Control compounds for preclinical drug-induced liver injury assessment: Consensus-driven systematic review by the ProEuroDILI network.

BACKGROUND & AIMS: Idiosyncratic drug-induced liver injury (DILI) is a complex and unpredictable event caused by drugs, and herbal or dietary supplements. Early identification of human hepatotoxicity at preclinical stages remains a major challenge, in which the selection of validated in vitro systems and test drugs has a significant impact. In this systematic review, we analyzed the compounds used in hepatotoxicity assays and established a list of DILI-positive and -negative control drugs for validation of in vitro models of DILI, supported by literature and clinical evidence and endorsed by an expert committee from the COST Action ProEuroDILI Network (CA17112). METHODS: Following 2020 PRISMA guidelines, original research articles focusing on DILI which used in vitro human models and performed at least one hepatotoxicity assay with positive and negative control compounds, were included. Bias of the studies was assessed by a modified 'Toxicological Data Reliability Assessment Tool'. RESULTS: A total of 51 studies (out of 2,936) met the inclusion criteria, with 30 categorized as reliable without restrictions. Although there was a broad consensus on positive compounds, the selection of negative compounds lacked clarity. 2D monoculture, short exposure times and cytotoxicity endpoints were the most tested, although there was no consensus on drug concentrations. CONCLUSIONS: Extensive analysis highlighted the lack of agreement on control compounds for in vitro DILI assessment. Following comprehensive in vitro and clinical data analysis together with input from the expert committee, an evidence-based consensus-driven list of 10 positive and negative control drugs for validation of in vitro models of DILI is proposed. IMPACT AND IMPLICATIONS: Prediction of human toxicity early in the drug development process remains a major challenge, necessitating the development of more physiologically relevant liver models and careful selection of drug-induced liver injury (DILI)-positive and -negative control drugs to better predict the risk of DILI associated with new drug candidates. Thus, this systematic study has crucial implications for standardizing the validation of new in vitro models of DILI. By establishing a consensus-driven list of positive and negative control drugs, the study provides a scientifically justified framework for enhancing the consistency of preclinical testing, thereby addressing a significant challenge in early hepatotoxicity identification. Practically, these findings can guide researchers in evaluating safety profiles of new drugs, refining in vitro models, and informing regulatory agencies on potential improvements to regulatory guidelines, ensuring a more systematic and efficient approach to drug safety assessment.

Toxicologic Pathology Forum: Opinion on Interpretive Challenges for Procedure-Related Effects Associated With Direct Central Nervous System Delivery of Oligonucleotides to Rodents, Dogs, and Nonhuman Primates.

Direct delivery of therapeutics to the central nervous system (CNS) greatly expands opportunities to treat neurological diseases but is technically challenging. This opinion outlines principal technical aspects of direct CNS delivery via intracerebroventricular (ICV) or intrathecal (IT) injection to common nonclinical test species (rodents, dogs, and nonhuman primates) and describes procedure-related clinical and histopathological effects that confound interpretation of test article-related effects. Direct dosing is by ICV injection in mice due to their small body size, while other species are dosed IT in the lumbar cistern. The most frequent procedure-related functional effects are transient absence of lower spinal reflexes after IT injection or death soon after ICV dosing. Common procedure-related microscopic findings in all species include leukocyte infiltrates in CNS meninges or perivascular (Virchow-Robin) spaces; nerve fiber degeneration in the spinal cord white matter (especially dorsal and lateral tracts compressed by dosing needles or indwelling catheters), spinal nerve roots, and sciatic nerve; meningeal fibrosis at or near IT injection sites; hemorrhage; and gliosis. Findings typically are minimal to occasionally mild. Findings tend to be more severe and/or have a higher incidence in the spinal cord segments and spinal nerve roots at or close to the site of administration.

City checking: Piloting the UK's first community-based drug safety testing (drug checking) service in 2 city centres.

AIMS: To explore the feasibility of delivering community-based drug safety testing (drug checking), to trial service design characteristics and to compare with festival-based testing. METHODS: In total, 171 substances of concern were submitted on 5 dates at 3 venues in 2 UK cities and tested using up to 6 analytical techniques. Test results and harm reduction advice were distributed directly to over 200 service users through 144 tailored healthcare consultations, to stakeholders, and through early warning systems, media and social media alerts. RESULTS: The 171 samples were submitted and identified as MDMA (43.3%), cocaine (12.9%), ketamine (12.9%), various psychedelics submitted by students, and heroin and a synthetic cannabinoid submitted by rough sleeping communities, with 76% of samples' test results as expected. The 144 primary service users identified as 91.7% white, 68.1% male, with an average age of 26.7 years. Reported harm reduction intentions included alerting friends and acquaintances (37.5%), being more careful mixing that substance (35.4%), lowered dosage (27.8%), disposal of further substances (6.9%) and additionally 2.8% handed over further substances for verified destruction. CONCLUSION: Community-based drug safety testing (drug checking) was piloted for the first time in the UK-within a drugs service, a community centre and a church-with consideration given to meso-level operational feasibility and micro-level behavioural outcomes. Service design characteristics such as venue, day of week, prior publicity, service provider, and direct and indirect dissemination of results all may impact on outcomes. Future studies should consider cost-benefit analyses of community and event-based testing and context-appropriate macro, meso and micro-level evaluations.

HepaRG culture in tethered spheroids as an in vitro three-dimensional model for drug safety screening.

Conventional two-dimensional (2D) monolayer cultures of HepaRG cells allow in vitro maintenance of many liver-specific functions. However, cellular dedifferentiation and functional deterioration over an extended culture period in the conventional 2D HepaRG culture have hampered its applications in drug testing. To address this issue, we developed tethered spheroids of HepaRG cells on Arg-Gly-Asp (RGD) and galactose-conjugated substratum with an optimized hybrid ratio as an in vitro three-dimensional (3D) human hepatocyte model. The liver-specific gene expression level and drug metabolizing enzyme activities in HepaRG-tethered spheorids were markedly higher than those in 2D cultures throughout the culture period of 7 days. The inducibility of three major cytochrome P450 (CYP) enzymes, namely CYP1A2, CYP2B6 and CYP3A4, was improved in both mRNA and activity level in tethered spheroids. Drug-induced cytotoxic responses to model hepatotoxins (acetaminophen, chlorpromazine and ketoconazole) in tethered spheroids were comparable to 2D cultures as well as other studies in the literature. Our results suggested that the HepaRG-tethered spheroid would be an alternative in vitro model suitable for drug safety screening.

Drug checking services for people who use drugs: a systematic review.

BACKGROUND AND AIMS: Drug checking services provide people who use drugs with chemical analysis results of their drug samples while simultaneously monitoring the unregulated drug market. We sought to identify and synthesize literature on the following domains: (a) the influence of drug checking services on the behaviour of people who use drugs; (b) monitoring of drug markets by drug checking services; and (c) outcomes related to models of drug checking services. METHODS: Systematic review. A systematic literature search was conducted in MEDLINE, Embase, Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, PsycINFO, Scopus, Web of Science and Dissertations and Theses Global. Eligible studies were peer-reviewed articles and conference abstracts or grey literature, published in any language since 1990 and including original data on the domains. We assessed risk of bias for quantitative peer-reviewed articles reporting on behaviour or models of drug checking services using National Institutes of Health tools. RESULTS: We screened 2463 titles and abstracts and 156 full texts, with 90 studies meeting inclusion criteria. Most (n = 65, 72.2%) were from Europe and used cross-sectional designs (n = 79, 87.7%). Monitoring of drug markets by drug checking services (n = 63, 70%) was the most reported domain, followed by the influence of drug checking services on behaviour (n = 31, 34.4%), including intent to use, actual use and disposal of the drug, and outcomes related to models of drug checking services (n = 17, 18.9%). The most common outcome measures were detection of unexpected substances (n = 50, 55.6%), expected substances (n = 44, 48.9%), new psychoactive substances (n = 40, 44.4%) and drugs of concern (n = 32, 36.5%) by drug checking services. CONCLUSIONS: Drug checking services appear to influence behavioural intentions and the behaviour of people who use drugs, particularly when results from drug checking services are unexpected or drugs of concern. Monitoring of drug markets by drug checking services is well established in Europe, and increasingly in North America. Concerns about drug contents and negative health consequences facilitate the use of drug checking services; lack of concern; trust in drug sellers; lack of accessibility of drug checking services; and legal and privacy concerns are barriers to use.

Visitors of the Dutch drug checking services: Profile and drug use experience.

BACKGROUND: Drug checking services (DCS) provide information about drug content and purity, alongside personalized feedback, to people who use drugs; however, the demographic and drug use characteristics of DCS clients are rarely reported. This paper describes these characteristics for clients of the Dutch DCS, the Drug Information and Monitoring System (DIMS). METHODS: 1,530 participants completed a pen-and-paper questionnaire at one of eight participating DCS in the Netherlands in 2018. RESULTS: The participants were mostly highly educated males in their twenties with no migration background. Experience with drugs prior to coming to the DCS was common. Only 0.7% indicated they had never used any of the twenty drugs studied. 93% of participants reported use of ecstasy or MDMA with an average of 6.3 years since first use. CONCLUSIONS: These results indicate that drug checking can be a valuable tool for public health services as it facilitates access to more difficult-to-reach communities who use drugs. It is unlikely that DCS encourage drug initiation, since almost all people who visit the Dutch DCS already report experience with drugs. However, DCS should be aware that their services might not be easily accessible or attractive to all demographic groups.

Drug safety testing, disposals and dealing in an English field: Exploring the operational and behavioural outcomes of the UK's first onsite 'drug checking' service.

BACKGROUND: In a year when UK drug-related deaths and festival drug-related deaths reached their highest on record, a pilot festival drug safety testing service was introduced with the aim of reducing drug-related harm. This paper describes the operational and behavioural outcomes of this pilot and explores the relationship between drug use, supply and policing within festival grounds. METHODS: Chemists in a temporary laboratory analysed 247 substances submitted by the public to a free, confidential testing service across four days at a UK festival in July 2016. Test results were returned to service users embedded in 230 healthcare consultations delivered to approximately 900 festival-goers (one in five drug using festival-goers) that included harm reduction advice and the opportunity to use a disposal service for further substances of concern. Consultation data were collected at point of care, matched with test results, coded and analysed using SPSS RESULTS: Test results revealed that one in five substances was not as sold or acquired. One in five service users utilised the disposal service for further substances of concern in their possession and another one in six moderated their consumption. Two thirds of those whose sample was missold disposed of further substances, compared with under one in ten whose sample was as sold. Service users who acquired substances onsite at the festival were more than twice as likely to have been missold them as those acquired offsite, were nearly twice as likely to use the disposal service and were on average two years younger. Women were more likely to be using the drug for the first time and more likely to use the disposal service. Test results were shared with emergency services; alerts issued across site and an unanticipated feedback loop occurred to some drug suppliers. CONCLUSION: This pilot suggests that festival-goers engage productively with onsite drug safety testing services when given the opportunity, such services can access harder-to-reach and new user groups and can play a part in reducing drug-related harm by identifying and informing service users, emergency services and offsite drug using communities about substances of concern. Disposals to the testing service for onward police destruction provide an externally corroborated measure of impact, reducing harm to the individual and others by removing such substances from site. Evidence of differential dealing onsite and its potential negative consequences has implications for future research and policing.

High-Throughput Analysis of Mitochondrial Oxygen Consumption.

Interest in the investigation of mitochondrial dysfunction has seen a resurgence over recent years due to the implication of such dysfunction in both drug-induced toxicity and a variety of disease states. Here we describe a methodology to assist in such investigations whereby the oxygen consumption of isolated mitochondria is assessed in a high-throughput fashion using a phosphorescent oxygen-sensitive probe , standard microtiter plates, and plate reader detection. The protocols provided describe the required isolation procedures, initial assay optimization, and subsequent compound screening. Typical data is also provided illustrating the expected activity levels as well as recommended plate maps and data analysis approaches.

Development and regulatory application of microRNA biomarkers.

MicroRNAs, a class of regulatory small non-coding RNAs, are emerging as promising biomarkers for different health outcomes. Due to their tissue specificity, stability in extracellular space and high conservation between preclinical test species, applications of novel miRNA-based biomarkers for drug safety testing regarding hepatotoxicity and cardiotoxicity are investigated. Furthermore, miRNA expression is altered by environmental exposure such as cigarette smoke or polychlorinated biphenyls. As a consequence, miRNAs potentially influence tumor suppressor genes and oncogenes and may influence carcinogenesis. This has raised the interest in the use of miRNA profiles for health risk assessment. This review summarizes the recent developments in miRNA research with focus on biomarkers for drug safety testing and biomarkers for health outcomes related to environmental exposures.