thing as bad news.

How can small-to-medium pharma and biotech companies enhance the chances of running a successful drug project and maximise the return on a limited number of assets? Having a full appreciation of the safety risks associated with proposed drug targets is a crucial element in understanding the unwanted side-effects that might stop a project in its tracks. Having this information is necessary to complement knowledge about the probable efficacy of a future drug. However, the lack of data-rich insight into drug-target safety is one of the major causes of drug-project failure today. Conducting comprehensive target-safety reviews early in the drug discovery process enables project teams to make the right decisions about which drug targets to take forward.

Translational Biomarkers of Neurotoxicity: A Health and Environmental Sciences Institute Perspective on the Way Forward.

Neurotoxicity has been linked to a number of common drugs and chemicals, yet efficient and accurate methods to detect it are lacking. There is a need for more sensitive and specific biomarkers of neurotoxicity that can help diagnose and predict neurotoxicity that are relevant across animal models and translational from nonclinical to clinical data. Fluid-based biomarkers such as those found in serum, plasma, urine, and cerebrospinal fluid (CSF) have great potential due to the relative ease of sampling compared with tissues. Increasing evidence supports the potential utility of fluid-based biomarkers of neurotoxicity such as microRNAs, F2-isoprostanes, translocator protein, glial fibrillary acidic protein, ubiquitin C-terminal hydrolase L1, myelin basic protein, microtubule-associated protein-2, and total tau. However, some of these biomarkers such as those in CSF require invasive sampling or are specific to one disease such as Alzheimer's, while others require further validation. Additionally, neuroimaging methodologies, including magnetic resonance imaging, magnetic resonance spectroscopy, and positron emission tomography, may also serve as potential biomarkers and have several advantages including being minimally invasive. The development of biomarkers of neurotoxicity is a goal shared by scientists across academia, government, and industry and is an ideal topic to be addressed via the Health and Environmental Sciences Institute (HESI) framework which provides a forum to collaborate on key challenging scientific topics. Here we utilize the HESI framework to propose a consensus on the relative potential of currently described biomarkers of neurotoxicity to assess utility of the selected biomarkers using a nonclinical model.

Skin sensitization induced Langerhans' cell mobilization: variable requirements for tumour necrosis factor-α.

Upon antigen/allergen recognition, epidermal Langerhans' cells (LC) are mobilized and migrate to the local lymph node where they play a major role in initiating or regulating immune responses. It had been proposed that all chemical allergens induce LC migration via common cytokine signals delivered by TNF-α and IL-1ß. Here the dependence of LC migration on TNF-α following treatment of mice with various chemical allergens has been investigated. It was found that under standard conditions the allergens oxazolone, paraphenylene diamine, and trimellitic anhydride, in addition to the skin irritant sodium lauryl sulfate, were unable to trigger LC mobilization in the absence of TNF-α signalling. In contrast, two members of the dinitrohalobenezene family (2,4-dinitrochlorobenzene [DNCB] and 2,4-dinitrofluorobenzene [DNFB]) promoted LC migration independently of TNF-R2 (the sole TNF-α receptor expressed by LC) and TNF-α although the presence of IL-1ß was still required. However, increasing doses of oxazolone overcame the requirement of TNF-α for LC mobilization, whereas lower doses of DNCB were still able to induce LC migration in a TNF-α-independent manner. These novel findings demonstrate unexpected heterogeneity among chemical allergens and furthermore that LC can be induced to migrate from the epidermis via different mechanisms that are either dependent or independent of TNF-α. Although the exact mechanisms with regard to the signals that activate LC have yet to be elucidated, these differences may translate into functional speciation that will likely impact on the extent and quality of allergic sensitization.

De novo LINE-1 retrotransposition in HepG2 cells preferentially targets gene poor regions of chromosome 13.

Long interspersed nuclear elements (Line-1 or L1s) account for ~17% of the human genome. While the majority of human L1s are inactive, ~80-100 elements remain retrotransposition competent and mobilize through RNA intermediates to different locations within the genome. De novo insertions of L1s account for polymorphic variation of the human genome and disruption of target loci at their new location. In the present study, fluorescence in situ hybridization and DNA sequencing were used to characterize retrotransposition profiles of L1(RP) in cultured human HepG2 cells. While expression of synthetic L1(RP) was associated with full-length and truncated insertions throughout the entire genome, a strong preference for gene-poor regions, such as those found in chromosome 13 was observed for full-length insertions. These findings shed light into L1 targeting mechanisms within the human genome and question the putative randomness of L1 retrotransposition.

An integrated characterization of serological, pathological, and functional events in doxorubicin-induced cardiotoxicity.

Many efficacious cancer treatments cause significant cardiac morbidity, yet biomarkers or functional indices of early damage, which would allow monitoring and intervention, are lacking. In this study, we have utilized a rat model of progressive doxorubicin (DOX)-induced cardiomyopathy, applying multiple approaches, including cardiac magnetic resonance imaging (MRI), to provide the most comprehensive characterization to date of the timecourse of serological, pathological, and functional events underlying this toxicity. Hannover Wistar rats were dosed with 1.25 mg/kg DOX weekly for 8 weeks followed by a 4 week off-dosing "recovery" period. Electron microscopy of the myocardium revealed subcellular degeneration and marked mitochondrial changes after a single dose. Histopathological analysis revealed progressive cardiomyocyte degeneration, hypertrophy/cytomegaly, and extensive vacuolation after two doses. Extensive replacement fibrosis (quantified by Sirius red staining) developed during the off-dosing period. Functional indices assessed by cardiac MRI (including left ventricular ejection fraction (LVEF), cardiac output, and E/A ratio) declined progressively, reaching statistical significance after two doses and culminating in "clinical" LV dysfunction by 12 weeks. Significant increases in peak myocardial contrast enhancement and serological cardiac troponin I (cTnI) emerged after eight doses, importantly preceding the LVEF decline to <50%. Troponin I levels positively correlated with delayed and peak gadolinium contrast enhancement, histopathological grading, and diastolic dysfunction. In summary, subcellular cardiomyocyte degeneration was the earliest marker, followed by progressive functional decline and histopathological manifestations. Myocardial contrast enhancement and elevations in cTnI occurred later. However, all indices predated "clinical" LV dysfunction and thus warrant further evaluation as predictive biomarkers.

Reducing attrition in drug development: smart loading preclinical safety assessment.

Entry into the crucial preclinical good laboratory practice (GLP) stage of toxicology testing triggers significant R&D investment yet >20% of AstraZeneca's potential new medicines have been stopped for safety reasons in this GLP phase alone. How could we avoid at least some of these costly failures? An analysis of historical toxicities that caused stopping ('stopping toxicities') showed that >50% were attributable to target organ toxicities emerging within 2 weeks of repeat dosing or to acute cardiovascular risks. By frontloading 2-week repeat-dose toxicity studies and a comprehensive assessment of cardiovascular safety, we anticipate a potential 50% reduction in attrition in the GLP phase. This will reduce animal use overall, save significant R&D costs and improve drug pipeline quality.

Reprogramming of the HepG2 genome by long interspersed nuclear element-1.

Long Interspersed Nuclear Element-1 (LINE-1 or L1) is an autonomous, mobile element within the human genome that transposes via a "copy and paste" mechanism and relies upon L1-encoded endonuclease and reverse transcriptase (RT) activities to compromise genome integrity. L1 has been implicated in various forms of cancer, but its role in the regulation of the oncogenic phenotype is not understood. The present studies were conducted to evaluate mechanisms of genetic regulatory control in HepG2 cells by human L1, or a D702Y mutant deficient in RT activity, and their influence on cellular phenotype. Forced expression of synthetic L1 ORF1p and ORF2p was associated with formation of cytoplasmic foci and minor association with the nuclear compartment. While de novo L1 mobilizations were only identified in cells expressing wild type L1, and were absent in the D702Y mutant, changes in gene expression profiles involved RT dependent as well as RT independent mechanisms. Synthetic L1 altered the expression of 24 in silico predicted genetic targets; ten of which showed RT-dependence, ten RT-independence, and four reciprocal regulatory control by both wild type and RT mutant. Of five targets examined, only VCAM1 and PTPRB colocalized with newly retrotransposed wild type L1. Biological discretization to partition patterns of gene expression into unique frequencies identified adhesion, inflammation, and cellular metabolism as key processes targeted for molecular interference with disruption of epithelial-to-mesenchymal programming seen irrespective of the RT phenotype. These findings establish L1 as a key regulator of genome plasticity and EMT via mechanisms independent of RT activity.

Target organ toxicities in studies conducted to support first time in man dosing: an analysis across species and therapy areas.

An analysis of target organ toxicities in first time in man (FTiM) toxicity studies for 77 AstraZeneca candidate drugs (CDs) was conducted across a range of therapy areas. In the rodent, the most frequently affected organ was the liver followed by adrenal glands, kidney, spleen, bone marrow and thymus. In non-rodent, liver and thymus were the most frequently affected organs, followed closely by the testis and GI tract. The profile of affected organs was largely similar across the therapy areas of respiratory and inflammation, cardiovascular/gastrointestinal and CNS/pain. The oncology/infection therapy area differed with a larger range of organs affected. For the 75 CDs for which both rodent and non-rodent studies were conducted, new target organs were identified in non-rodents for 43 of the CDs. Notably, the changes seen only in non-rodents included organ systems of high relevance for human risk assessment such as the liver, male reproductive tissues and CNS. Additionally, profiles were similar for those CDs that progressed into human trials and those that did not. Overall, our data provide new insights into drug toxicity profiles in pre-clinical species and additionally confirm the value of using non-rodents as a second species in toxicity testing to support human safety.