Animal Alternatives OK'd by New Law.

The FDA Modernization Act 2.0 allows companies to submit nonanimal data using certain alternative technologies to demonstrate the safety and efficacy of investigational drugs prior to human trials. Animal rights supporters hope the law represents a shift away from animal use, but researchers caution that organ-chips and other innovations, although potentially valuable, cannot replace animal models to test drugs in development.

Reproducibility, relevance and reliability as barriers to efficient and credible biomedical technology translation.

Biomedical research accuracy and relevance for improving healthcare are increasingly identified as costly problems. Basic research data quality, reporting and methodology, and reproducibility are common factors implicated in this challenge. Preclinical models of disease and therapy, largely conducted in rodents, have known deficiencies in replicating most human conditions. Their translation to human results is acknowledged to be poor for decades. Clinical data quality and quantity is also recognized as deficient; gold standard randomized clinical trials are expensive. Few solid conclusions from clinical studies are replicable and many remain unpublished. The translational pathway from fundamental biomedical research through to innovative solutions handed to clinical practitioners is therefore highly inefficient and costly in terms of wasted resources, early claims from fundamental discoveries never witnessed in humans, and few new, improved solutions available clinically for myriad diseases. Improving this biomedical research strategy and resourcing for reliability, translational relevance, reproducibility and clinical impact requires careful analysis and consistent enforcement at both funding and peer review levels.

De-risking in Tier I CNS safety assessments is the primary function of study design and technical training of laboratory staff observers.

Derisking is not a pharmaceutical industry strategy to reduce time, effort, or costs in drug development. Derisking strategies originated within the National Institutes of Health as a predicate to good science. There is a growing sentiment within drug development programs to diminish the importance of behavioral measures in toxicological studies and in the Tiered Safety assessment plans of the U.S. Regulatory Agencies and the International Commission on Harmonization. The validity and reliability of the Functional Observational Batter (FOB) is critically dependent on consistency and technical quality in each risk assessment plan. US Federal and International drug approval organizations have universally adopted the concept of principles of test construction rather than delineating specific behavioral assay endpoints for inclusion of the FOB in nonclinical safety protocols. The validity and reliability of behavioral observations in standardized neurotoxicity screening is critically dependent on the FOB developed by the Study Director with the Sponsor throughout all stages of testing.. The individual risk factors selected for observation to be included in the early Tier 1 safety program should be determined by the mechanism and mode of action of the test article. The results of Tier I testing are the basis for Tier II testing designs. Critical to the compliance with Good Laboratory Practices is the documentation of training of the operational staff scheduled to conduct all aspects of the established protocol.

Performance Metrics of Sunscreen Formulations: In Vitro and In Vivo Techniques.

Effective skin protection of consumers by sunscreens can only be achieved when meaningful and reliable test methods are available to objectively measure the protection of sunscreen products. Quantitative, scientifically sound, and valid methods to detect UVB and UVA light protection as well as methods to assess sunscreen substantivity to water are required. Continuous improvement and, if necessary, extension of the test methods are important to provide optimum protection from harmful sun rays to the consumer. This work documents the historical background of the development of sunscreen test methods and provides the actual worldwide status of applied methods. Future developments and trends are discussed as far as they actually become apparent.

Assessment of Natural Sunlight Protection Provided by 10 High-SPF Broad-Spectrum Sunscreens and Sun-Protective Fabrics.

In 1978, the FDA Advisory Panel proposed both indoor and natural sunlight SPF testing methods but reverted to indoor testing only in 1993. Today's sunscreen sun protection and broad-spectrum claims are based on mandated clinical tests using solar simulators and in vitro spectrophotometers. This research evaluated the protection of 10 high-SPF (30-110), broad-spectrum sunscreen products, as well as 6 sun-protective fabrics against natural sunlight in Arequipa, Peru. Each of the 17 subjects was exposed to natural sunlight for 1 h and 59 min under clear skies, with temperatures and humidity similar to those in an indoor clinical laboratory. Test sites were photographed 16-24 h later. Four dermatologists evaluated the photographs for erythema and persistent pigment darkening (PPD). Perceptible sun-induced skin injury (sunburn and/or pigmentation) was detected at 97% of the sunscreen-protected scores. The most sun-sensitive subjects obtained the least erythema protection. The higher the SPF was, the higher the erythema protection, but the intensity of PPD was also higher. The 2 sunscreens using only FDA-approved sunscreen filters rated 30 SPF and 45+ SPF performed poorly: Eighty-one percent of the 136 scores were graded 1 minimal erythema dose or higher erythema, achieving, at a maximum, SPF of 5-7 in natural sunlight. Sun-protective fabrics tested provided excellent sun protection. The erythema and PPD observed through the sunscreens in less than 2 h are incongruous with the broad-spectrum, high-SPF sunscreen claims. Reapplying these sunscreens and staying in the sun longer, as stated on the product labels, would have subjected the subjects to even more UV exposure. High-SPF, broad-spectrum sunscreen claims based on indoor solar simulator testing do not agree with the natural sunlight protection test results.

A Review of the Main Considerations for Formulation Development in Preclinical Toxicology Studies.

The main considerations for the development of a formulation for preclinical safety assessment testing are explored. Intravenous, inhalation, oral and dermal dosing are given focus and although different dose routes do present their own individual challenges there are common themes that emerge. In each case it is necessary to maximise exposure to achieve high doses to satisfy regulatory requirements for safety assessment testing. This often involves producing formulations that are at the limits of solubility and maximum volumes possible for administration to different test species by the chosen route. It is concluded that for all routes it is important to thoroughly explore the stability of the test item in the proposed formulation matrix well ahead of dosing any animals, giving careful consideration to which excipients are used and what their underlying toxicity profile may be for the relevant preclinical species. In addition, determining the maximum achievable concentrations and weighing that against the maximum volumes that can be given by the chosen route in all the test species at an early stage will also give a read on whether it would be theoretically possible to achieve suitably high enough doses to support clinical work. Not doing so can cause delays in the development programme and may have ethical repercussions.

Current Trends of Practices in Nonclinical Toxicology: An Industry Survey.

The growth in drug development over the past years reflects significant advancements in basic sciences and a greater understanding of molecular pathways of disease. Benchmarking industry practices has been important to enable a critical reflection on the path to evolve pharmaceutical testing, and the outcome of past industry surveys has had some impact on best practices in testing. A survey was provided to members of SPS, ACT, and STP. The survey consisted of 37 questions and was provided to 2550 participants with a response rate of 24%. Most respondents (∼75%) came from the US and Europe. The survey encompassed multiple topics encountered in nonclinical testing of pharmaceuticals. The most frequent target indications were oncology (69%), inflammation (55%), neurology/psychiatry/pain (46%), cardiovascular (44%), and metabolic diseases (39%). The most frequent drug-induced toxicology issues confronted were hepatic, hematopoietic, and gastrointestinal. Toxicological effects that impacted the no observed adverse effect level (NOAEL) were most frequently based on histopathology findings. The survey comprised topics encountered in the use of biomarkers in nonclinical safety assessment, most commonly those used to assess inflammation, cardiac/vascular, renal, and hepatic toxicity as well as common practices related to the assessment of endocrine effects, carcinogenicity, genotoxicity, juvenile and male-mediated developmental and female reproductive toxicity. The survey explored the impact of regulatory meetings on program design, application of the 3 Rs, and reasons for program delays. Overall, the survey results provide a broad perspective of current practices based on the experience of the scientific community engaged in nonclinical safety assessment.

Preparation and preliminary quality evaluation of aspirin/L-glutamate compound pellets.

L-glutamate is an important component of protein. It can prevent gastrointestinal damage caused by NSAIDs. We constructed two-phase enteric-coated granules of aspirin and L-glutamate compound by extrusion spheronization method and fluidized bed coating. The subliminal effective dose of L-glutamate is 100 mg/kg tested by model of gastric ulcer of rats induced by aspirin and drug administration. HPLC-UV and UV-Vis methods were adopted to determine content and cumulative release of aspirin and L-glutamate as quality analysis method indexes. The prescription and process optimization were carried out with yield, sphericity and dissolution. The two-phase compound granules have good sphericity of 0.93 ± 0.05 (aspirin pellets) and 0.94 ± 0.02 (L-glutamate pellets), content of salicylic acid (0.24 ± 0.03)%, dissolution of aspirin (2.36 ± 0.11)%. Quality evaluation and preliminary stability meet the commercial requirements. The stored environment of compound preparation should be sealed in a cool and dark place.

Ensemble learning application to discover new trypanothione synthetase inhibitors.

Trypanosomatid-caused diseases are among the neglected infectious diseases with the highest disease burden, affecting about 27 million people worldwide and, in particular, socio-economically vulnerable populations. Trypanothione synthetase (TryS) is considered one of the most attractive drug targets within the thiol-polyamine metabolism of typanosomatids, being unique, essential and druggable. Here, we have compiled a dataset of 401 T. brucei TryS inhibitors that includes compounds with inhibitory data reported in the literature, but also in-house acquired data. QSAR classifiers were derived and validated from such dataset, using publicly available and open-source software, thus assuring the portability of the obtained models. The performance and robustness of the resulting models were substantially improved through ensemble learning. The performance of the individual models and the model ensembles was further assessed through retrospective virtual screening campaigns. At last, as an application example, the chosen model-ensemble has been applied in a prospective virtual screening campaign on DrugBank 5.1.6 compound library. All the in-house scripts used in this study are available on request, whereas the dataset has been included as supplementary material.

International Consensus on Minimum Preclinical Testing Requirements for the Development of Innovative Therapies For Children and Adolescents with Cancer.

Cancer remains the leading cause of disease-related death in children. For the many children who experience relapses of their malignant solid tumors, usually after very intensive first-line therapy, curative treatment options are scarce. Preclinical drug testing to identify promising treatment elements that match the molecular make-up of the tumor is hampered by the fact that (i) molecular genetic data on pediatric solid tumors from relapsed patients and thus our understanding of tumor evolution and therapy resistance are very limited to date and (ii) for many of the high-risk entities, no appropriate and molecularly well-characterized patient-derived models and/or genetic mouse models are currently available. However, recent regulatory changes enacted by the European Medicines Agency (class waiver changes) and the maturation of the RACE for Children act with the FDA, will require a significant increase in preclinical pediatric cancer research and clinical development must occur. We detail the outcome of a pediatric cancer international multistakeholder meeting whose output aims at defining an international consensus on minimum preclinical testing requirements for the development of innovative therapies for children and adolescents with cancer. Recommendations based on the experience of the NCI funded PPTP/C (www.ncipptc.org) and the EU funded ITCC-P4 public private partnership (www.itccp4.eu) are provided for the use of cell-based and mouse models for pediatric solid malignancies, as well as guidance on the scope and content of preclinical proof-of-concept data packages to inform clinical development dependent on clinical urgency. These recommendations can serve as a minimal guidance necessary to jumpstart preclinical pediatric research globally.

Introduction to the EQIPD quality system.

While high risk of failure is an inherent part of developing innovative therapies, it can be reduced by adherence to evidence-based rigorous research practices. Supported through the European Union's Innovative Medicines Initiative, the EQIPD consortium has developed a novel preclinical research quality system that can be applied in both public and private sectors and is free for anyone to use. The EQIPD Quality System was designed to be suited to boost innovation by ensuring the generation of robust and reliable preclinical data while being lean, effective and not becoming a burden that could negatively impact the freedom to explore scientific questions. EQIPD defines research quality as the extent to which research data are fit for their intended use. Fitness, in this context, is defined by the stakeholders, who are the scientists directly involved in the research, but also their funders, sponsors, publishers, research tool manufacturers, and collaboration partners such as peers in a multi-site research project. The essence of the EQIPD Quality System is the set of 18 core requirements that can be addressed flexibly, according to user-specific needs and following a user-defined trajectory. The EQIPD Quality System proposes guidance on expectations for quality-related measures, defines criteria for adequate processes (i.e. performance standards) and provides examples of how such measures can be developed and implemented. However, it does not prescribe any pre-determined solutions. EQIPD has also developed tools (for optional use) to support users in implementing the system and assessment services for those research units that successfully implement the quality system and seek formal accreditation. Building upon the feedback from users and continuous improvement, a sustainable EQIPD Quality System will ultimately serve the entire community of scientists conducting non-regulated preclinical research, by helping them generate reliable data that are fit for their intended use.

Epidemiology and reporting characteristics of preclinical systematic reviews.

In an effort to better utilize published evidence obtained from animal experiments, systematic reviews of preclinical studies are increasingly more common-along with the methods and tools to appraise them (e.g., SYstematic Review Center for Laboratory animal Experimentation [SYRCLE's] risk of bias tool). We performed a cross-sectional study of a sample of recent preclinical systematic reviews (2015-2018) and examined a range of epidemiological characteristics and used a 46-item checklist to assess reporting details. We identified 442 reviews published across 43 countries in 23 different disease domains that used 26 animal species. Reporting of key details to ensure transparency and reproducibility was inconsistent across reviews and within article sections. Items were most completely reported in the title, introduction, and results sections of the reviews, while least reported in the methods and discussion sections. Less than half of reviews reported that a risk of bias assessment for internal and external validity was undertaken, and none reported methods for evaluating construct validity. Our results demonstrate that a considerable number of preclinical systematic reviews investigating diverse topics have been conducted; however, their quality of reporting is inconsistent. Our study provides the justification and evidence to inform the development of guidelines for conducting and reporting preclinical systematic reviews.

3D cell culture models: Drug pharmacokinetics, safety assessment, and regulatory consideration.

Nonclinical testing has served as a foundation for evaluating potential risks and effectiveness of investigational new drugs in humans. However, the current two-dimensional (2D) in vitro cell culture systems cannot accurately depict and simulate the rich environment and complex processes observed in vivo, whereas animal studies present significant drawbacks with inherited species-specific differences and low throughput for increased demands. To improve the nonclinical prediction of drug safety and efficacy, researchers continue to develop novel models to evaluate and promote the use of improved cell- and organ-based assays for more accurate representation of human susceptibility to drug response. Among others, the three-dimensional (3D) cell culture models present physiologically relevant cellular microenvironment and offer great promise for assessing drug disposition and pharmacokinetics (PKs) that influence drug safety and efficacy from an early stage of drug development. Currently, there are numerous different types of 3D culture systems, from simple spheroids to more complicated organoids and organs-on-chips, and from single-cell type static 3D models to cell co-culture 3D models equipped with microfluidic flow control as well as hybrid 3D systems that combine 2D culture with biomedical microelectromechanical systems. This article reviews the current application and challenges of 3D culture systems in drug PKs, safety, and efficacy assessment, and provides a focused discussion and regulatory perspectives on the liver-, intestine-, kidney-, and neuron-based 3D cellular models.

The New S7B/E14 Question and Answer Draft Guidance for Industry: Contents and Commentary.

In August 2020, the International Council on Harmonisation (ICH) released a new draft document, which for the first time combined nonclinical (S7B) and clinical (E14) Questions and Answers (Q&As) into 1 document. FDA describes the revision as a "value proposition": if the human ether-à-go-go assay and the in vivo study are performed in a standardized way, the number of dedicated thorough QT (TQT) studies can be reduced. In this article, we describe and discuss the Q&As that relate to clinical ECG evaluation. If supported by negative standardized nonclinical assays, Q&A 5.1 will obviate the need for a TQT study in the case that a >2-fold exposure margin vs high clinical scenario cannot be obtained. Q&A 6.1 addresses drugs that are poorly tolerated in healthy subjects and cannot be studied at high doses or in placebo-controlled studies; it therefore mainly applies to oncology drugs. It will enable sponsors to claim that a new drug has a "low likelihood of proarrhythmic effects" in the case that the mean corrected QT effect is <10 milliseconds at the time of market application. The E14 2015 revision allowed application of concentration-corrected QT analysis on data from routinely performed clinical pharmacology studies, for example, the first-in-human study and the proportion of dedicated TQT studies has since steadily decreased. It can be foreseen that the proposed new revision will further reduce the number of TQT studies. To achieve harmonization across regulatory regions, it seems important to reach consensus within the International Council on Harmonisation group on the new threshold proposed in 6.1. For this purpose, the Implementation Working Group has asked for public comments.

Reducing False Positives through the Application of Fluorescence Lifetime Technology: A Comparative Study Using TYK2 Kinase as a Model System.

The predominant assay detection methodologies used for enzyme inhibitor identification during early-stage drug discovery are fluorescence-based. Each fluorophore has a characteristic fluorescence decay, known as the fluorescence lifetime, that occurs throughout a nanosecond-to-millisecond timescale. The measurement of fluorescence lifetime as a reporter for biological activity is less common than fluorescence intensity, even though the latter has numerous issues that can lead to false-positive readouts. The confirmation of hit compounds as true inhibitors requires additional assays, cost, and time to progress from hit identification to lead drug-candidate optimization. To explore whether the use of fluorescence lifetime technology (FLT) can offer comparable benefits to label-free-based approaches such as RapidFire mass spectroscopy (RF-MS) and a superior readout compared to time-resolved fluorescence resonance energy transfer (TR-FRET), three equivalent assays were developed against the clinically validated tyrosine kinase 2 (TYK2) and screened against annotated compound sets. FLT provided a marked decrease in the number of false-positive hits when compared to TR-FRET. Further cellular screening confirmed that a number of potential inhibitors directly interacted with TYK2 and inhibited the downstream phosphorylation of the signal transducer and activator of transcription 4 protein (STAT4).

Behavioral Battery for Testing Candidate Analgesics in Mice. II. Effects of Endocannabinoid Catabolic Enzyme Inhibitors and ∆9-Tetrahydrocannabinol.

Enhanced signaling of the endocannabinoid (eCB) system through inhibition of the catabolic enzymes monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) has received increasing interest for development of candidate analgesics. This study compared effects of MAGL and FAAH inhibitors with effects of ∆9-tetrahydrocannabinol (THC) using a battery of pain-stimulated, pain-depressed, and pain-independent behaviors in male and female mice. Intraperitoneal injection of dilute lactic acid (IP acid) served as an acute visceral noxious stimulus to stimulate two behaviors (stretching, facial grimace) and depress two behaviors (rearing, nesting). Nesting and locomotion were also assessed in the absence of IP acid as pain-independent behaviors. THC and a spectrum of six eCB catabolic enzyme inhibitors ranging from MAGL- to FAAH-selective were assessed for effectiveness to alleviate pain-related behaviors at doses that did not alter pain-independent behaviors. The MAGL-selective inhibitor MJN110 produced the most effective antinociceptive profile, with 1.0 mg/kg alleviating IP acid effects on stretching, grimace, and nesting without altering pain-independent behaviors. MJN110 effects on IP acid-depressed nesting had a slow onset and long duration (40 minutes to 6 hours), were blocked by rimonabant, and tended to be greater in females. As inhibitors increased in FAAH selectivity, antinociceptive effectiveness decreased. PF3845, the most FAAH-selective inhibitor, produced no antinociception up to doses that disrupted locomotion. THC decreased IP acid-stimulated stretching and grimace at doses that did not alter pain-independent behaviors; however, it did not alleviate IP acid-induced depression of rearing or nesting. These results support further consideration of MAGL-selective inhibitors as candidate analgesics for acute inflammatory pain. SIGNIFICANCE STATEMENT: This study characterized a spectrum of endocannabinoid catabolic enzyme inhibitors ranging in selectivity from monoacylglycerol lipase-selective to fatty acid amide hydrolase-selective in a battery of pain-stimulated, pain-depressed, and pain-independent behaviors previously pharmacologically characterized in a companion paper. This battery provides a method for prioritizing candidate analgesics by effectiveness to alleviate pain-related behaviors at doses that do not alter pain-independent behaviors, with inclusion of pain-depressed behaviors increasing translational validity and decreasing susceptibility to motor-depressant false positives.

Behavioral Battery for Testing Candidate Analgesics in Mice. I. Validation with Positive and Negative Controls.

This study evaluated a battery of pain-stimulated, pain-depressed, and pain-independent behaviors for preclinical pharmacological assessment of candidate analgesics in mice. Intraperitoneal injection of dilute lactic acid (IP acid) served as an acute visceral noxious stimulus to produce four pain-related behaviors in male and female ICR mice: stimulation of 1) stretching, 2) facial grimace, 3) depression of rearing, and 4) depression of nesting. Additionally, nesting and locomotion in the absence of the noxious stimulus were used to assess pain-independent drug effects. These six behaviors were used to compare effects of two mechanistically distinct but clinically effective positive controls (ketoprofen and oxycodone) and two negative controls that are not clinically approved as analgesics but produce either general motor depression (diazepam) or motor stimulation (amphetamine). We predicted that analgesics would alleviate all IP acid effects at doses that did not alter pain-independent behaviors, whereas negative controls would not. Consistent with this prediction, ketoprofen (0.1-32 mg/kg) produced the expected analgesic profile, whereas oxycodone (0.32-3.2 mg/kg) alleviated all IP acid effects except depression of rearing at doses lower than those that altered pain-independent behaviors. For the negative controls, diazepam (1-10 mg/kg) failed to block IP acid-induced depression of either rearing or nesting and only decreased IP acid-stimulated behaviors at doses that also decreased pain-independent behaviors. Amphetamine (0.32-3.2 mg/kg) alleviated all IP acid effects but only at doses that also stimulated locomotion. These results support utility of this model as a framework to evaluate candidate-analgesic effects in a battery of complementary pain-stimulated, pain-depressed, and pain-independent behavioral endpoints. SIGNIFICANCE STATEMENT: Preclinical assays of pain and analgesia often yield false-positive effects with candidate analgesics. This study used two positive-control analgesics (ketoprofen, oxycodone) and two active negative controls (diazepam, amphetamine) to validate a strategy for distinguishing analgesics from nonanalgesics by profiling drug effects in a battery of complementary pain-stimulated, pain-depressed, and pain-independent behaviors in male and female mice.

In vivo safety testing of Antibody Drug Conjugates.

Antibody Drug Conjugates (ADCs) are complex multi-domain biotherapeutics which combine, with the aid of a chemical linker, tumor-targeting antibodies with potent small molecule cytotoxicants (also called warhead or payload) for the treatment of cancer. ADCs are a rapidly growing class of pharmaceuticals with nine FDA-approved drugs already on the market and over eighty at different stages of clinical development, and also an increasing number under evaluation for non-oncological indications. Off-target toxicity and a narrow therapeutic index has been a problem with ADCs. This has driven the search for better targeting (disease models, cell surface antigens), linker stability, and payload specificity. Analysis of regulatory approval documents, scientific publications and ICH guidance shows that safety evaluation of ADCs requires novel integrated strategies different from both standard chemotherapy and antibody-based products, e.g. development and validation of ADC analytical assays. There is no ADC-specific guidance on safety evaluation; current guidance emphasises the need for an adaptive approach but more ADC-specific guidance is now arguably possible. The data now available will help to optimize primary target specificity, select appropriate combination partners, develop in silico models, and provide guidance for preclinical and clinical safety evaluation for the next generation of this class of multi-domain therapeutics.

Evaluation of the carcinogenicity of dichloromethane in rats, mice, hamsters and humans.

Dichloromethane (DCM) is a high production volume chemical (>1000 t/a) mainly used as an industrial solvent. Carcinogenicity studies in rats, mice and hamsters have demonstrated a malignant tumor inducing potential of DCM only in the mouse (lung and liver) at 1000-4000 ppm whereas human data do not support a conclusion of cancer risk. Based on this, DCM has been classified as a cat. 2 carcinogen. Dose-dependent toxicokinetics of DCM suggest that DCM is a threshold carcinogen in mice, initiating carcinogenicity via the low affinity/high capacity GSTT1 pathway; a biotransformation pathway that becomes relevant only at high exposure concentrations. Rats and hamsters have very low activities of this DCM-metabolizing GST and humans have even lower activities of this enzyme. Based on the induction of specific tumors selectively in the mouse, the dose- and species-specific toxicokinetics in this species, and the absence of a malignant tumor response by DCM in rats and hamsters having a closer relationship to DCM toxicokinetics in humans and thus being a more relevant animal model, the current classification of DCM as human carcinogen cat. 2 remains appropriate.

The use of emerging safety biomarkers in nonclinical and clinical safety assessment - The current and future state: An IQ DruSafe industry survey.

Pharmaceutical and biotechnology companies rarely disclose their use of translational emerging safety biomarkers (ESBs) during drug development, and the impact of ESB use on the speed of drug development remains unclear. A cross-industry survey of 20 companies of varying size was conducted to understand current trends in ESB use and future use prospects. The objectives were to: (1) determine current ESB use in nonclinical and clinical drug development and impact on asset advancement; (2) identify opportunities, gaps, and challenges to greater ESB implementation; and (3) benchmark perspectives on regulatory acceptance. Although ESBs were employed in only 5-50% of studies/programs, most companies used ESBs to some extent, with larger companies demonstrating greater nonclinical use. Inclusion of ESBs in investigational new drug applications (INDs) was similar across all companies; however, differences in clinical trial usage could vary among the prevailing health authority (HA). Broader implementation of ESBs requires resource support, cross-industry partnerships, and collaboration with HAs. This includes generating sufficient foundational data, demonstrating nonclinical to clinical translatability and practical utility, and clearly written criteria by HAs to enable qualification. If achieved, ESBs will play a critical role in the development of next-generation, translationally-tailored standard laboratory tests for drug development.