The Spontaneous Incidence of Neurological Clinical Signs in Preclinical Species Using Cage-side Observations or High-definition Video Monitoring: A Retrospective Analysis.

When conducting toxicology studies, the interpretation of drug-related neurological clinical signs such as convulsions, myoclonus/myoclonic jerks, tremors, ataxia, and salivation requires an understanding of the spontaneous incidence of those observations in commonly used laboratory animal species. The spontaneous incidence of central nervous system clinical signs in control animals from a single facility using cage-side observations or high definition video monitoring was retrospectively analyzed. Spontaneous convulsions were observed at low incidence in Beagle dogs and Sprague-Dawley rats but were not identified in cynomolgus monkeys and Göttingen minipigs. Spontaneous myoclonic jerks and muscle twitches were observed at low incidence in Beagle dogs, cynomolgus monkeys, and Sprague-Dawley rats but were not seen in Göttingen minipigs. Spontaneous ataxia/incoordination was identified in all species and generally with a higher incidence when using video monitoring. Salivation and tremors were the two most frequent spontaneous clinical signs and both were observed in all species. Data from the current study unveil potential limitations when using control data obtained from a single study for toxicology interpretation related to low incidence neurological clinical signs while providing historical control data from Beagle dogs, cynomolgus monkeys, Sprague-Dawley rats, and Göttingen minipigs.

The impact of anaphylaxis on the absorption of intranasal epinephrine in anaesthetized non-naive beagle dogs.

Background: Epinephrine delivery via an intranasal spray (neffy) is being evaluated as an additional option to treat severe allergic reaction and may provide clinical benefit by reducing the time to dosing in community settings by avoiding needles. Given that hypotension is a hallmark symptom of severe allergic reactions, a preclinical study was conducted to evaluate the impact of this factor on epinephrine absorption via neffy. Objective: The objective of this study was to evaluate the absorption of epinephrine via neffy in a dog model of anaphylaxis with severe hypotension. Methods: Epinephrine absorption via neffy was evaluated in anesthetized beagle dogs under both normal conditions and hypotension associated with anaphylaxis. A total of 14 dogs (10 males and 4 females) were dosed with neffy, 1.0 mg, under normal conditions, followed by neffy, 1.0 mg, under conditions of anaphylaxis. Results: The mean maximum concentration of epinephrine was higher during anaphylaxis than under normal conditions (2,670 ± 2,150 pg/mL and 1,330 ± 739 pg/mL [P < .05]). Relative to normal conditions, anaphylaxis resulted in higher overall epinephrine exposure (area under the curve from 0 to 45 minutes = 54,400 ± 18,100 min × pg/mL and 34,300 ± 21,500 minutes × pg/mL [P < .05]), which is likely due to the increase in vascular permeability commonly observed during severe allergic reactions. Conclusion: Taken together with real-world evidence from nasal naloxone treatment for opioid overdose demonstrating that the reduced blood flow or hypotension associated with overdose does not appear to suppress naloxone's efficacy, the current findings demonstrate that epinephrine is well absorbed following neffy delivery during the hypotension associated with severe anaphylaxis reactions.

An Overview of Gene Editing Modalities and Related Non-clinical Testing Considerations.

Gene therapy has become an important modality for a wide range of therapeutic indications with a rapid increase in the number of therapeutic candidates being developed in this field. Understanding the molecular biology underlying the gene therapy is often critical to develop appropriate safety assessment strategies. We aimed to discuss some of the commonly used gene therapy modalities and common preclinical toxicology testing considerations when developing gene therapies. Non-viral gene delivery methods such as electroporation, microinjection, peptide nanoparticles and lipid nanoparticles are deployed as innovative molecular molecular construct which are included in the design of novel gene therapies and the associated molecular biology mechanisms have become relevant knowledge to non-clinical toxicology. Viral gene delivery methodologies including Adenovirus vectors, Adeno-Associated virus vectors and Lentivirus gene therapy vectors have also advanced considerably across numerous therapeutic areas, raising unique non-clinical toxicology and immunological considerations. General toxicology, biodistribution and tumorigenicity are the pillars of non-clinical safety testing in gene therapies. Evaluating the tumorigenicity potential of a gene editing therapy often leverages molecular pathology while some translational challenges remain. Toxicology study design is entering a new era where science-driven customized approaches and program specific considerations have become the norm.

Polysorbate 80-Induced Anaphylactoid Reaction and the Effects on Cardiovascular Function: Dose Threshold and Species Comparison.

Polysorbate 80 (PS80) is commonly used in pre-clinical formulations. The dose threshold for cardiovascular (CV) changes and hypersensitivity reaction in the dog was assessed and compared to other species. PS80 was administered by intravenous (IV) bolus (.5, 1 mg/kg), IV infusion (.3, .5, 1, 3 mg/kg), subcutaneous (SC) injection (5, 10, 15 mg/kg) and oral gavage (10 mg/kg) to dogs with CV monitoring. Monkeys and minipigs received PS80 by IV infusion at 3 mg/kg. Plasma histamine concentration was measured following PS80 IV infusion and with diphenhydramine pre-treatment in dogs only. In dogs, PS80 was not associated with CV changes at doses up to 15 mg/kg SC and 10 mg/kg oral, but decreased blood pressure and increased heart rate with IV bolus at ≥ .5 mg/kg and IV infusion at ≥ 1.0 mg/kg and decreased body temperature with IV infusion at 3 mg/kg was observed. Transient edema and erythema were noted with all administration routes, in all three species including doses that were devoid of CV effects. In monkeys and minipigs, PS80 did not induce CV, cutaneous or histamine concentration changes. These results suggest that mild, transient skin changes occur following PS80 administration at doses that are not associated with CV effects in the dogs. In dogs, the cardiovascular effect threshold was <.5 mg/kg for IV bolus, .3 mg/kg for IV infusion, 15 mg/kg for SC injection, and 10 mg/kg for oral administration. Monkey and minipig were refractory to PS80-induced histamine release at 3 mg/kg by IV infusion over 15 minutes.

Human Immunodeficiency Virus Type 1 Vpr Mediates Degradation of APC1, a Scaffolding Component of the Anaphase-Promoting Complex/Cyclosome.

HIV-1 encodes several accessory proteins-Nef, Vif, Vpr, and Vpu-whose functions are to modulate the cellular environment to favor immune evasion and viral replication. While Vpr was shown to mediate a G2/M cell cycle arrest and provide a replicative advantage during infection of myeloid cells, the mechanisms underlying these functions remain unclear. In this study, we defined HIV-1 Vpr proximity interaction network using the BioID proximity labeling approach and identified 352 potential Vpr partners/targets, including several complexes, such as the cell cycle-regulatory anaphase-promoting complex/cyclosome (APC/C). Herein, we demonstrate that both the wild type and cell cycle-defective mutants of Vpr induce the degradation of APC1, an essential APC/C scaffolding protein, and show that this activity relies on the recruitment of DCAF1 by Vpr and the presence of a functional proteasome. Vpr forms a complex with APC1, and the APC/C coactivators Cdh1 and Cdc20 are associated with these complexes. Interestingly, we found that Vpr encoded by the prototypic HIV-1 NL4.3 does not interact efficiently with APC1 and is unable to mediate its degradation as a result of a N28S-G41N amino acid substitution. In contrast, we show that APC1 degradation is a conserved feature of several primary Vpr variants from transmitted/founder virus. Functionally, Vpr-mediated APC1 degradation did not impact the ability of the protein to induce a G2 cell cycle arrest during infection of CD4+ T cells or enhance HIV-1 replication in macrophages, suggesting that this conserved activity may be important for other aspects of HIV-1 pathogenesis. IMPORTANCE The function of the Vpr accessory protein during HIV-1 infection remains poorly defined. Several cellular targets of Vpr were previously identified, but their individual degradation does not fully explain the ability of Vpr to impair the cell cycle or promote HIV-1 replication in macrophages. Here, we used the unbiased proximity labeling approach, called BioID, to further define the Vpr proximity interaction network and identified several potentially new Vpr partners/targets. We validated our approach by focusing on a cell cycle master regulator, the APC/C complex, and demonstrated that Vpr mediated the degradation of a critical scaffolding component of APC/C called APC1. Furthermore, we showed that targeting of APC/C by Vpr did not impact the known activity of Vpr. Since degradation of APC1 is a conserved feature of several primary variants of Vpr, it is likely that the interplay between Vpr and APC/C governs other aspects of HIV-1 pathogenesis.

The Biology of Vasopressin.

Vasopressins are evolutionarily conserved peptide hormones. Mammalian vasopressin functions systemically as an antidiuretic and regulator of blood and cardiac flow essential for adapting to terrestrial environments. Moreover, vasopressin acts centrally as a neurohormone involved in social and parental behavior and stress response. Vasopressin synthesis in several cell types, storage in intracellular vesicles, and release in response to physiological stimuli are highly regulated and mediated by three distinct G protein coupled receptors. Other receptors may bind or cross-bind vasopressin. Vasopressin is regulated spatially and temporally through transcriptional and post-transcriptional mechanisms, sex, tissue, and cell-specific receptor expression. Anomalies of vasopressin signaling have been observed in polycystic kidney disease, chronic heart failure, and neuropsychiatric conditions. Growing knowledge of the central biological roles of vasopressin has enabled pharmacological advances to treat these conditions by targeting defective systemic or central pathways utilizing specific agonists and antagonists.

Hof1 plays a checkpoint-related role in MMS-induced DNA damage response in Candida albicans.

Cells depend on robust DNA damage recognition and repair systems to maintain genomic integrity for survival in a mutagenic environment. In the pathogenic yeast Candida albicans, a subset of genes involved in the response to DNA damage-induced genome instability and morphological changes has been found to regulate virulence. To better understand the virulence-linked DNA repair network, we screened for methyl methane sulfonate (MMS) sensitivity within the GRACE conditional expression collection and identified 56 hits. One of these potential DNA damage repair-associated genes, a HOF1 conditional mutant, unexpectedly had a previously characterized function in cytokinesis. Deletion of HOF1 resulted in MMS sensitivity and genome instability, suggesting Hof1 acts in the DNA damage response. By probing genetic interactions with distinct DNA repair pathways, we found that Hof1 is genetically linked to the Rad53 pathway. Furthermore, Hof1 is down-regulated in a Rad53-dependent manner and its importance in the MMS response is reduced when Rad53 is overexpressed or when RAD4 or RAD23 is deleted. Together, this work expands our understanding of the C. albicans DNA repair network and uncovers interplay between the cytokinesis regulator Hof1 and the Rad53-mediated checkpoint.

Characterization of a novel separase-interacting protein and candidate new securin, Eip1p, in the fungal pathogen Candida albicans.

Proper chromosome segregation is crucial for maintaining genomic stability and dependent on separase, a conserved and essential cohesin protease. Securins are key regulators of separases, but remain elusive in many organisms due to sequence divergence. Here, we demonstrate that the separase homologue Esp1p in the ascomycete Candida albicans, an important pathogen of humans, is essential for chromosome segregation. However, C. albicans lacks a sequence homologue of securins found in model ascomycetes. We sought a functional homologue through identifying Esp1p interacting factors. Affinity purification of Esp1p and mass spectrometry revealed Esp1p-Interacting Protein1 (Eip1p)/Orf19.955p, an uncharacterized protein specific to Candida species. Functional analyses demonstrated that Eip1p is important for chromosome segregation but not essential, and modulated in an APCCdc20-dependent manner, similar to securins. Eip1p is strongly enriched in response to methyl methanesulfate (MMS) or hydroxyurea (HU) treatment, and its depletion partially suppresses an MMS or HU-induced metaphase block. Further, Eip1p depletion reduces Mcd1p/Scc1p, a cohesin subunit and separase target. Thus, Eip1p may function as a securin. However, other defects in Eip1p-depleted cells suggest additional roles. Overall, the results introduce a candidate new securin, provide an approach for identifying these divergent proteins, reveal a putative anti-fungal therapeutic target, and highlight variations in mitotic regulation in eukaryotes.