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The diagnosis and management of drug-induced liver injury (DILI) remains a challenge in clinical trials in drug development. The qualification of emerging biomarkers capable of predicting DILI soon after the initiation of treatment, differentiating DILI from underlying liver disease, identifying the causal entity, and assigning appropriate treatment options after DILI is diagnosed are needed. Qualification efforts have been hindered by lack of properly stored and consented biospecimens that are linked to clinical data relevant to a specific context of use. Recommendations are made for biospecimen collection procedures, with the focus on clinical trials, and for specific emerging biomarkers to focus qualification efforts.

Clinical pathology reporting practices are diverse among individuals and organizations involved in nonclinical toxicology studies. Clear, informative, and consistent reporting of clinical pathology results increases their value and avoids misinterpretation, resulting in decreased drug development costs. In recent years, certain common practices in clinical pathology reporting have been embraced by industry leaders and more consistently utilized across the pharmaceutical industry. The purpose of this manuscript is to review current clinical pathology reporting practices and to provide nonbinding suggestions to improve consistency, quality, and value of clinical pathology reports generated in support of nonclinical toxicology studies.

Microsampling techniques enable the minimization of blood collection volume from animals and subsequent handling of the blood samples or their derived plasma or serum samples. This offers advantages over conventional large-volume sampling, such as eliminating the need for satellite animals and improving animal welfare aspects, and providing the opportunity for additional assessments in small animals where blood volume constraints limit endpoints. This study evaluated the feasibility of implementation of capillary microsampling (CMS) in a single-dose study in mice with the ultimate goal of enabling its use in toxicology studies. The focus was on the impact of microsampling on toxicokinetic assessment and on the subsequent hematology assessment in the same animal. A seventy (70)-μL blood collection via CMS from the tail vein had a minimal effect on the hematology parameters of mice (strain C57BL/6) in samples taken within 24 h of blood collection. TK parameters were similar in plasma samples collected via CMS and cardiac puncture sampling. A bioanalytical assay was developed which enabled the quantification of concentration of both the parent drug and a metabolite using only 5-μL plasma sample per analysis. Incurred sample reanalysis (ISR), unexpected event investigation, and re-assay were successfully performed on the limited samples (≤ 20 μL) collected from CMS. The results of this study confirmed the feasibility of implementing CMS in regulated mouse toxicity studies and demonstrated that it is possible to eliminate or reduce satellite animals.

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.

Sickle cell disease (SCD) is a severe genetic disorder caused by a mutation in hemoglobin (b6Glu-Val), which allows the mutant hemoglobin to assemble into long polymers when deoxygenated. Over time, these polymers build up and deform red blood cells, leading to hemolytic anemia, vaso-occlusion, and end organ damage. A number of recent therapies for SCD have focused on modulating the mutant hemoglobin directly, however, reduction or elimination of 2,3-DPG to reduce Hb S polymerization and RBC sickling has recently been proposed as a therapeutic strategy for SCD. Current clinical studies focus on activation of pyruvate kinase to reduce 2,3-DPG, however, direct targeting of the enzyme which produces 2,3-DPG; Bisphosphoglycerate Mutase (BPGM) may also be possible. In this study we evaluate the impact of elimination of 2,3-DPG on SCD pathology by complete knockout of BPGM in Townes model mice. Animals with complete knockout of BPGM (BPGM -/-) have no detectable 2,3-DPG, while animals that are heterozygous for BPGM (BPGM -/+) have 2,3-DPG levels comparable to Townes mice. Western Blot analysis confirms that BPGM -/- animals completely lack BPGM, while BPGM -/+ animals have BPGM levels that are nearly equivalent to Townes mice. As expected from the lack of 2,3-DPG, BPGM -/- animals have increased oxygen affinity, observed as a 39% decrease in p50 relative to Townes mice. Complete elimination of 2,3-DPG has significant effects on markers of hemolytic anemia in BPGM -/- mice. Mice lacking 2,3-DPG have a 60% increase in hemoglobin (3.7 g/dL), a 53% increase in red blood cell count, and a 29% increase in hematocrit relative to Townes mice. The BPGM -/- mice also have a 57% decrease in reticulocytes, and a 61% decrease in spleen weight relative to Townes animals, consistent with decreased extramedullary hematopoiesis. Consistent with the reduction in hemolysis, BPGM -/- animals had a 59% reduction in red blood cell sickling under robust hypoxic conditions. BPGM -/+ animals had hemoglobin, RBC, and hematocrit levels that were similar to Townes animals, and a similar degree of RBC sickling to Townes mice. Liver phenotype was similar across all variants, with areas of random necrosis observed in BPGM -/-, BPGM -/+ and Townes mice. Higher percentages of microcytic and/or hyperchromic RBCs were observed in BPGM -/- animals relative to BPGM -/+ or Townes animals. These results suggest that modulation of 2,3-DPG has a positive effect on RBC sickling and hemolytic anemia, which may have therapeutic benefits for SCD patients. However, the lack of improvement in organ damage suggests that modulation of 2,3-DPG alone may not be sufficient for complete elimination of SCD phenotypes, and further investigation of this therapeutic avenue may be necessary. Disclosures No relevant conflicts of interest to declare.

The continuing education course on integrating clinical and anatomical pathology data was designed to communicate the importance of using a weight of evidence approach to interpret safety findings in toxicology studies. This approach is necessary, as neither clinical nor anatomic pathology data can be relied upon in isolation to fully understand the relationship between study findings and the test article. Basic principles for correlating anatomic pathology and clinical pathology findings and for integrating these with other study end points were reviewed. To highlight these relationships, a series of case examples, presented jointly by a clinical pathologist and an anatomic pathologist, were used to illustrate the collaborative effort required between clinical and anatomical pathologists. In addition, the diagnostic utility of traditional liver biomarkers was discussed using results from a meta-analysis of rat hepatobiliary marker and histopathology data. This discussion also included examples of traditional and novel liver and renal biomarker data implementation in nonclinical toxicology studies to illustrate the relationship between discrete changes in biochemistry and tissue morphology.

The Society of Toxicologic Pathology formed a working group in collaboration with the American Society for Veterinary Clinical Pathology to provide recommendations for the appropriate inclusion of clinical pathology evaluation in recovery arms of nonclinical toxicity studies but not on when to perform recovery studies. Evaluation of the recovery of clinical pathology findings is not required routinely but provides useful information on risk assessment in nonclinical toxicity studies and is recommended when the ability of the organ to recover is uncertain. The study design generally requires inclusion of concurrent controls to separate procedure-related changes from test article–related changes, but return of clinical pathology values toward baseline may be sufficient in some cases. Evaluation of either a select or full panel of standard hematology, coagulation, and serum and urine chemistry biomarkers can be scientifically justified. It is also acceptable to redesignate dosing phase animals to the recovery phase or vice versa to optimize data interpretation. Assessment of delayed toxicity during the recovery phase is not required but may be appropriate in development programs with unique concerns. Evaluation of the recovery of clinical pathology data for vaccine development is required and, for efficacy markers, is recommended if it furthers pharmacologic understanding.

The toxicologic pathologist plays a vital role in the scientific community, using their unique blend of diagnostic and investigative skills to advance biomedical research, public health, drug discovery, or regulatory practices. But what exactly do toxicologic pathologists contribute? Where do these specialized professionals work? How can toxicologic pathologists maximize their efficiency and potential? To enlighten students and trainees, as well as early- or mid-career toxicologic pathologists, or even those approaching retirement, the Career Development and Outreach Committee of the Society of Toxicologic Pathology (STP) sponsored a career development workshop entitled “Practical Strategies for Navigating Toxicologic Pathology in One’s Early Career…and Beyond!” in conjunction with the STP 37th annual symposium. The workshop featured toxicologic pathologists from contract research organizations and the pharmaceutical industry, who provided their perspectives on career preparation, evolving veterinary pathologist roles within various sectors of toxicologic pathology, the fundamentals of safety assessment, logistics of projects involving good laboratory practices, tools for effective interpretation and communication of anatomic and clinical pathology results, and a recap of scientific resources available to support the toxicologic pathologist in his or her journey. This article provides brief summaries of the talks presented during this career development workshop.

There is limited direction in the literature or regulatory guidance on determination of adversity for clinical pathology (CP) biomarkers in preclinical safety studies. Toxicologic clinical pathologists representing the American Society for Veterinary Clinical Pathology—Regulatory Affairs Committee and Society of Toxicologic Pathology—Clinical Pathology Interest Group identified principles, overall approach, and unique considerations for assessing adversity in CP data interpretation to provide a consensus opinion. Emphasized is the need for pathophysiologic context and a weight-of-evidence approach. Most CP biomarkers do not have the potential to be adverse in isolation, regardless of magnitude of change. Rather, they quantify or describe the impact of effects, provide adjunct or supportive information regarding a process or pathogenesis, and provide translational biomarkers of effect. Most often, CP changes are part of a constellation of findings that collectively are adverse. Thus, most CP changes must be interpreted in conjunction with other study findings and require contextual and integrative interpretation. Exceptions include critical CP changes without correlates that indicate a health risk in the tested species. Overall, CP changes should not be interpreted in isolation and their adversity is best addressed with an integrated approach.

In preclinical safety assessment, blood volume requirements for various endpoints pose a major challenge. The goal of this working group was to review current practices for clinical pathology (CP) testing in preclinical toxicologic studies, and to discuss advantages and disadvantages of methods for reducing blood volume requirements. An industry-wide survey was conducted to gather information on CP instrumentation and blood collection practices for hematology, clinical biochemistry, and coagulation evaluation in laboratory animals involved in preclinical studies. Based on the survey results and collective experience of the authors, the working group proposes the following "points to consider" for CP testing: (1) For most commercial analyzers, 0.5 mL and 0.8 mL of whole blood are sufficient for hematology and biochemistry evaluation, respectively. (2) Small analyzers with low volume requirements and low throughput have limited utility in preclinical studies. (3) Sample pooling or dilution is inappropriate for many CP methods. (4) Appropriate collection sites should be determined based on blood volume requirements and technical expertise. (5) Microsampling does not provide sufficient volume given current analyzer and quality assurance requirements. (6) Study design considerations include: the use of older/larger animals (rodents), collection of CP samples before toxicokinetic samples, use of separate subsets of mice for hematology and clinical biochemistry testing, use of a priority list for clinical biochemistry, and when possible, eliminating coagulation testing.

Ipilimumab is a fully human immunoglobulin G1 (IgG1) monoclonal antibody against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), an inhibitory receptor expressed on activated effector T cells and regulatory T cells (Treg), that inhibits the binding of CTLA4 to B7 ligands. BMS-986218 and BMS-986249 are second generation molecules that share the same amino acid sequence and ligand blocking properties as ipilimumab, but are mechanistically distinct. BMS-986218 is non-fucosylated (NF) and has an increased affinity for the activating Fcγ receptor (FcγR, CD16) affording the possibility of increased anti-tumor activity via depletion of Treg in the tumor. In contrast, BMS-986249 is a Probody™ of ipilimumab that has a masking peptide covering the active antigen-binding site of the antibody which is clipped by specific proteases within tumors, exposing the fully active antibody, and potentially offering reduced systemic toxicity liabilities with comparable efficacy to ipilimumab. In 1-month toxicity studies in monkeys (n = 5/sex/group) at weekly doses of 3, 15, or 75 mg/kg IV of BMS-986218 or 10 or 50 mg/kg IV of BMS-986249 or ipilimumab, profound enhancement of peripheral T-cell activation occurred in a dose-dependent manner for all 3 compounds following neoantigen immunization (keyhole limpet hemocyanin [KLH], HIV necessary and enforcing factor [Nef], and HIV group specific antigen [Gag] peptides), consistent with target pharmacology. Consistent with the intended mechanistic differences, peripheral T cell activation was generally increased at corresponding doses of BMS-986218 compared to ipilimumab, and was delayed and reduced in monkeys given BMS-986249. BMS-986218, ipilimumab, and BMS-986249 were clinically tolerated by monkeys at doses up to 3, 10, and 50 mg/kg, respectively, with generally mild, loose feces in some monkeys and/or minimal body weight decrease. At higher doses, early euthanasia occurred for 1 and 6 monkeys at 15 and 75 mg/kg BMS-986218, respectively, from Days 22-53 and 1 monkey at 50 mg/kg ipilimumab on Day 55 due to profound clinical toxicity. The predominant microscopic finding was generally dose-related lymphohistiocytic inflammation within a variety of tissues at all doses for all compounds, with BMS-986218 resulting in the greatest incidence, severity, and distribution of tissues and BMS-986249 having the least effects. The GI tract (stomach, cecum, and colon) and the kidney were the most severely and consistently affected, whereas additional organs were affected at higher doses. Most changes were partially or fully reversible during an 8-week recovery period with the exceptions of one monkey at 75 mg/kg BMS-986218 and one monkey at 50 mg/kg ipilimumab that were euthanatized on Days 53 or 55 due to unresolved GI toxicity, persistent lymphohistocytic inflammation, and/or unchanged or progressive increases in AST and ALT. Based on the tolerability and generally mild severity of lymphohistiocytic tissue inflammation, the highest non-severely toxic doses (HNSTD) for BMS-986218, ipilimumab, and BMS-986249 in monkeys following 1 month of dosing were 3 mg/kg (mean AUC 0-168h = 11,300 μg•h/mL), 10 mg/kg (AUC[0-168h] =44,600 µg·h/mL), and 50 mg/kg (AUC[0-168h] =205,000 µg·h/mL), respectively. Overall, these results support the potential of these 2nd generation anti-CTLA4 antibodies to offer an improved risk /benefit profile with increased activity of the NF variant and improved safety of the Probody™ relative to ipilimumab. Citation Format: Karen D. Price, Frank Simutis, Anthony Fletcher, Lila Ramaiah, Rima Srour, John Kozlosky, Jean Sathish, John Engelhardt, Annette Capozzi, James Crona, Courtni Newsome, Jennifer Wheeler, Daniel Szatkowski, Austin Thekkumthala, Bojing Wang, Wendy Freebern, Helen Haggerty, Todd Bunch, Michael Graziano. Nonclinical safety evaluation of two distinct second generation variants of anti-CTLA4 monoclonal antibody, ipilimumab, in monkeys [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B33.

The purpose of this paper by the Regulatory Affairs Committee (RAC) of the American Society for Veterinary Clinical Pathology (ASVCP) is to review the current regulatory guidances (eg, guidelines) and published recommendations for best practices in veterinary toxicologic clinical pathology, particularly in the pharmaceutical and biotechnology industries, and to utilize the combined experience of ASVCP RAC to provide updated recommendations. Discussion points include (1) instrumentation, validation, and sample collection, (2) routine laboratory variables, (3) cytologic laboratory variables, (4) data interpretation and reporting (including peer review, reference intervals and statistics), and (5) roles and responsibilities of clinical pathologists and laboratory personnel. Revision and improvement of current practices should be in alignment with evolving regulatory guidance documents, new technology, and expanding understanding and utility of clinical pathology. These recommendations provide a contemporary guide for the refinement of veterinary toxicologic clinical pathology best practices.