Chemistry-Based Modeling on Phenotype-Based Drug-Induced Liver Injury Annotation: From Public to Proprietary Data.

Drug-induced liver injury (DILI) is an important safety concern and a major reason to remove a drug from the market. Advancements in recent machine learning methods have led to a wide range of in silico models for DILI predictive methods based on molecule chemical structures (fingerprints). Existing publicly available DILI data sets used for model building are based on the interpretation of drug labels or patient case reports, resulting in a typical binary clinical DILI annotation. We developed a novel phenotype-based annotation to process hepatotoxicity information extracted from repeated dose in vivo preclinical toxicology studies using INHAND annotation to provide a more informative and reliable data set for machine learning algorithms. This work resulted in a data set of 430 unique compounds covering diverse liver pathology findings which were utilized to develop multiple DILI prediction models trained on the publicly available data (TG-GATEs) using the compound's fingerprint. We demonstrate that the TG-GATEs compounds DILI labels can be predicted well and how the differences between TG-GATEs and the external test compounds (Johnson & Johnson) impact the model generalization performance.

The mitochondrial fission protein Drp1 in liver is required to mitigate NASH and prevents the activation of the mitochondrial ISR.

OBJECTIVE: The mitochondrial fission protein Drp1 was proposed to promote NAFLD, as inhibition of hepatocyte Drp1 early in life prevents liver steatosis induced by high-fat diet in mice. However, whether Drp1-knockdown in older mice can reverse established NASH is unknown. METHODS: N-acetylgalactosamine-siRNA conjugates, an FDA approved method to deliver siRNA selectively to hepatocytes, were used to knockdown hepatocyte-Drp1 in mice (NAG-Drp1si). NASH was induced in C57BL/6NTac mice by Gubra-Amylin-NASH diet (D09100310, 40% fat, 22% fructose and 2% cholesterol) and treatment with NAG-Drp1si was started at week 24 of diet. Circulating transaminases, liver histology, gene expression of fibrosis and inflammation markers, and hydroxyproline synthesis determined NASH severity. Liver NEFA and triglycerides were quantified by GC/MS. Mitochondrial function was determined by respirometry. Western blots of Oma1, Opa1, p-eIf2α, as well as transcriptional analyses of Atf4-regulated genes determined ISR engagement. RESULTS: NAG-Drp1si treatment decreased body weight and induced liver inflammation in adult healthy mice. Increased hepatic Gdf15 production was the major contributor to body-weight loss caused by NAG-Drp1si treatment, as Gdf15 receptor deletion (Gfral KO) prevented the decrease in food intake and mitigated weight loss. NAG-Drp1si activated the Atf4-controlled integrated stress response (ISR) to increase hepatic Gdf15 expression. NAG-Drp1si in healthy mice caused ER stress and activated the mitochondrial protease Oma1, which are the ER and mitochondrial triggers that activate the Atf4-controlled ISR. Remarkably, induction of NASH was not sufficient to activate Oma1 in liver. However, NAG-Drp1si treatment was sufficient to activate Oma1 in adult mice with NASH, as well as exacerbating NASH-induced ER stress. Consequently, NAG-Drp1si treatment in mice with NASH led to higher ISR activation, exacerbated inflammation, fibrosis and necrosis. CONCLUSION: Drp1 mitigates NASH by decreasing ER stress, preventing Oma1 activation and ISR exacerbation. The elevation in Gdf15 actions induced by NAG-Drp1si might represent an adaptive response decreasing the nutrient load to liver when mitochondria are misfunctional. Our study argues against blocking Drp1 in hepatocytes to combat NASH.

Special Issue on the Pathobiology of Laboratory Nonhuman Primates: A Review of Species, Substrain, Geographical Origin, Age, and Modality-Related Factors.

Nonhuman primates (NHPs) are utilized in nonclinical safety testing due to their phylogenetic proximity to humans and similarity in physiology and anatomy. However, ethical considerations and the increased demand for NHPs, coupled with the current shortage in their supply, have increased the calls to minimize their use. In addition, the increased demand and supply shortage of NHPs have increased the use of animals sourced from different geographical origins, and animals of different ages, which can complicate the interpretation of study results. Coupled with the relative uniqueness of findings induced by novel therapeutic modalities, there is an increasing need for a deeper understanding of the systemic pathobiology of NHPs. Here we provide a brief preview of the two main themes discussed in this special issue, which include the influence of geographical origin, age, and sex on background pathology, clinical pathology reference values, other relevant toxicology endpoints, and organ system pathology.

Cytotoxic T lymphocyte antigen-4 regulates development of xenogenic graft versus host disease in mice via modulation of host immune responses induced by changes in human T cell engraftment and gene expression.

Graft versus host disease (GvHD) is a major clinical problem with a significant unmet medical need. We examined the role of cytotoxic T lymphocyte antigen-4 (CTLA-4) in a xenogenic GvHD (xeno-GvHD) model induced by injection of human peripheral mononuclear cells (hPBMC) into irradiated non-obese diabetic (NOD) SCID gamma (NSG) mice. Targeting the CTLA-4 pathway by treatment with CTLA-4 immunoglobulin (Ig) prevented xeno-GvHD, while anti-CTLA-4 antibody treatment exacerbated the lethality and morbidity associated with GvHD. Xeno-GvHD is associated with infiltration of hPBMCs into the lungs, spleen, stomach, liver and colon and an increase in human proinflammatory cytokines, including interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-5. Infiltration of donor cells and increases in cytokines were attenuated by treatment with CTLA-4 Ig, but remained either unaffected or enhanced by anti-CTLA-4 antibody. Further, splenic human T cell phenotyping showed that CTLA-4 Ig treatment prevented the engraftment of human CD45+ cells, while anti-CTLA-4 antibody enhanced donor T cell expansion, particularly CD4+ (CD45RO+ ) subsets, including T box transcription factor TBX21 (Tbet)+ CXCR3+ and CD25+ forkhead box protein 3 (FoxP3) cells. Comprehensive analysis of transcriptional profiling of human cells isolated from mouse spleen identified a set of 417 differentially expressed genes (DEGs) by CTLA-4 Ig treatment and 13 DEGs by anti-CTLA-4 antibody treatment. The CTLA-4 Ig regulated DEGs mapped to down-regulated apoptosis, inflammasome, T helper type 17 (Th17) and regulatory T cell (Treg ) pathways and enhanced Toll-like receptor (TLR) receptor signaling, TNF family signaling, complement system and epigenetic and transcriptional regulation, whereas anti-CTLA-4 antibody produced minimal to no impact on these gene pathways. Our results show an important role of co-inhibitory CTLA-4 signaling in xeno-GvHD and suggest the therapeutic utility of other immune checkpoint co-inhibitory pathways in the treatment of immune-mediated diseases driven by hyperactive T cells.

Outcomes of the European Federation of Pharmaceutical Industries and Associations Oligonucleotide Working Group Survey on Nonclinical Practices and Regulatory Expectations for Therapeutic Oligonucleotide Safety Assessment.

The Oligonucleotide Working Group of the European Federation of Pharmaceutical Industries and Associations (EFPIA) conducted a survey of companies to understand the trends in nonclinical practices and regulatory expectations for oligonucleotide drug safety assessment. Twenty-two companies of different types, with varying oligonucleotide experience levels in the field, participated. The survey identified key regulatory challenges and areas of perceived health authority (HA) concern regarding nonclinical safety strategies for oligonucleotides, such as the choice of toxicology species, approaches to dose setting in toxicity studies, dose scaling from animals to humans, the implementation (and regulatory acceptability) of lean packages, and methods for dealing with impurities and human-specific off-targets. The perceived oligonucleotide experience of HAs and the relevance of guidance to oligonucleotide development were also assessed. The results showed a general lack of consensus on nonclinical safety assessment approaches being used for this growing class of medicines and highlight the need for continuing collaboration between sponsors and HAs to better define best practices.

Normal Anatomy, Histology, and Spontaneous Pathology of the Kidney, and Selected Renal Biomarker Reference Ranges in the Cynomolgus Monkey.

To further our understanding of the nonhuman primate kidney anatomy, histology, and incidences of spontaneous pathology, we retrospectively examined kidneys from a total of 505 control Cynomolgus monkeys (Macaca fascicularis; 264 male and 241 females) aged 2 to 6 years, from toxicity studies. Kidney weights, urinalysis, and kidney-related clinical biochemistry parameters were also evaluated. Although the functional anatomy of the monkey kidney is relatively similar to that of other laboratory animals and humans, a few differences and species-specific peculiarities exist. Unlike humans, the macaque kidney is unipapillate, with a relatively underdeveloped papilla, scarce long loops of Henle, and a near-equivalent cortical to medullary ratio. The most common spontaneous microscopic findings were interstitial infiltrates or interstitial nephritis and other tubular lesions, but several forms of glomerulopathy that may be interpreted as drug-induced were occasionally observed. Common incidental findings of little pathological significance included: papillary mineralization, epithelial pigment, multinucleate cells, cuboidal metaplasia of the Bowman's capsule, and urothelial inclusions. Kidney weights, and some clinical chemistry parameters, showed age- and sex-related variations. Taken together, these data will aid the toxicologic pathologist to better evaluate the nonhuman primate kidney and assess the species' suitability as a model for identifying and characterizing drug-induced injury.

Morphological characterization of canine retrobulbar lymphoma.

This report characterized seven cases of canine retrobulbar lymphoma that have been diagnosed during 2008 to 2014 by immunophenotyping of CD3 and Pax5. Classification of lymphoma were performed according to the revised WHO guidelines. Four retrobulbar lymphomas were of T-cell origin, while the others were of B-cell. Out of 7 cases, four subtypes were diagnosed in this study; T-cell-rich large B-cell lymphoma (3/7), T-cell lymphoblastic lymphoma (2/7), peripheral T-cell lymphoma (1/7), and cutaneous nonepitheliotropic lymphoma (1/7). T-cell-rich large B-cell lymphoma was found to be the most frequent subtype found.

A Translational Model for Diet-related Atherosclerosis: Effect of Statins on Hypercholesterolemia and Atherosclerosis in a Minipig.

Models of atherosclerosis are used in preclinical studies but often fail to translate to humans. A model that better reflects human atherosclerosis is necessary. We recently engineered the ExeGen™ low-density lipoprotein receptor (LDLR) miniswine, in which the LDL receptor gene is modified to drive hypercholesterolemia and atherosclerosis, and showed diet-related exacerbation of these phenotypes. Five groups of animals, either wild type (+/+) or heterozygous (+/-), were fed either a normal or high-fat diet for 6 months. One group of heterozygous pigs fed a high-fat diet was also administered atorvastatin at 3 mg/kg/day. Clinical chemistry and anatomic pathology parameters were measured biweekly and at termination. The high-fat diet resulted in increased adiposity and interspersion of adipocytes within the salivary glands. The heterozygous pigs on the high-fat diet gained more weight and had significant increases in total cholesterol, high-density lipoprotein, and LDL compared to wild-type animals or heterozygous animals fed a normal diet. Atorvastatin attenuated these parameters, indicating the statin had a beneficial effect, even in a high-fat diet scenario. Atorvastatin treatment also reduced the intensity of Oil Red O staining in pigs on high-fat diet. Atorvastatin-related amelioration of several indices of cardiovascular pathophysiology in this model underscores its utility for drug discovery.

Evaluation of insulin-like growth factor acid-labile subunit as a potential biomarker of effect for deoxynivalenol-induced proinflammatory cytokine expression.

Consumption of the trichothecene deoxynivalenol (DON) suppresses growth in experimental animals - an adverse effect that was used to establish the tolerable daily intake for this toxin. DON ingestion has been recently found to suppress plasma insulin-like growth factor acid-labile subunit (IGFALS), a protein essential for growth. Studies were conducted to explore the feasibility of using plasma IGFALS as a biomarker of effect for DON. In the first study, weanling mice were fed 0, 1, 2.5, 5 and 10 ppm DON and weight and plasma IGFALS determined at intervals over 9 wk. Reduced body weight gains were detectable beginning at wk 5 in the 10 ppm dose and wk 7 at the 5 ppm dose. Plasma IGFALS was significantly depressed at wk 5 in the 5 and 10 ppm groups at wk 9 in the 10 ppm group. Depressed IGFALS significantly correlated with reduced body weight at wk 5 and 9. Benchmark dose modeling revealed the BMDL and BMD for plasma IGFALS reduction were 1.1 and 3.0 ppm DON and for weight reduction were 2.1 and 4.5 ppm DON. In the second study, it was demonstrated that mice fed 15 ppm DON diet had significantly less plasma IGFALS than mice fed identical amounts of control diet. Thus DON's influence on IGFALS likely reflects the combined effects of reduced food intake as well as its physiological action involving suppressors of cytokine signaling. Taken together, these findings suggest that plasma IGFALS might be a useful biomarker for DON's adverse effects on growth.