Antisense Oligonucleotide-Related Macrovesicular Vacuolation of Hippocampal Neurons in Nonhuman Primates.

2'-methoxyethyl (MOE) antisense oligonucleotides (ASOs) tested in multidose intrathecal nonhuman primate (NHP) toxicity studies have consistently revealed the presence of single large vacuoles in pyramidal neurons of the hippocampus in the absence of any cellular response. Termed "macrovesicular," these vacuoles were characterized by immunohistochemistry and transmission electron microscopy which showed that these vacuoles are dilated lysosomes in neurons containing accumulated ASO. Additionally, two NHP studies were conducted to investigate the role of tissue fixation on their histogenesis. In Fixation Study 1, 6 doses of 5 mg 2'-MOE ASO with a full phosphorothioate backbone were administered by lumbar puncture over 5 weeks; in Fixation Study 2, 5 doses of 35 mg 2'-MOE ASO with a mixed phosphorothioate/phosphodiester backbone were administered over 12 weeks. At necropsy in each study, brain slices were either immersion fixed in neutral buffered 10% formalin or Carnoy's fixative; frozen at -80 °C; or perfusion fixed with modified Karnovsky's fixative. Fixed samples were processed to paraffin, sectioned, and stained with hematoxylin and eosin (H&E) and compared with H&E cryosections prepared from the frozen tissue of the same brain. The presence of vacuoles in fixed brain tissue but never in fresh frozen tissue showed that they arose during postmortem tissue fixation, and as such represent a processing artifact that is not relevant to human safety assessment of intrathecally administered 2'-MOE ASOs.

LRRK2 inhibitors induce reversible changes in nonhuman primate lungs without measurable pulmonary deficits.

The kinase-activating mutation G2019S in leucine-rich repeat kinase 2 (LRRK2) is one of the most common genetic causes of Parkinson's disease (PD) and has spurred development of LRRK2 inhibitors. Preclinical studies have raised concerns about the safety of LRRK2 inhibitors due to histopathological changes in the lungs of nonhuman primates treated with two of these compounds. Here, we investigated whether these lung effects represented on-target pharmacology and whether they were reversible after drug withdrawal in macaques. We also examined whether treatment was associated with pulmonary function deficits. We conducted a 2-week repeat-dose toxicology study in macaques comparing three different LRRK2 inhibitors: GNE-7915 (30 mg/kg, twice daily as a positive control), MLi-2 (15 and 50 mg/kg, once daily), and PFE-360 (3 and 6 mg/kg, once daily). Subsets of animals dosed with GNE-7915 or MLi-2 were evaluated 2 weeks after drug withdrawal for lung function. All compounds induced mild cytoplasmic vacuolation of type II lung pneumocytes without signs of lung degeneration, implicating on-target pharmacology. At low doses of PFE-360 or MLi-2, there was ~50 or 100% LRRK2 inhibition in brain tissue, respectively, but histopathological lung changes were either absent or minimal. The lung effect was reversible after dosing ceased. Lung function tests demonstrated that the histological changes in lung tissue induced by MLi-2 and GNE-7915 did not result in pulmonary deficits. Our results suggest that the observed lung effects in nonhuman primates in response to LRRK2 inhibitors should not preclude clinical testing of these compounds for PD.

Incidental Ultrastructural Findings in the Sural Nerve and Dorsal Root Ganglion of Aged Control Sprague Dawley Rats in a Nonclinical Carcinogenicity Study.

Xenobiotic-induced peripheral nerve damage is a growing concern. Identifying relative risks that a new drug may cause peripheral nerve injury over long periods of administration is gathering importance in the evaluation of animal models. Separating out age-related changes in peripheral nerves of rats caused by compression injury from drug-induced effects has been difficult. Biopsy of the sural nerve is utilized in humans for investigations of peripheral neuropathy, because it is largely removed from the effects of nerve compression. This study used transmission electron microscopy to identify incidental findings in the sural nerves and dorsal root ganglia of aged control rats over time. The goal was to establish a baseline understanding of the range of possible changes that could be noted in controls compared to rats treated with any new investigative drug. In this evaluation, most sural nerve fibers from aged control rats had few ultrastructural abnormalities of pathologic significance. However, glycogenosomes, polyglucosan bodies, swollen mitochondria, autolysosomes, split myelin, Schwann cell processes, and endoneural macrophages with phagocytosed debris (considered an indication of ongoing degenerative changes) were occasionally noted.

Myocarditis in Cynomolgus Monkeys Following Treatment with Immune Checkpoint Inhibitors.

PURPOSE: Immune checkpoint inhibitors (ICI) targeting PD1, PDL1, or CTLA4 are associated with immune-related adverse events (irAE) in multiple organ systems including myocarditis. The pathogenesis and early diagnostic markers for ICI-induced myocarditis are poorly understood, and there is currently a lack of laboratory animal model to enhance our understanding. We aimed to develop such a model using cynomolgus monkeys. EXPERIMENTAL DESIGN: Chinese-origin cynomolgus monkeys were dosed intravenously with vehicle or nivolumab 20 mg/kg plus ipilimumab 15 mg/kg once weekly and euthanized on day 29. RESULTS: Multiple organ toxicities were observed in cynomolgus monkeys, and were characterized by loose feces, lymphadenopathy, and mononuclear cell infiltrations of varying severity in heart, colon, kidneys, liver, salivary glands, and endocrine organs. Increased proliferation of CD4+ and CD8+ T lymphocytes as well as an increase in activated T cells and central memory T cells in the blood, spleen, and lymph nodes, were observed. Transcriptomic analysis suggested increased migration and activation of T cells and increased phagocytosis and antigen presentation in the heart. Mononuclear cell infiltration in myocardium was comprised primarily of T cells, with lower numbers of macrophages and occasional B cells, and was associated with minimal cardiomyocyte degeneration as well as increases in cardiac troponin-I and NT-pro-BNP. Morphologically, cardiac lesions in our monkey model are similar to the reported ICI myocarditis in humans. CONCLUSIONS: We have developed a monkey model characterized by multiple organ toxicities including myocarditis. This model may provide insight into the immune mechanisms and facilitate biomarker identification for ICI-associated irAEs.

Evaluation of cardiovascular changes in dogs administered three positive controls using jacketed external telemetry-blood pressure (JET-BP).

INTRODUCTION: Nonclinical safety studies are increasingly incorporating cardiac safety endpoints to discover potential cardiovascular liabilities. This trend for more thorough cardiovascular nonclinical safety evaluation is prudent given the high attrition rate of potential therapeutics due to unexpected cardiovascular liabilities discovered in late-stage clinical trials or post-market approval. In particular, the causal relationship of blood pressure changes that lead to risk of major adverse cardiac events suggests hemodynamic changes should be critically evaluated in preclinical studies of novel therapeutics. METHODS: Jacketed external telemetry with an implanted miniature blood pressure transmitter (JET-BP) was used to characterize the tolerability, functionality, and sensitivity of this study design in dogs. Thirty-six male or female beagles (n=6 dogs/sex/group) were administered vehicle control (reverse osmosis water) or etilefrine (1, 10mg/kg), sotalol (3, 30mg/kg), and hydralazine (1, 10mg/kg) on separate days. Telemetry data were evaluated for positive control article-related changes and retrospective power analysis was also completed. Animals were evaluated for instrumentation-related changes in clinical and anatomic pathology endpoints. RESULTS: All three positive controls elicited the expected pharmacologic responses that were statistically different at high and low doses. Retrospective power analysis confirmed this study design was able to statistically differentiate minor (approximately 5 to 15%) changes in electrocardiography and blood pressure values. This study also demonstrated the potential advantages of combining cardiovascular data across sex when the test article exposure and pharmacodynamics were consistent. Data collection using miniature telemetry blood pressure transmitters did not result in anatomic or clinical pathology findings that would prevent their use in general toxicology studies. DISCUSSION: This characterization study indicates that JET-BP in dogs offers a scientifically-robust method to evaluate novel therapeutics for potential cardiovascular liabilities.

Cytologic identification of immature endospores in a dog with rhinosporidiosis.

An 8-year-old, intact, male Labrador Retriever was presented to the Boren Veterinary Medical Teaching Hospital at Oklahoma State University with a 2-month history of severe sneezing episodes that resulted in epistaxis and bilateral sanguineous discharge. Rhinoscopy revealed a small polypoid mass, and specimens were obtained for histopathology. Microscopic examination of formalin-fixed tissue specimens revealed organisms consistent with Rhinosporidium seeberi. The mass was surgically excised and impression smears were made for cytology examination. Smears revealed high numbers of endospores, typical of those previously described for R seeberi. In addition, numerous smaller structures, presumed to be immature endospores, were noted. The immature endospores were morphologically distinct from mature endospores and have not been described previously. Recognition of immature forms of Rhinosporidium may help prevent misidentification of the organism or misdiagnosis of a dual infection.

Cytokines and synthetic double-stranded RNA augment the T helper 1 immune response of swine to porcine reproductive and respiratory syndrome virus.

Immunization of pigs with a modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine initially elicits a weak interferon (IFN)-gamma response. To improve the immune response, an adjuvant consisting of plasmid encoding either porcine interleukin (IL)-12 or IFN-alpha was co-administered during vaccination. In the presence of either adjuvant, at least a three-fold increase in the primary virus-specific IFN-gamma response was observed. While this enhancement was only transient (1 week) when the IL-12 expressing plasmid was used, the effect was not only still apparent at 6 weeks after vaccination in the presence of the IFN-alpha expressing plasmid but even after challenge with a virulent genetically divergent PRRSV. In contrast, no effect of either adjuvant on the production of anti-virus antibodies was noticed throughout the study. Despite the apparent augmentation of a T helper (Th) 1 type response by the inclusion of IFN-alpha or IL-12 during vaccination, this modulation did not necessarily correlate with a reduction in viremia. Since a similar increase in the degree of the IFN-gamma response to the PRRSV vaccine could be achieved by substituting polyinosinic-polycytidylic acid in lieu of either cytokine, exposure to PRRSV in the presence of a variety of Th 1 polarizing molecules can positively influence the development of the cell-mediated immune response of swine to this pathogen. Conceivably, such intervention could be applied to improve the formulation of anti-PRRSV vaccines.

Gradual development of the interferon-gamma response of swine to porcine reproductive and respiratory syndrome virus infection or vaccination.

Infection of swine with virulent porcine reproductive and respiratory syndrome (PRRS) virus induced a rapid, robust antibody response that comprised predominantly nonneutralizing antibodies and waned after approximately 3 months. In contrast, the initial onset of virus-specific interferon (IFN)-gamma-secreting cells (SC) in the pig lymphocyte population remained at a fairly low level during this period and then increased gradually in frequency, plateauing at 6 months postinfection. A similar polarization of the host humoral and cellular immune responses was also observed in pigs immunized with a PRRS-modified live virus (MLV) vaccine. Even coadministration of an adjuvant that enhanced the immune response to a pseudorabies (PR) MLV vaccine failed to alter the induction of PRRS virus-specific IFN-gamma SC (comprising predominantly CD4/CD8 alpha double positive memory T cells with a minority being typical CD4(-)/CD8 alpha beta(+) T cells) and the generation of neutralizing antibodies. Moreover, unlike inactivated PR virus, nonviable PRRS virus did not elicit virus-neutralizing antibody production. Presumably, an intrinsic property of this pathogen delays the development of the host IFN-gamma response and preferentially stimulates the synthesis of antibodies incapable of neutralization.

Age-associated loss of bone marrow hematopoietic cells is reversed by GH and accompanies thymic reconstitution.

Deterioration of the thymus gland during aging is accompanied by a reduction in plasma GH. Here we report gross and microscopic results from 24-month-old Wistar-Furth rats treated with rat GH derived from syngeneic GH3 cells or with recombinant human GH. Histological evaluation of aged rats treated with either rat or human GH displayed clear morphologic evidence of thymic regeneration, reconstitution of hematopoietic cells in the bone marrow, and multiorgan extramedullary hematopoiesis. Quantitative evaluation of formalin-fixed, hematoxylin and eosin-stained sections of bone marrow from aged rats revealed at least a 50% reduction in the number hematopoietic bone marrow cells, compared with that of young 3-month-old rats. This age-associated decline in bone marrow leukocytes, as well as the increase in bone marrow adipocytes, was significantly reversed by in vivo treatment with GH. Restoration of bone marrow cellularity was caused primarily by erythrocytic and granulocytic cells, but all cell lineages were represented and their proportions were similar to those in aged control rats. On a per-cell basis, GH treatment in vivo significantly increased the number of in vitro myeloid colony forming units in both bone marrow and spleen. Morphological evidence of enhanced extramedullary hematopoiesis was observed in the spleen, liver, and adrenal glands from animals treated with GH. These results confirm that GH prevents thymic aging. Furthermore, these data significantly extend earlier findings by establishing that GH dramatically promotes reconstitution of another primary hematopoietic tissue by reversing the accumulation of bone marrow adipocytes and by restoring the number of bone marrow myeloid cells of both the erythrocytic and granulocytic lineages.