Unexpected neurotoxicity in chronic toxicity studies with a HBV viral expression inhibitor.

The non-clinical safety profile of the small molecule hepatitis B virus viral expression inhibitor RG7834 was studied in a package consisting of safety pharmacology, genotoxicity, repeat dose toxicity and reproductive toxicity studies. The chronic monkey toxicity study identified dose- and time-dependent symptoms of polyneuropathy, with correlating nerve conduction velocity reductions and axonal degeneration in peripheral nerves and spinal cord, in all compound treatment groups with no evidence of reversibility after approximately 3 months of treatment cessation. Similar histopathological findings were observed in the chronic rat toxicity study. Subsequent in vitro neurotoxicity investigations and ion channel electrophysiology did not elucidate a potential mechanism for the late toxicity. However, based on similar findings observed with a structurally different molecule, an inhibition of their common pharmacological targets, PAPD5 & PAPD 7, was considered as a possible mechanism of toxicity. In conclusion, the marked neuropathies, only observed after chronic dosing, did not support further clinical development of RG7834 because of its foreseen clinical treatment duration of up to 48 weeks in chronic HBV patients.

Toxicologic Pathology Forum: A Roadmap for Building State-of-the-Art Digital Image Data Resources for Toxicologic Pathology in the Pharmaceutical Industry.

Digitization of histologic slides brings with it the promise of enhanced toxicologic pathology practice through the increased application of computational methods. However, the development of these advanced methods requires access to substrate image data, that is, whole slide images (WSIs). Deep learning methods, in particular, rely on extensive training data to develop robust algorithms. As a result, pharmaceutical companies interested in leveraging computational methods in their digital pathology workflows must first invest in data infrastructure to enable data access for both data scientists and pathologists. The process of building robust image data resources is challenging and includes considerations of generation, curation, and storage of WSI files, and WSI access including via linked metadata. This opinion piece describes the collective experience of building resources for WSI data in the Roche group. We elaborate on the challenges encountered and solutions developed with the goal of providing examples of how to build a data resource for digital pathology analytics in the pharmaceutical industry.

Immunohistochemical Characterization of Proliferative Lesions in the Thymus of Aging CD-1 Mice From Two Studies on the RITA Database, With Special Reference to the Perivascular Space.

Thymic lymphoid hyperplasia is a common age-related finding, which occurs particularly in female CD-1 mice. The main differential diagnoses are malignant lymphoma and thymoma. A systematic investigation of control groups from two carcinogenicity studies was performed including measurements of thymic size, and the immunohistochemistry (IHC) markers pan-Cytokeratin (pan-CK) for thymic epithelial cells; CD3 and CD45R/B220 for T and B lymphocytes, respectively; CD31 for endothelial cells; and F4/80 for macrophages. Thymoma can be differentiated by increased numbers of proliferating epithelial cells demonstrated by pan-CK IHC staining. Differentiation between lymphoid hyperplasia and lymphoma is more challenging as a mixture of B and T lymphocytes can be present in both findings. The present investigation showed that the thymic perivascular space is the compartment where the increased numbers of lymphocytes in hyperplasia are localized and not the medulla, as previously thought. The lymphoepithelial compartment is atrophic to the same extent in thymi diagnosed with age-related involution or lymphoid hyperplasia. Both diagnoses are thus related to variations in lymphoid cellularity of the nonepithelial perivascular space, which is continuous with the perithymic tissue. Likewise, lymphomas have a predilection to colonize the perivascular space and to spare the lymphoepithelial compartment.

Selective pharmacological inhibition of DDR1 prevents experimentally-induced glomerulonephritis in prevention and therapeutic regime.

BACKGROUND: Discoidin domain receptor 1 (DDR1) is a collagen-activated receptor tyrosine kinase extensively implicated in diseases such as cancer, atherosclerosis and fibrosis. Multiple preclinical studies, performed using either a gene deletion or a gene silencing approaches, have shown this receptor being a major driver target of fibrosis and glomerulosclerosis. METHODS: The present study investigated the role and relevance of DDR1 in human crescentic glomerulonephritis (GN). Detailed DDR1 expression was first characterized in detail in human GN biopsies using a novel selective anti-DDR1 antibody using immunohistochemistry. Subsequently the protective role of DDR1 was investigated using a highly selective, novel, small molecule inhibitor in a nephrotoxic serum (NTS) GN model in a prophylactic regime and in the NEP25 GN mouse model using a therapeutic intervention regime. RESULTS: DDR1 expression was shown to be mainly limited to renal epithelium. In humans, DDR1 is highly induced in injured podocytes, in bridging cells expressing both parietal epithelial cell (PEC) and podocyte markers and in a subset of PECs forming the cellular crescents in human GN. Pharmacological inhibition of DDR1 in NTS improved both renal function and histological parameters. These results, obtained using a prophylactic regime, were confirmed in the NEP25 GN mouse model using a therapeutic intervention regime. Gene expression analysis of NTS showed that pharmacological blockade of DDR1 specifically reverted fibrotic and inflammatory gene networks and modulated expression of the glomerular cell gene signature, further validating DDR1 as a major mediator of cell fate in podocytes and PECs. CONCLUSIONS: Together, these results suggest that DDR1 inhibition might be an attractive and promising pharmacological intervention for the treatment of GN, predominantly by targeting the renal epithelium.

An innovative investigative approach to characterize the effects observed in a combined fertility study in male and female rats.

This paper describes the characterization of male- and female-mediated effects in a standard ICH rat fertility and early embryonic development study with a discontinued clinical small molecule. In the standard study, the test item had no effect on the number of treated females becoming pregnant, but litter sizes were reduced at the high dose level. In the treated male rats, increased incidences of abnormal sperm, decreases in average sperm path and straight line velocities, and minimal retention of mature sperm in the seminiferous tubules were observed at all dose-levels tested. These findings were unexpected in view of a lack of histopathological changes in the reproductive organs of either gender in 4-week repeat dose studies in rats and monkeys. A follow-up fertility study was conducted using an innovative flexible study design and a single high-dose level. In the first instance, treated male rats were mated with untreated females, followed by necropsy of a subset of males. The intention was then to re-mate the males after an 8-week wash-out period with either treated or untreated females depending on the outcome of the first mating. On this occasion, litter sizes were not affected, but the testicular effects were reproduced. A second mating with treated females reproduced the reduced litter sizes due to increased pre- and post-implantation loss, demonstrating that the effect on fecundity was female-mediated. The testicular changes in males were shown to be reversible after a 12-week recovery period.

Image analysis for TSH mRNA in situ hybridization in pituitary glands from rats with thyroid follicular cell hypertrophy after treatment with three different test compounds.

The goal of this in situ hybridization and image analysis technique is to study the effects of new pharmacological/chemical entities on the thyroid and pituitary gland in rats, reveal the pathogenesis of thyroid follicular cell hypertrophy and to retrospectively exclude the risk of thyroid tumor development in humans. In the present study, we describe the increase of thyroid-stimulating hormone- (TSH-) beta subunit mRNA in the pars distalis of the pituitary gland and the quantitative measurement of TSH mRNA positive cells from rats of three 4-week toxicity studies treated with three different test compounds inducing thyroid follicular cell and hepatocellular hypertrophy in rats. Compared to immunohistochemistry (IHC), in situ hybridization (ISH) for TSH was found to be more sensitive. With this technique we are able to exclude a direct effect of the test compound on the thyroid gland by showing the activation of thyrotrope cells from the pituitary gland and therefore this technique retrospectively enables us to exclude a possible risk for humans at an early stage of drug development. Also in case blood serum samples for evaluation of TSH are not available anymore or hepatocellular hypertrophy is not present (close metabolic relationship between thyroid gland and liver in rodents), the described method allows retrospective investigations on thyroid follicular cell hypertrophy or hyperplasia. This can be of high relevance in human safety assessment for certain drugs in order to exclude a primary effect on the thyroid gland especially when it comes to thyroid neoplasia in rodents as previously described.

Use of early phenotypic in vivo markers to assess human relevance of an unusual rodent non-genotoxic carcinogen in vitro.

Foci of altered hepatocytes (FAH) are considered putative, pre-neoplastic lesions that can occur spontaneously in aging rodents, but can also be induced by chemicals or drugs. Progression of FAH to hepatocellular neoplasms has been reported repeatedly but increases in foci in rodents do not necessarily lead to tumors in carcinogenicity studies and the relevance for humans often remains unclear. Here we present the case of RG3487, a molecule which induced FAH and, later on, tumors in rats. Because the molecule was negative in genotoxicity assays it was classified as a non-genotoxic carcinogen. In order to assess the potential for liver tumor formation in humans, we analyzed treatment-induced changes in vivo to establish a possible mode of action (MoA). In vivo and in vitro gene expression analysis revealed that nuclear receptor signaling was unlikely to be the relevant MoA and no other known mechanism could be established. We therefore took an approach comparing phenotypic markers, including mRNA changes, proliferation and glycogen accumulation, in vitro using cells of different species to assess the human relevance of this finding. Since the alterations observed in rats were not seen in the liver of mice or dogs in vivo, we could validate the relevance of the cell models chosen by use of hepatocytes from these species in vitro. This ultimately allowed for a cross-species comparison, which suggested that the formation of FAH and liver tumors was rat specific and unlikely to translate to human. Our work showed that phenotypic species comparison in vitro is a useful approach for assessment of the human relevance of pre-clinical findings where no known mechanism can be established.

From the Cover: The Minipig is a Suitable Non-Rodent Model in the Safety Assessment of Single Stranded Oligonucleotides.

Non-human primates (NHPs) are currently considered to be the non-rodent species of choice for the preclinical safety assessment of single-stranded oligonucleotide (SSO) drugs. We evaluated minipigs as a potential alternative to NHPs to test the safety of this class of compounds. Four different phosphorothioated locked nucleic acid-based SSOs (3 antisense and 1 anti-miR), all with known safety profiles, were administered to minipigs using similar study designs and read-outs as in earlier NHP studies with the same compounds. The studies included toxicokinetic investigations, in-life monitoring, clinical and anatomic pathology. In the minipig, we demonstrated target engagement by the SSOs where relevant, and a similar toxicokinetic behavior in plasma, kidney, and liver when compared with NHPs. Clinical tolerability was similar between minipig and NHPs. For the first time, we showed similar and dose-dependent effects on the coagulation and complement cascade after intravenous dosing similar to those observed in NHPs. Similar to NHPs, morphological changes were seen in proximal tubular epithelial cells of the kidney, Kupffer cells, hepatocytes, and lymph nodes. Minipigs appeared more sensitive to the high-dose kidney toxicity of most of the selected SSOs than NHPs. No new target organ or off-target toxicities were identified in the minipig. The minipig did not predict the clinical features of human injection site reactions better than the NHPs, but histopathological similarities were observed between minipigs and NHPs. We conclude that there is no impediment, as default, to the use of minipigs as the non-rodent species in SSO candidate non-clinical safety packages.

Bioprinted 3D Primary Liver Tissues Allow Assessment of Organ-Level Response to Clinical Drug Induced Toxicity In Vitro.

Modeling clinically relevant tissue responses using cell models poses a significant challenge for drug development, in particular for drug induced liver injury (DILI). This is mainly because existing liver models lack longevity and tissue-level complexity which limits their utility in predictive toxicology. In this study, we established and characterized novel bioprinted human liver tissue mimetics comprised of patient-derived hepatocytes and non-parenchymal cells in a defined architecture. Scaffold-free assembly of different cell types in an in vivo-relevant architecture allowed for histologic analysis that revealed distinct intercellular hepatocyte junctions, CD31+ endothelial networks, and desmin positive, smooth muscle actin negative quiescent stellates. Unlike what was seen in 2D hepatocyte cultures, the tissues maintained levels of ATP, Albumin as well as expression and drug-induced enzyme activity of Cytochrome P450s over 4 weeks in culture. To assess the ability of the 3D liver cultures to model tissue-level DILI, dose responses of Trovafloxacin, a drug whose hepatotoxic potential could not be assessed by standard pre-clinical models, were compared to the structurally related non-toxic drug Levofloxacin. Trovafloxacin induced significant, dose-dependent toxicity at clinically relevant doses (≤ 4uM). Interestingly, Trovafloxacin toxicity was observed without lipopolysaccharide stimulation and in the absence of resident macrophages in contrast to earlier reports. Together, these results demonstrate that 3D bioprinted liver tissues can both effectively model DILI and distinguish between highly related compounds with differential profile. Thus, the combination of patient-derived primary cells with bioprinting technology here for the first time demonstrates superior performance in terms of mimicking human drug response in a known target organ at the tissue level.

Advanced Clinical Imaging and Tissue-based Biomarkers of the Eye for Toxicology Studies in Minipigs.

There is increased interest to use minipigs in ocular toxicology studies due to their anatomical similarities with human eyes and as a substitute for nonhuman primates. This requires adaptation of enhanced optical coherence tomography (OCT) techniques and of ocular relevant immunohistochemistry (IHC) or in situ hybridization (ISH) markers to porcine eyes. In this study, OCT and OCT angiography (AngioOCT) were performed on adult Göttingen minipigs. To increase structural information on retinal and choroidal vasculature, OCT data were speckle denoized and choroidal blood vessels were segmented with threshold filtering. In addition, we established a set of IHC and ISH markers on Davidson's fixed paraffin-embedded minipig eyes: neurofilament-160, neuronal nuclei, calretinin, protein kinase C-α, vimentin, glial fibrillary acidic protein, glutamine synthetase, ionized calcium-binding adaptor molecule-1, rhodopsin, synaptophysin, postsynaptic density protein-95, retinal pigment epithelium (RPE)-specific protein-65, von Willebrand factor, α-smooth muscle actin, desmin, and Ki-67, thus enabling visualization of retinal neuronal and glial cells, photoreceptors, synapses, RPE, blood vessels, myocytes, macrophages, or cell proliferation. Using ISH, transcripts of vascular endothelial growth factor A, angiopoietin-2, and endothelial tyrosine kinase were visualized. This article describes for the first time in minipig eyes speckle noise-free OCT, AngioOCT, and a set of IHC/ISH markers on Davidson's fixed paraffin-embedded tissues and helps to establish the minipig for ocular toxicology and pharmacology studies.

A proposal for a novel rationale for critical effect size in dose-response analysis based on a multi-endpoint in vivo study with methyl methanesulfonate.

Methyl methanesulfonate, a well-known direct-acting genotoxicant, was assessed in a multi-endpoint study in rats using six closely spaced dose levels. The main goal of the study was to investigate the genotoxic response at very low doses and to analyse this response with dedicated statistical tools in order to find a Point of Departure (PoD) and related metrics. Software packages like PROAST or EPA-BMDS require the toxicologist to define a so-called critical effect size (CES) or benchmark response (BMR) and this choice has a large impact on the result of the PoD calculation. Currently, increases of 5%, 10% or 1 standard deviation over concurrent vehicle controls have been proposed for CES/BMR, values that may or may not be suited for all genotoxicity endpoints. Based on the data obtained in this study, we propose an endpoint specific CES approach that reflects the typical evaluation process of a regulatory acceptable genotoxicology study. However, we are aware that this ratio-based CES strategy will need to be more fully developed with additional experimentation and should be mainly seen as a starting point for scientific discussion.

The distributional nexus of choroid plexus to cerebrospinal fluid, ependyma and brain: toxicologic/pathologic phenomena, periventricular destabilization, and lesion spread.

Bordering the ventricular cerebrospinal fluid (CSF) are epithelial cells of choroid plexus (CP), ependyma and circumventricular organs (CVOs) that contain homeostatic transporters for mediating secretion/reabsorption. The distributional pathway ("nexus") of CP-CSF-ependyma-brain furnishes peptides, hormones, and micronutrients to periventricular regions. In disease/toxicity, this nexus becomes a conduit for infectious and xenobiotic agents. The sleeping sickness trypanosome (a protozoan) disrupts CP and downstream CSF-brain. Piperamide is anti-trypanosomic but distorts CP epithelial ultrastructure by engendering hydropic vacuoles; this reflects phospholipidosis and altered lysosomal metabolism. CP swelling by vacuolation may occlude CSF flow. Toxic drug tools delineate injuries to choroidal compartments: cyclophosphamide (vasculature), methylcellulose (interstitium), and piperazine (epithelium). Structurally perturbed CP allows solutes to penetrate the ventricles. There, CSF-borne pathogens and xenobiotics may permeate the ependyma to harm neurogenic stem cell niches. Amoscanate, an anti-helmintic, potently injures rodent ependyma. Ependymal/brain regions near CP are vulnerable to CSF-borne toxicants; this proximity factor links regional barrier breakdown to nearby periventricular pathology. Diverse diseases (e.g., African sleeping sickness, multiple sclerosis) take early root in choroidal, circumventricular, or perivascular loci. Toxicokinetics informs on pathogen, anti-parasitic agent, and auto-antibody distribution along the CSF nexus. CVOs are susceptible to plasma-borne toxicants/pathogens. Countering the physico-chemical and pathogenic insults to the homeostasis-mediating ventricle-bordering cells sustains brain health and fluid balance.

Primary endothelial damage is the mechanism of cardiotoxicity of tubulin-binding drugs.

The use of tubulin binders (TBs) in the treatment of cancer often is associated with cardiotoxicity, the mechanism of which has not been elucidated. To test the hypothesis that interstitial cells of the myocardium are the primary target of TBs, we evaluated the acute effects of a single iv administration of three reference TBs: colchicine (0.2 and 2 mg/kg), vinblastine (0.5 and 3 mg/kg), and vincristine (0.1 and 1 mg/kg) 6 and 24 h after dosing. Mitotic arrest was identified at 24 h in all high-dose groups based on an increase in the number of mitotic figures in the interstitium coupled with a decrease in the number of Ki67-positive interstitial cells. Analysis of the myocardial transcriptomic data further supported G2/M cell cycle arrest 6 h after dosing with the high-dose groups of all three compounds. Apoptotic figures and an increase in the number of cleaved caspase 3-positive cells were identified at 6 and 24 h at the highest dose of each compound predominantly in interstitial cells, whereas a few cardiomyocytes were affected as well. Transcriptomic profiling of the myocardium further suggested that some of the affected interstitial cells were endothelial cells based on the upregulation of genes typically associated with vascular damage and downregulation of endothelial cell-specific molecule 1 and apelin. Taken together, these data identify endothelial cells of the myocardium as the primary target of the cardiotoxicity of TBs and identify cell cycle arrest as the mechanism of this toxicity.