Assessing Longitudinal Treatment Efficacies and Alterations in Molecular Markers Associated with Glutamatergic Signaling and Immune Checkpoint Inhibitors in a Spontaneous Melanoma Mouse Model.

Previous work done by our laboratory described the use of an immunocompetent spontaneous melanoma-prone mouse model, TGS (TG-3/SKH-1), to evaluate treatment outcomes using inhibitors of glutamatergic signaling and immune checkpoint for 18 weeks. We showed a significant therapeutic efficacy with a notable sex-biased response in male mice. In this follow-up 18-week study, the dose of the glutamatergic signaling inhibitor was increased (from 1.7 mg/kg to 25 mg/kg), which resulted in improved responses in female mice but not male mice. The greatest reduction in tumor progression was observed in male mice treated with single-agent troriluzole and anti-PD-1. Furthermore, a randomly selected group of mice was removed from treatment after 18 weeks and maintained for up to an additional 48 weeks demonstrating the utility of the TGS mouse model to perform a ≥1-year preclinical therapeutic study in a physiologically relevant tumor-host environment. Digital spatial imaging analyses were performed in tumors and tumor microenvironments across treatment modalities using antibody panels for immune cell types and immune cell activation. The results suggest that immune cell populations and cytotoxic activities of T cells play critical roles in treatment responses in these mice. Examination of a group of molecular protein markers based on the proposed mechanisms of action of inhibitors of glutamatergic signaling and immune checkpoint showed that alterations in expression levels of xCT, γ-H2AX, EAAT2, PD-L1, and PD-1 are likely associated with the loss of treatment responses. These results suggest the importance of tracking changes in molecular markers associated with the mechanism of action of therapeutics over the course of a longitudinal preclinical therapeutic study in spatial and temporal manners.

eTRANSAFE: Building a sustainable framework to share reproducible drug safety knowledge with the public domain.

Integrative drug safety research in translational health informatics has rapidly evolved and included data that are drawn in from many resources, combining diverse data that are either reused from (curated) repositories, or newly generated at source. Each resource is mandated by different sets of metadata rules that are imposed on the incoming data. Combination of the data cannot be readily achieved without interference of data stewardship and the top-down policy guidelines that supervise and inform the process for data combination to aid meaningful interpretation and analysis of such data. The eTRANSAFE Consortium's effort to drive integrative drug safety research at a large scale hereby present the lessons learnt and the proposal of solution at the guidelines in practice at this Innovative Medicines Initiative (IMI) project. Recommendations in these guidelines were compiled from feedback received from key stakeholders in regulatory agencies, EFPIA companies, and academic partners. The research reproducibility guidelines presented in this study lay the foundation for a comprehensive data sharing and knowledge management plans accounting for research data management in the drug safety space - FAIR data sharing guidelines, and the model verification guidelines as generic deliverables that best practices that can be reused by other scientific community members at large. FAIR data sharing is a dynamic landscape that rapidly evolves with fast-paced technology advancements. The research reproducibility in drug safety guidelines introduced in this study provides a reusable framework that can be adopted by other research communities that aim to integrate public and private data in biomedical research space.

Perspectives on statistical strategies for the regulatory biomarker qualification process.

Qualification of a biomarker for use in a medical product development program requires a statistical strategy that aligns available evidence with the proposed context of use (COU), identifies any data gaps to be filled and plans any additional research required to support the qualification. Accumulating, interpreting and analyzing available data is outlined, step-by-step, illustrated by a qualified enrichment biomarker example and a safety biomarker in the process of qualification. The detailed steps aid requestors seeking qualification of biomarkers, allowing them to organize the available evidence and identify potential gaps. This provides a statistical perspective for assessing evidence that parallels clinical considerations and is intended to guide the overall evaluation of evidentiary criteria to support a specific biomarker COU.

Current state of cardiac troponin testing in Duchenne muscular dystrophy cardiomyopathy: review and recommendations from the Parent Project Muscular Dystrophy expert panel.

Cardiac disease is now the leading cause of death in Duchenne muscular dystrophy (DMD). Clinical evaluations over time have demonstrated asymptomatic cardiac troponin elevations and acute elevations are associated with symptoms and cardiac dysfunction in DMD. Clinicians require a better understanding of the relationship of symptoms, troponin levels and progression of cardiac disease in DMD. As clinical trials begin to assess novel cardiac therapeutics in DMD, troponin levels in DMD are important for safety monitoring and outcome measures. The Parent Project Muscular Dystrophy convened an expert panel of cardiologists, scientists, and regulatory and industry specialists on 16 December 2019 in Silver Spring, Maryland and reviewed published and unpublished data from their institutions. The panel recommended retrospective troponin data analyses, prospective longitudinal troponin collection using high-sensitivity cardiac troponin I assays, inclusion of troponin in future clinical trial outcomes and future development of clinical guidelines for monitoring and treating troponin elevations in DMD.

Qualification of translational safety biomarkers.

Safety biomarkers are important drug development tools, both preclinically and clinically. It is a straightforward process to correlate the performance of nonclinical safety biomarkers with histopathology, and ideally, the biomarker is useful in all species commonly used in safety assessment. In clinical validation studies, where histopathology is not feasible, safety biomarkers are compared to the response of standard biomarkers and/or to clinical adjudication. Worldwide, regulatory agencies have put in place processes to qualify biomarkers to provide confidence in the manner of use and interpretation of biomarker data in drug development studies. This paper describes currently qualified safety biomarkers which can be utilized to monitor for nephrotoxicity and cardiotoxicity and ongoing projects to qualify safety biomarkers for liver, skeletal muscle, and vascular injury. In many cases, the development and use of these critical drug development tools is dependent upon partnerships and the precompetitive sharing of data to support qualification efforts.

The Engineered Antibiotic Peptide PLG0206 Eliminates Biofilms and Is a Potential Treatment for Periprosthetic Joint Infections.

Antimicrobial peptides (AMPs) have recently gained attention for their potential to treat diseases related to bacterial and viral infections, as many traditional antimicrobial drugs have reduced efficacy in treating these infections due to the increased prevalence of drug-resistant pathogens. PLG0206, an engineered cationic antibiotic peptide that is 24 residues long, has been designed to address some limitations of other natural AMPs, such as toxicity and limited activity due to pH and ion concentrations. Nonclinical studies have shown that PLG0206 is highly selective for targeting bacterial cells and is not toxic to human blood cells. Antibiofilm experiments demonstrated that PLG0206 is effective at reducing both biotic and abiotic biofilm burdens following direct biofilm contact. PLG0206 has rapid and broad-spectrum activity against both Gram-positive and Gram-negative bacteria that are implicated as etiologic agents in periprosthetic joint infections, including multidrug-resistant ESKAPE pathogens and colistin-resistant isolates. A recent first-in-human study demonstrated that PLG0206 is well tolerated and safe as an intravenous infusion in healthy volunteers. Studies are planned to determine the efficacy of PLG0206 in patients for the treatment of periprosthetic joint infections. This review summarizes the chemistry, pharmacology, and microbiology of PLG0206 and explores its current preclinical, clinical, and regulatory status.

Biomarkers of Crohn's Disease to Support the Development of New Therapeutic Interventions.

BACKGROUND: Currently, 2 coprimary end points are used by health authorities to determine the effectiveness of therapeutic interventions in patients with Crohn's disease (CD): symptomatic remission (patient-reported outcome assessment) and endoscopic remission (ileocolonoscopy). However, there is lack of accepted biomarkers to facilitate regulatory decision-making in the development of novel therapeutics for the treatment of CD. METHODS: With support from the Helmsley Charitable Trust, Critical Path Institute formed the Crohn's Disease Biomarkers preconsortium (CDBpC) with members from the pharmaceutical industry, academia, and nonprofit organizations to evaluate the CD biomarker landscape. Biomarkers were evaluated based on biological relevance, availability of biomarker assays, and clinical validation data. RESULTS: The CDBpC identified the most critical need as pharmacodynamic/response biomarkers to monitor disease activity in response to therapeutic intervention. Fecal calprotectin (FC) and serum C-reactive protein (CRP) were identified as biomarkers ready for the regulatory qualification process. A number of exploratory biomarkers and potential panels of these biomarkers was also identified for additional development. Given the different factors involved in CD and disease progression, a combination of biomarkers, including inflammatory, tissue injury, genetic, and microbiome-associated biomarkers, will likely have the most utility. CONCLUSIONS: The primary focus of the Inflammatory Bowel Disease Regulatory Science Consortium will be development of exploratory biomarkers and the qualification of FC and CRP for IBD. The Inflammatory Bowel Disease Regulatory Science Consortium, focused on tools to support IBD drug development, will operate in the precompetitive space to share data, biological samples for biomarker testing, and assay information for novel biomarkers.

Serum glutamate dehydrogenase activity enables early detection of liver injury in subjects with underlying muscle impairments.

Serum activities of alanine and aspartate aminotransferases (ALT and AST) are used as gold standard biomarkers for the diagnosis of hepatocellular injury. Since ALT and AST lack liver specificity, the diagnosis of the onset of hepatocellular injury in patients with underlying muscle impairments is severely limited. Thus, we evaluated the potential of glutamate dehydrogenase (GLDH) as a liver specific alternative biomarker of hepatocellular injury. In our study, serum GLDH in subjects with Duchene muscular dystrophy (DMD) was equivalent to serum GLDH in age matched healthy subjects, while serum ALT was increased 20-fold in DMD subjects. Furthermore, serum GLDH in 131 subjects with variety of muscle impairments was similar to serum GLDH of healthy subjects while serum ALT corelated with serum creatine kinase, a widely accepted biomarker of muscle impairment. In addition, significant elevations of ALT, AST, and CK were observed in a case of a patient with rhabdomyolysis, while serum GLDH stayed within the normal range until the onset of hypoxia-induced liver injury. In a mouse model of DMD (DMDmdx), serum GLDH but not serum ALT clearly correlated with the degree of acetaminophen-induced liver injury. Taken together, our data support the utility of serum GLDH as a liver-specific biomarker of liver injury that has a potential to improve diagnosis of hepatocellular injury in patients with underlying muscle impairments. In drug development, GLDH may have utility as a biomarker of drug induced liver injury in clinical trials of new therapies to treat muscle diseases such as DMD.

Candidate biomarkers for the diagnosis and prognosis of drug-induced liver injury: An international collaborative effort.

Current blood biomarkers are suboptimal in detecting drug-induced liver injury (DILI) and predicting its outcome. We sought to characterize the natural variabilty and performance characteristics of 14 promising DILI biomarker candidates. Serum or plasma from multiple cohorts of healthy volunteers (n = 192 and n = 81), subjects who safely took potentially hepatotoxic drugs without adverse effects (n = 55 and n = 92) and DILI patients (n = 98, n = 28, and n = 143) were assayed for microRNA-122 (miR-122), glutamate dehydrogenase (GLDH), total cytokeratin 18 (K18), caspase cleaved K18, glutathione S-transferase α, alpha-fetoprotein, arginase-1, osteopontin (OPN), sorbitol dehydrogenase, fatty acid binding protein, cadherin-5, macrophage colony-stimulating factor receptor (MCSFR), paraoxonase 1 (normalized to prothrombin protein), and leukocyte cell-derived chemotaxin-2. Most candidate biomarkers were significantly altered in DILI cases compared with healthy volunteers. GLDH correlated more closely with gold standard alanine aminotransferase than miR-122, and there was a surprisingly wide inter- and intra-individual variability of miR-122 levels among healthy volunteers. Serum K18, OPN, and MCSFR levels were most strongly associated with liver-related death or transplantation within 6 months of DILI onset. Prediction of prognosis among DILI patients using the Model for End-Stage Liver Disease was improved by incorporation of K18 and MCSFR levels. Conclusion: GLDH appears to be more useful than miR-122 in identifying DILI patients, and K18, OPN, and MCSFR are promising candidates for prediction of prognosis during an acute DILI event. Serial assessment of these biomarkers in large prospective studies will help further delineate their role in DILI diagnosis and management.

Preclinical biomarker qualification.

Biomarkers are ubiquitously used within drug development programs in both nonclinical species and in humans to assess safety and efficacy of novel compounds. To routinely apply such novel biomarkers with certainty, a well-defined data package is necessary for review and endorsement by regulatory agencies including the US Food and Drug Administration, European Medicines Agency, and Japanese Pharmaceuticals and Medical Devices Agency. This type of endorsement is known as regulatory qualification. Novel approaches are being applied to speed the process, lower the resource intensity, and increase the accessibility of biomarker qualification data and it is likely that consortia will continue to play a fundamental role in the qualification process by bringing together like-minded stakeholders focused on specific tools to accelerate drug development. This article will focus on learnings from the previous three nonclinical biomarker qualification projects, as well as discuss the progression of preclinical biomarker projects into the clinical qualification space and the current strategy for the use of nonclinical biomarker data in the translational qualification of clinical biomarkers; much like nonclinical information is used in the approval of drug development candidates. Impact statement This minireview provides an overview of the history of preclinical biomarker qualification by summarizing the three examples of this type of qualification with US Food and Drug Administration, European Medicines Agency, and Japanese Pharmaceuticals and Medical Devices Agency. In addition, an overview of the biomarker qualification process is included to educate key stakeholders with links to relevant white papers that provide information on current evidentiary considerations. The manuscript also provides new information on the evolution of the role that preclinical qualification plays in clinical qualification of biomarkers and the novel approaches that are being utilized to improve the process.

What evidence do we need for biomarker qualification?

Biomarkers can facilitate all aspects of the drug development process. However, biomarker qualification-the use of a biomarker that is accepted by the U.S. Food and Drug Administration-needs a clear, predictable process. We describe a multistakeholder effort including government, industry, and academia that proposes a framework for defining the amount of evidence needed for biomarker qualification. This framework is intended for broad applications across multiple biomarker categories and uses.

Net Reclassification Index and Integrated Discrimination Index Are Not Appropriate for Testing Whether a Biomarker Improves Predictive Performance.

One of the goals of the Critical Path Institute's Predictive Safety Testing Consortium (PSTC) is to promote best practices for evaluating novel markers of drug induced injury. This includes the use of sound statistical methods. For rat studies, these practices have centered around comparing the area under the receiver-operator characteristic curve for each novel injury biomarker to those for the standard markers. In addition, the PSTC has previously used the net reclassification index (NRI) and integrated discrimination index (IDI) to assess the increased certainty provided by each novel injury biomarker when added to the information already provided by the standard markers. Due to their relatively simple interpretations, NRI and IDI have generally been popular measures of predictive performance. However recent literature suggests that significance tests for NRI and IDI can have inflated false positive rates and thus, tests based on these metrics should not be relied upon. Instead, when parametric models are employed to assess the added predictive value of a new marker, following (Pepe, M. S., Kerr, K. F., Longton, G., and Wang, Z. (2013). Testing for improvement in prediction model performance. Stat. Med. 32, 1467-1482), the PSTC recommends that likelihood based methods be used for significance testing.

Rat Urinary Osteopontin and Neutrophil Gelatinase-Associated Lipocalin Improve Certainty of Detecting Drug-Induced Kidney Injury.

Traditional kidney biomarkers are insensitive indicators of acute kidney injury, with meaningful changes occurring late in the course of injury. The aim of this work was to demonstrate the diagnostic potential of urinary osteopontin (OPN) and neutrophil gelatinase-associated lipocalin (NGAL) for drug-induced kidney injury (DIKI) in rats using data from a recent regulatory qualification submission of translational DIKI biomarkers and to compare performance of NGAL and OPN to five previously qualified DIKI urinary biomarkers. Data were compiled from 15 studies of 11 different pharmaceuticals contributed by Critical Path Institute's Predictive Safety Testing Consortium (PSTC) Nephrotoxicity Working Group (NWG). Rats were given doses known to cause DIKI or other target organ toxicity, and urinary levels of the candidate biomarkers were assessed relative to kidney histopathology and serum creatinine (sCr) and blood urea nitrogen (BUN).OPN and NGAL outperformed sCr and BUN in identifying DIKI manifested as renal tubular epithelial degeneration or necrosis. In addition, urinary OPN and NGAL, when used with sCr and BUN, increased the ability to detect renal tubular epithelial degeneration or necrosis. NGAL and OPN had comparable or improved performance relative to Kim-1, clusterin, albumin, total protein, and beta-2 microglobulin. Given these data, both urinary OPN and NGAL are appropriate for use with current methods for assessing nephrotoxicity to identify and monitor DIKI in regulatory toxicology studies in rats. These data also support exploratory use of urinary OPN and NGAL in safety monitoring strategies of early clinical trials to aid in the assurance of patient safety.

Advancing Risk Assessment through the Application of Systems Toxicology.

Risk assessment is the process of quantifying the probability of a harmful effect to individuals or populations from human activities. Mechanistic approaches to risk assessment have been generally referred to as systems toxicology. Systems toxicology makes use of advanced analytical and computational tools to integrate classical toxicology and quantitative analysis of large networks of molecular and functional changes occurring across multiple levels of biological organization. Three presentations including two case studies involving both in vitro and in vivo approaches described the current state of systems toxicology and the potential for its future application in chemical risk assessment.

Evaluation of the Relative Performance of Drug-Induced Skeletal Muscle Injury Biomarkers in Rats.

Novel skeletal muscle (SKM) injury biomarkers that have recently been identified may outperform or add value to the conventional SKM injury biomarkers aspartate transaminase (AST) and creatine kinase (CK). The relative performance of these novel biomarkers of SKM injury including skeletal troponin I (sTnI), myosin light chain 3 (Myl3), CK M Isoform (Ckm), and fatty acid binding protein 3 (Fabp3) was assessed in 34 rat studies including both SKM toxicants and compounds with toxicities in tissues other than SKM. sTnI, Myl3, Ckm, and Fabp3 all outperformed CK or AST and/or added value for the diagnosis of drug-induced SKM injury (ie, myocyte degeneration/necrosis). In addition, when used in conjunction with CK and AST, sTnI, Myl3, CKm, and Fabp3 individually and collectively improved diagnostic sensitivity and specificity, as well as diagnostic certainty, for SKM injury and responded in a sensitive manner to low levels of SKM degeneration/necrosis in rats. These findings support the proposal that sTnI, Myl3, Ckm, and Fabp3 are suitable for voluntary use, in conjunction with CK and AST, in regulatory safety studies in rats to monitor drug-induced SKM injury and the potential translational use of these exploratory biomarkers in early clinical trials to ensure patient safety.

Promoting Adoption of the 3Rs through Regulatory Qualification.

One mechanism to advance the application of novel safety assessment methodologies in drug development, including in silico or in vitro approaches that reduce the use of animals in toxicology studies, is regulatory qualification. Regulatory qualification, a formal process defined at the the U. S. Food and Drug Administration and the European Medicines Agency, hinges on a central concept of stating an appropriate "context of use" for a novel drug development tool (DDT) that precisely defines how that DDT can be used to support decision making in a regulated drug development setting. When accumulating the data to support a particular "context-of-use," the concept of "fit-for-purpose" often guides assay validation, as well as the type and amount of data or evidence required to evaluate the tool. This paper will review pathways for regulatory acceptance of novel DDTs and discuss examples of safety projects considered for regulatory qualification. Key concepts to be considered when defining the evidence required to formally adopt and potentially replace animal-intensive traditional safety assessment methods using qualified DDTs are proposed. Presently, the use of qualified translational kidney safety biomarkers can refine and reduce the total numbers of animals used in drug development. We propose that the same conceptual regulatory framework will be appropriate to assess readiness of new technologies that may eventually replace whole animal models.

Systems Toxicology: The Future of Risk Assessment.

Risk assessment, in the context of public health, is the process of quantifying the probability of a harmful effect to individuals or populations from human activities. With increasing public health concern regarding the potential risks associated with chemical exposure, there is a need for more predictive and accurate approaches to risk assessment. Developing such an approach requires a mechanistic understanding of the process by which xenobiotic substances perturb biological systems and lead to toxicity. Supplementing the shortfalls of traditional risk assessment with mechanistic biological data has been widely discussed but not routinely implemented in the evaluation of chemical exposure. These mechanistic approaches to risk assessment have been generally referred to as systems toxicology. This Symposium Overview article summarizes 4 talks presented at the 35th Annual Meeting of the American College of Toxicology.

The predictive safety testing consortium and the coalition against major diseases.

The Predictive Safety Testing Consortium and the Coalition Against Major Diseases, both launched by the Critical Path Institute, provide valuable examples of the outcomes and lessons learned by different types of consortia working on new drug development tools.

Modeling and simulation for medical product development and evaluation: highlights from the FDA-C-Path-ISOP 2013 workshop.

Medical-product development has become increasingly challenging and resource-intensive. In 2004, the Food and Drug Administration (FDA) described critical challenges facing medical-product development by establishing the critical path initiative [1]. Priorities identified included the need for improved modeling and simulation tools, further emphasized in FDA's 2011 Strategic Plan for Regulatory Science [Appendix]. In an effort to support and advance model-informed medical-product development (MIMPD), the Critical Path Institute (C-Path) [www.c-path.org], FDA, and International Society of Pharmacometrics [www.go-isop.org] co-sponsored a workshop in Washington, D.C. on September 26, 2013, to examine integrated approaches to developing and applying model- MIMPD. The workshop brought together an international group of scientists from industry, academia, FDA, and the European Medicines Agency to discuss MIMPD strategies and their applications. A commentary on the proceedings of that workshop is presented here.

Design and synthesis of hydroxyethylamine (HEA) BACE-1 inhibitors: prime side chromane-containing inhibitors.

The structure activity relationship of the prime region of conformationally restricted hydroxyethylamine (HEA) BACE inhibitors is described. Variation of the P1' region provided selectivity over Cat-D with a series of 2,2-dioxo-isothiochromanes and optimization of the P2' substituent of chromane-HEA(s) with polar substituents provided improvements in the compound's in vitro permeability. Significant potency gains were observed with small aliphatic substituents such as methyl, n-propyl, and cyclopropyl when placed at the C-2 position of the chromane.