Drug Safety Testing Research Articles

The Neonatal and Juvenile Pig in Pediatric Drug Discovery and Development.

Pharmacotherapy in pediatric patients is challenging in view of the maturation of organ systems and processes that affect pharmacokinetics and pharmacodynamics. Especially for the youngest age groups and for pediatric-only indications, neonatal and juvenile animal models can be useful to assess drug safety and to better understand the mechanisms of diseases or conditions. In this respect, the use of neonatal and juvenile pigs in the field of pediatric drug discovery and development is promising, although still limited at this point. This review summarizes the comparative postnatal development of pigs and humans and discusses the advantages of the juvenile pig in view of developmental pharmacology, pediatric diseases, drug discovery and drug safety testing. Furthermore, limitations and unexplored aspects of this large animal model are covered. At this point in time, the potential of the neonatal and juvenile pig as nonclinical safety models for pediatric drug development is underexplored.

Generation of human liver organoids from pluripotent stem cell-derived hepatic endoderms.

BackgroundThe use of a personalized liver organoid derived from human-induced pluripotent stem cells (HuiPSCs) is advancing the use of in vitro disease models for the design of specific, effective therapies for individuals. Collecting patient peripheral blood cells for HuiPSC generation is preferable because it is less invasive; however, the capability of blood cell-derived HuiPSCs for hepatic differentiation and liver organoid formation remains uncertain. Moreover, the currently available methods for liver organoid formation require a multistep process of cell differentiation or a combination of hepatic endodermal, endothelial and mesenchymal cells, which is a major hurdle for the application of personalized liver organoids in high-throughput testing of drug toxicity and safety. To demonstrate the capability of blood cell-derived HuiPSCs for liver organoid formation without support from endothelial and mesenchymal cells.MethodsThe peripheral blood-derived HuiPSCs first differentiated into hepatic endoderm (HE) in two-dimensional (2D) culture on Matrigel-coated plates under hypoxia for 10 days. The HE was then collected and cultured in 3D culture using 50% Matrigel under ambient oxygen. The maturation of hepatocytes was further induced by adding hepatocyte growth medium containing HGF and oncostatin M on top of the 3D culture and incubating the culture for an additional 12–17 days. The function of the liver organoids was assessed using expression analysis of hepatocyte-specific gene and proteins. Albumin (ALB) synthesis, glycogen and lipid storage, and metabolism of indocyanine were evaluated. The spatial distribution of albumin was examined using immunofluorescence and confocal microscopy.ResultsCD34+ hematopoietic cell-derived HuiPSCs were capable of differentiating into definitive endoderm expressing SOX17 and FOXA2, hepatic endoderm expressing FOXA2, hepatoblasts expressing AFP and hepatocytes expressing ALB. On day 25 of the 2D culture, cells expressed SOX17, FOXA2, AFP and ALB, indicating the presence of cellular heterogeneity. In contrast, the hepatic endoderm spontaneously formed a spherical, hollow structure in a 3D culture of 50% Matrigel, whereas hepatoblasts and hepatocytes could not form. Microscopic observation showed a single layer of polygonal-shaped cells arranged in a 3D structure. The hepatic endoderm-derived organoid synthesis ALB at a higher level than the 2D culture but did not express definitive endoderm-specific SOX17, indicating the greater maturity of the hepatocytes in the liver organoids. Confocal microscopic images and quantitative ELISA confirmed albumin synthesis in the cytoplasm of the liver organoid and its secretion. Overall, 3D culture of the hepatic endoderm is a relatively fast, simple, and less laborious way to generate liver organoids from HuiPSCs that is more physiologically relevant than 2D culture.

Modelling and Simulation for Preclinical Cardiac Safety Assessment of Drugs with Human iPSC-Derived Cardiomyocytes

As a potentially life threatening side effect, pharmaceutical compounds may trigger cardiac arrhythmias by impeding the heart’s electrical and mechanical function. For this reason, any new compound needs to be tested since 2005 for its proarrhythmic risk both during the preclinical and the clinical phase of the drug development process. While intensive monitoring of cardiac activity during clinical tests with human volunteers constitutes a major cost factor, preclinical in vitro tests with non cardiac cells and in vivo tests with animals are currently under serious debate because of their poor extrapolation to drug cardiotoxicity in humans. For about five years now, regulatory agencies, industry and academia are working on an overhaul of the cardiac drug safety paradigm that is built a) on human heart muscle cells, that can be abundantly bioengineered from donor stem cells without ethical concerns (human induced pluripotent stem cell derived cardiomyocytes, hiPSC-CMs), and b) on computational models of human cardiac electrophysiology both at the cellular and the organ level. The combined use of such human in vitro and human in silico models during the preclinical phase is expected to improve proarrhythmia test specificity (i.e. to lower the false-positive rate), to better inform about the need of thorough heart monitoring in the clinic, and to reduce or even replace animal experiments. This review article starts by concisely informing about the electrical activity of the human heart, about its possible impairment due to drug side effects, and about hiPSC-CM assays for cardiac drug safety testing. It then summarizes the mathematical description of human cardiac electrophysiology in terms of mechanistic ODE and PDE models, and illustrates how their numerical analysis may provide insight into the genesis of drug induced arrhythmias. Finally, this paper surveys proarrhythmic risk estimation methods, that involve the simulation of human heart muscle cells, and addresses opportunities and challenges for future interdisciplinary research.

Integrating in vitro data and physiologically based kinetic modeling-facilitated reverse dosimetry to predict human cardiotoxicity of methadone

Development of novel testing strategies to detect adverse human health effects is of interest to replace in vivo-based drug and chemical safety testing. The aim of the present study was to investigate whether physiologically based kinetic (PBK) modeling-facilitated conversion of in vitro toxicity data is an adequate approach to predict in vivo cardiotoxicity in humans. To enable evaluation of predictions made, methadone was selected as the model compound, being a compound for which data on both kinetics and cardiotoxicity in humans are available. A PBK model for methadone in humans was developed and evaluated against available kinetic data presenting an adequate match. Use of the developed PBK model to convert concentration–response curves for the effect of methadone on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) in the so-called multi electrode array (MEA) assay resulted in predictions for in vivo dose–response curves for methadone-induced cardiotoxicity that matched the available in vivo data. The results also revealed differences in protein plasma binding of methadone to be a potential factor underlying variation between individuals with respect to sensitivity towards the cardiotoxic effects of methadone. The present study provides a proof-of-principle of using PBK modeling-based reverse dosimetry of in vitro data for the prediction of cardiotoxicity in humans, providing a novel testing strategy in cardiac safety studies.

Reconstructed human skin shows epidermal invagination towards integrated neopapillae indicating early hair follicle formation in vitro.

Application of reconstructed human Skin (RhS) is a promising approach for the treatment of extensive wounds and for drug efficacy and safety testing. However, incorporating appendages, such as hair follicles, into RhS still remains a challenge. The hair follicle plays a critical role in thermal regulation, dispersion of sweat and sebum, sensory and tactile functions, skin regeneration, and repigmentation. The aim of this study was to determine whether human neopapilla could be incorporated into RhS (differentiated epidermis on fibroblast and endothelial cell populated dermis) and whether the neopapillae maintain their inductive follicular properties in vitro. Neopapillae spheroids, constructed from expanded and self‐aggregating dermal papilla cells, synthesized extracellular matrix typically found in follicular papillae. Compared with dermal fibroblasts, neopapillae showed increased expression of multiple genes (Wnt5a, Wnt10b, and LEF1) known to regulate hair development and also increased secretion of CXCL1, which is a strong keratinocyte chemoattractant. When neopapillae were incorporated into the dermis of RhS, they stimulated epidermal down‐growth resulting in engulfment of the neopapillae sphere. Similar to the native hair follicle, the differentiated invaginating epidermis inner side was keratin 10 positive and the undifferentiated outer side keratin 10 negative. The outer side was keratin 15 positive confirming the undifferentiated nature of these keratinocytes aligning a newly formed collagen IV, laminin V positive basement membrane within the hydrogel. In conclusion, we describe a RhS model containing neopapillae with hair follicle‐inductive properties. Importantly, epidermal invagination occurred to engulf the neopapillae, thus demonstrating in vitro the first steps towards hair follicle morphogenesis in RhS.

Tethered primary hepatocyte spheroids on polystyrene multi-well plates for high-throughput drug safety testing

Hepatocyte spheroids are useful models for mimicking liver phenotypes in vitro because of their three-dimensionality. However, the lack of a biomaterial platform which allows the facile manipulation of spheroid cultures on a large scale severely limits their application in automated high-throughput drug safety testing. In addition, there is not yet a robust way of controlling spheroid size, homogeneity and integrity during extended culture. This work addresses these bottlenecks to the automation of hepatocyte spheroid culture by tethering 3D hepatocyte spheroids directly onto surface-modified polystyrene (PS) multi-well plates. However, polystyrene surfaces are inert toward functionalization, and this makes the uniform conjugation of bioactive ligands very challenging. Surface modification of polystyrene well plates is achieved herein using a three-step sequence, resulting in a homogeneous distribution of bioactive RGD and galactose ligands required for spheroid tethering and formation. Importantly, treatment of polystyrene tethered spheroids with vehicle and paradigm hepatotoxicant (chlorpromazine) treatment using an automated liquid handling platform shows low signal deviation, intact 3D spheroidal morphology and Z’ values above 0.5, and hence confirming their amenability to high-throughput automation. Functional analyses performance (i.e. urea and albumin production, cytochrome P450 activity and induction studies) of the polystyrene tethered spheroids reveal significant improvements over hepatocytes cultured as collagen monolayers. This is the first demonstration of automated hepatotoxicant treatment on functional 3D hepatocyte spheroids tethered directly on polystyrene multi-well plates, and will serve as an important advancement in the application of 3D tethered spheroid models to high throughput drug screening.

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

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. 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. 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. 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.

Features of macro- and microstructure of the liver, pancreas, stomach, small and large intestinesin the study of chronic toxicity of tablets "Trianol".

Background. Due to the increased requirement for evaluation of safety, drugsafety and toxicological testing of new drugs take one of the most important places in pre-clinical studies. Objective: to identify features of the macro – and microstructure of the liver, pancreas, stomach, small intestine and colon in the study of chronic toxicity of tablets Trianol. Methods. This study researched the characteristics of macro- and microstructure of the liver, pancreas, stomach, small intestine and colon in the study of chronic toxicity of tablets Trianol. Experiments performed on rats of Vistar line of both sexes weighing 180-230g, obtained from the nursery of the Institute of Pharmacology and toxicology of NAMS of Ukraine, contained in a vivarium and receiving standard diet. When working with experimental animals was guided by the requirements of the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes (Strasbourg, 18.03.86). Studied the toxicity of three doses of “Trianol”: minimum (therapeutic) dose for tablets amounted to 50.0 mg/kg, intermediate – 250,0 mg/kg, maximum (subtoxic) – 500,0 mg/kg. Results . It is established that chronic introduction of trianol in a therapeutic dose induces hypertrophy of the gastric mucosa and the appearance of liver hepatocytes with signs of degeneration in cytoplasm. Identified changes are reversible. Conclusion . Intermediate and subtoxic doses cause the appearance of toxic changes in the form of swelling of the mucous membrane of the stomach, degenerative changes in cells and destruction of liver cells that must be considered when prescribing the drug. Immunotoxicaction on Peyer’s patch is not revealed.

NHP-immunome: A translational research-oriented database of non-human primate immune system proteins

We are currently living the advent of a new age for medicine in which basic research is being quickly translated into marketable drugs, and the widespread access to genomics data is allowing the design and implementation of personalized solutions to medical conditions. Non-human primates (NHP) have gained an essential role in drug discovery and safety testing due to their close phylogenetic relationship to humans. In this study, a collection of well characterized genes of the human immune system was used to define the orthology-based immunome in four NHP species, with carefully curated annotations available based on multi-tissue RNA-seq datasets. A broad variation in the frequency of expressed protein isoforms was observed between species. Finally, this analysis also revealed the lack of expression of at least four different chemokines in new-world primates. In addition, transcripts corresponding to four genes including interleukin 12 subunit alpha were expressed in humans but no other primate species analyzed. Access to the non-human primate immunome is available in http://www.fidic.org.co:90/proyecto/.

Identification of Serum Biomarkers to Distinguish Hazardous and Benign Aminotransferase Elevations.

The standard circulating biomarker of liver injury in both clinical settings and drug safety testing is alanine aminotransferase (ALT). However, ALT elevations sometimes lack specificity for tissue damage. To identify novel serum biomarkers with greater specificity for injury, we combined unique animal models with untargeted proteomics, followed by confirmation with immunoblotting. Using proteomics, we identified 109 proteins in serum from mice with acetaminophen (APAP)-induced liver injury that were not detectable in serum from mice with benign ALT elevations due to high-dose dexamethasone (Dex). We selected 4 (alcohol dehydrogenase 1A1 [Aldh1a1], aldehyde dehydrogenase 1 [Adh1], argininosuccinate synthetase 1 [Ass1], and adenosylhomocysteinase [Ahcy]) with high levels for further evaluation. Importantly, all 4 were specific for injury when using immunoblots to compare serum from Dex-treated mice and mice with similar lower ALT elevations due to milder models of APAP or bromobenzene-induced liver injury. Immunoblotting for ALDH1A1, ADH1, and ASS1 in serum from APAP overdose patients without liver injury and APAP overdose patients with mild liver injury revealed that these candidate biomarkers can be detected in humans with moderate liver injury as well. Interestingly, further experiments with serum from rats with bile duct ligation-induced liver disease indicated that Aldh1a1 and Adh1 are not detectable in serum in cholestasis and may therefore be specific for hepatocellular injury and possibly even drug-induced liver injury, in particular. Overall, our results strongly indicate that ALDH1A1, ADH1, and ASS1 are promising specific biomarkers for liver injury. Adoption of these biomarkers could improve preapproval drug safety assessment.

Dry Eye: The Effect of Chi-Ju-Di-Huang-Wan Plus Si Wu Tang and the Underlying Mechanism.

Objectives: In this pilot study, the effect of 970 mg Chi-Ju-Di-Huang-Wan (CJDHW) plus 30 mg four-substance decoction (Si Wu Tang; CJDHWSWT) was evaluated, in terms of its ability to alleviate dry eye symptoms and its therapeutic mechanism. Methods: This double-masked prospective investigation has recruited dry eye patients who have been randomly selected into two groups, namely treatment (n = 15) versus nontreatment (n = 15). In the treatment group, a daily oral intake of CJDHWSWT plus eye drops systane ultra was given for 90 consecutive days. In the nontreatment group, only defined eye drops were prescribed. The examinations included Schirmer's test, fluorescein-stained superficial punctate keratitis (SPK), artificial tear consumption, tear vascular endothelium growth factor (VEGF) level, and ocular surface disease index. The drug safety tests included liver and kidney functions, and complete blood counts. The candidates were observed during the screening visit and the following three monthly follow-ups. The data were analyzed by unpaired Student's t-test. Results: Compared to no significance in the nontreatment group, CJDHWSWT significantly (p = 0.03) increased the tear secretion after 3 months of intake. Furthermore, in contrast to no significance in the treatment group, there were significant alterations, including (i) increased fluorescein-stained SPK areas (p = 0.03); (ii) increased artificial tear instillation amount (p = 0.03); (iii) elevated tear VEGF protein levels (p = 0.03) in the nontreatment group; and (iv) significant improvement in clinically relevant phenomenon (e.g., reading limit and uncomfortable feeling in windy conditions), after treatment of artificial tear plus oral intake of CJDHWSWT. As shown by the post-treatment normal defined laboratory data, there were no adverse drug effects. Conclusions: This study has supported that CJDHWSWT is safe and effective in relieving dry eye's clinically relevant symptoms/phenomena. CJDHWSWT avoided the tear VEGF upregulation probably induced by dry eye-associated hypoxia/ischemia.

Novel method for action potential measurements from intact cardiac monolayers with multiwell microelectrode array technology

The cardiac action potential (AP) is vital for understanding healthy and diseased cardiac biology and drug safety testing. However, techniques for high throughput cardiac AP measurements have been limited. Here, we introduce a novel technique for reliably increasing the coupling of cardiomyocyte syncytium to planar multiwell microelectrode arrays, resulting in a stable, label-free local extracellular action potential (LEAP). We characterized the reliability and stability of LEAP, its relationship to the field potential, and its efficacy for quantifying AP morphology of human induced pluripotent stem cell derived and primary rodent cardiomyocytes. Rise time, action potential duration, beat period, and triangulation were used to quantify compound responses and AP morphology changes induced by genetic modification. LEAP is the first high throughput, non-invasive, label-free, stable method to capture AP morphology from an intact cardiomyocyte syncytium. LEAP can accelerate our understanding of stem cell models, while improving the automation and accuracy of drug testing.

Development and validation of bioengineered intestinal tubules for translational research aimed at safety and efficacy testing of drugs and nutrients

Currently used intestinal cell models have limited translational value, therefore, development of novel in vitro intestinal models that recapitulate the human in vivo setting more closely are of interest. Here, an advanced intestinal model was developed by the incorporation of physiological parameters, such as extracellular matrix (ECM) elements and shear stress, to cultured Caco-2 cells in a 3-dimensional environment. Caco-2 cells grown on ECM-coated hollow fiber membranes (HFM) under physiological shear stress show an improved phenotype, as demonstrated by the presence of enterocytes, goblet, Paneth, enteroendocrine and stem cells. Additionally, this model showed signs of an improved morphology due to the appearance of villi-like structures. Similar to epithelial cells grown on Transwells™, the current model remains easy to use, cost efficient and allows apical and basolateral access. The bioengineered intestinal tubule was validated by exposure to Clostridium difficile toxin A, the leading cause of healthcare-associated diarrhea. The loss of the tight junction network was supported by an increase in inulin-FITC leakage and the number of goblet cells increased, in agreement with clinical findings. In addition to toxicity screening, the bioengineered intestinal tubules are considered useful for drug and nutrient safety and efficacy testing.

Tissue engineered models of healthy and malignant human bone marrow.

Tissue engineering is becoming increasingly successful in providing in vitro models of human tissues that can be used for ex vivo recapitulation of functional tissues as well as predictive testing of drug efficacy and safety. From simple tissue models to microphysiological platforms comprising multiple tissue types connected by vascular perfusion, these "tissues on a chip" are emerging as a fast track application for tissue engineering, with great potential for modeling diseases and supporting the development of new drugs and therapeutic targets. We focus here on tissue engineering of the hematopoietic stem and progenitor cell compartment and the malignancies that can develop in the human bone marrow. Our overall goal is to demonstrate the utility and interconnectedness of improvements in bioengineering methods developed in one area of bone marrow studies for the remaining, seemingly disparate, bone marrow fields.

Automated quantification study of human cardiomyocyte synchronization using holographic imaging.

This paper investigates the rhythm strip and parameters of synchronization of human induced pluripotent stem cell (iPS) derived cardiomyocytes. The synchronization is evaluated from quantitative phase images of beating cardiomyocytes which are obtained using the time-lapse digital holographic imaging method. By quantitatively monitoring the dry mass redistribution, digital holography provides the physical contraction-relaxation signal caused by autonomous cardiac action potential. In order to analyze the synchronicity at the cell-to-cell level, we extracted single cardiac muscle cells, which contain the nuclei, from the phase images of cardiomyocytes containing multiple cells resulting from the fusion of k-means clustering and watershed segmentation algorithms. We demonstrate that mature cardiomyocyte cell synchronization can be automatically evaluated by time-lapse microscopic holographic imaging. Our proposed method can be applied for studies on cardiomyocyte disorders and drug safety testing systems.

Cell culture conditions affect the ability of high content imaging assay to detect drug-induced changes in cellular parameters in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs)

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are widely used for drug safety and efficacy testing with various techniques, including high content imaging (HCI). Upon drug treatment, a significant number of hiPSC-CMs grown in regular 96-well plates coated with fibronectin detached from the bottom of the plate, complicating data acquisition. Several cell culture configurations were tested to improve cell adherence, and the effects of these configurations on total cell number, separation of feature values between the negative (DMSO 0.1%) and positive (antimycin, staurosporine) controls, scale of feature value differences, and data variability were statistically calculated. hiPSC-CMs were plated on fibronectin- (in “blanket” configuration) or MaxGel- (in “sandwich” configuration) coated plates and covered with a layer of either HydroMatrix or MaxGel 2, 7, or 11d after plating. After a total of 14d in culture, cells were treated with compounds, labeled with four fluorescent dyes (Hoechst, TMRM, NucView, and RedDot), and imaged with GE INCell2000. Based on the statistical parameters calculated, the MaxGel 25% 7d “sandwich” was superior to all other tested conditions when the cells were treated with 0.3 μM antimycin for 2 h and test compounds 10 μM crizotinib and 30 μM amiodarone for 48 h. For staurosporine treatment, the best culturing condition varied between MaxGel “sandwich” systems, depending on which parameters were under consideration. Thus, cell culturing conditions can significantly affect the ability of high content imaging to detect changes in cellular features during compound treatment and should be thoroughly evaluated before committing to compound testing.

Generation and Analysis of Pluripotent Stem Cell-Derived Cardiomyocytes and Endothelial Cells for High Content Screening Purposes.

Human-induced pluripotent stem cells (hiPSCs) and their differentiated derivatives became a new, promising source for in vitro screening techniques. Cell lines derived from healthy individuals can be applied for drug safety testing, while patient-derived cells provide a platform to model diseases in vitro and can be used as a tool for personalized medicine including specific drug efficacy testing and identification of new pharmacological targets as well as for tailoring pharmacological therapies. Efficient differentiation protocols yielding cardiomyocytes or endothelial cells derived from iPSCs have been developed recently. Phenotypic characterization and gene expression profiling of these derivatives can reveal clues for developmental and pathological questions. Moreover, functional analysis and cell-based assays using automated fluorescence imaging platform and high content analysis characterize cell type-specific profiles of hiPSC-derived cardiomyocytes (hiPSC-CM) and endothelial cells (hiPSC-EC) at the cellular and subcellular levels. This can be utilized in a platform which can provide multiple endpoint profiles of candidate compounds.

Early afterdepolarizations in cardiac action potentials as mixed mode oscillations due to a folded node singularity.

Early afterdepolarizations (EADs) are pathological voltage oscillations during the repolarization phase of cardiac action potentials. They are considered as potential precursors to cardiac arrhythmias and have recently gained much attention in the context of preclinical drug safety testing under the Comprehensive in vitro Proarrhythmia Assay (CiPA) paradigm. From the viewpoint of multiple time scales theory, the onset of EADs has previously been studied by means of mathematical action potential models with one slow ion channel gating variable. In this article, we for the first time associate EADs with mixed mode oscillations in dynamical systems with two slow gating variables and present a folded node singularity of the slow flow as a novel mechanism for EADs genesis. We derive regions of the pharmacology parameter space in which EADs occur using both the folded node analysis and a full system bifurcation analysis, and we suggest the normal distance to the boundary of the EADs region as a mechanism-based risk metric to computationally estimate a drug’s proarrhythmic liability.

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

BackgroundIn 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. MethodsChemists 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 ResultsTest 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. ConclusionThis 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 Measurement of Gut Transit Time Using Larval Zebrafish

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and pharmacological similarities to that of the mammals. GI intolerance during clinical testing of drug candidates is common and may pose a serious threat to human health. Testing for GI toxicity in preclinical mammalian models can be expensive in terms of time, test compound, and labor. The high-throughput method presented here may be used to predict GI safety issues. Compared to mammalian models, this method allows for more expedient assessment of test compound effects on GI transit while using low quantities of compound. In this method, larval zebrafish (7 days post fertilization) are fed food containing a fluorescent label. After feeding, each larval fish is placed into a well of a 96-conical-bottom-well plate and dosed with test compound (dissolved in water) or the vehicle. As gut transit occurs, fecal matter accumulates on the bottom of the wells, and the rate at which this happens is monitored by measuring fluorescence from the bottom of the well repeatedly over time using a plate spectrophotometer. The fluorescence from larvae in a given treatment group are averaged and these values are graphed along with standard error, for each measurement time, yielding a curve representing average transit of food over time. Effects on gut transit time are identified by comparing the area under the curve for each treatment group to that of the vehicle-treated group. This method detected changes in zebrafish GI transit time induced by drugs with known clinical GI effects; it can be employed to interrogate dozens of treatments for GI effects per day. As such, safer compounds can be quickly prioritized for mammalian testing, which expedites discovery and proffers 3Rs advancement.