Points to consider regarding the use and implementation of virtual controls in nonclinical general toxicology studies.

The replacement of a proportion of concurrent controls by virtual controls in nonclinical safety studies has gained traction over the last few years. This is supported by foundational work, encouraged by regulators, and aligned with societal expectations regarding the use of animals in research. This paper provides an overview of the points to consider for any institution on the verge of implementing this concept, with emphasis given on database creation, risks, and discipline-specific perspectives.

Practical Considerations in Determining Adversity and the No-Observed-Adverse-Effect-Level (NOAEL) in Nonclinical Safety Studies: Challenges, Perspectives and Case Studies.

Determining the adverse nature of findings from nonclinical safety studies often poses a challenge for the key stakeholders responsible for interpreting the results of definitive toxicity studies in support of pharmaceutical product development. Although there are instances in which responses to treatment clearly indicate intolerability or tissue injury associated with dysfunction; in practice, more often there is uncertainty in characterizing an effect of drug treatment as adverse or not. This is due to the inherent variability in responses of biological test systems to toxicological insults, leaving the ultimate analyses of adversity to individual interpretation and subjectivity. This article is a follow-up to the workshop entitled, "Adverse or Not Adverse?: Thinking process behind adversity determination during nonclinical drug development," conducted at the 58th Annual Meeting of the Society of Toxicology, March 2019 in Baltimore, MD. In this paper, we further discuss and incorporate the perspectives of authors representing different roles, such as Study Director, Study Pathologist, Pharmacology/Toxicology Reviewer (U.S. Food and Drug Administration), and Sponsor in the determination and use of adversity. We also present a practical stepwise approach as an aid in this assessment, and further apply these principles to discuss 10 case studies with different therapeutic modalities and unique challenges.

Old drugs, new uses: Drug repurposing in hematological malignancies.

Discovery and development of novel anti-cancer drugs are expensive and time consuming. Systems biology approaches have revealed that a drug being developed for a non-cancer indication can hit other targets as well, which play critical roles in cancer progression. Since drugs for non-cancer indications would have already gone through the preclinical and partial or full clinical development, repurposing such drugs for hematological malignancies would cost much less, and drastically reduce the development time, which is evident in case of thalidomide. Here, we have reviewed some of the drugs for their potential to repurpose for treating the hematological malignancies. We have also enlisted resources that can be helpful in drug repurposing.

The apoptotic mechanism of action of the sphingosine kinase 1 selective inhibitor SKI-178 in human acute myeloid leukemia cell lines.

We previously developed SKI-178 (N'-[(1E)-1-(3,4-dimethoxyphenyl)ethylidene]-3-(4-methoxxyphenyl)-1H-pyrazole-5-carbohydrazide) as a novel sphingosine kinase-1 (SphK1) selective inhibitor and, herein, sought to determine the mechanism-of-action of SKI-178-induced cell death. Using human acute myeloid leukemia (AML) cell lines as a model, we present evidence that SKI-178 induces prolonged mitosis followed by apoptotic cell death through the intrinsic apoptotic cascade. Further examination of the mechanism of action of SKI-178 implicated c-Jun NH2-terminal kinase (JNK) and cyclin-dependent protein kinase 1 (CDK1) as critical factors required for SKI-178-induced apoptosis. In cell cycle synchronized human AML cell lines, we demonstrate that entry into mitosis is required for apoptotic induction by SKI-178 and that CDK1, not JNK, is required for SKI-178-induced apoptosis. We further demonstrate that the sustained activation of CDK1 during prolonged mitosis, mediated by SKI-178, leads to the simultaneous phosphorylation of the prosurvival Bcl-2 family members, Bcl-2 and Bcl-xl, as well as the phosphorylation and subsequent degradation of Mcl-1. Moreover, multidrug resistance mediated by multidrug-resistant protein1 and/or prosurvival Bcl-2 family member overexpression did not affect the sensitivity of AML cells to SKI-178. Taken together, these findings highlight the therapeutic potential of SKI-178 targeting SphK1 as a novel therapeutic agent for the treatment of AML, including multidrug-resistant/recurrent AML subtypes.

Gambogic acid inhibits multiple myeloma mediated osteoclastogenesis through suppression of chemokine receptor CXCR4 signaling pathways.

Bone disease, characterized by the presence of lytic lesions and osteoporosis is the hallmark of multiple myeloma (MM). Stromal cell-derived factor 1α (SDF-1α) and its receptor, CXC chemokine receptor 4 (CXCR4), has been implicated as a regulator of bone resorption, suggesting that agents that can suppress SDF1α/CXCR4 signaling might inhibit osteoclastogenesis, a process closely linked to bone resorption. We, therefore, investigated whether gambogic acid (GA), a xanthone, could inhibit CXCR4 signaling and suppress osteoclastogenesis induced by MM cells. Through docking studies we predicted that GA directly interacts with CXCR4. This xanthone down-regulates the expression of CXCR4 on MM cells in a dose- and time-dependent manner. The down-regulation of CXCR4 was not due to proteolytic degradation, but rather GA suppresses CXCR4 mRNA expression by inhibiting nuclear factor-kappa B (NF-κB) DNA binding. This was further confirmed by quantitative chromatin immunoprecipitation assay, as GA inhibits p65 binding at the CXCR4 promoter. GA suppressed SDF-1α-induced chemotaxis of MM cells and downstream signaling of CXCR4 by inhibiting phosphorylation of Akt, p38, and Erk1/2 in MM cells. GA abrogated the RANKL-induced differentiation of macrophages to osteoclasts in a dose- and time-dependent manner. In addition, we found that MM cells induced differentiation of macrophages to osteoclasts, and that GA suppressed this process. Importantly, suppression of osteoclastogenesis by GA was mediated through IL-6 inhibition. Overall, our results show that GA is a novel inhibitor of CXCR4 expression and has a strong potential to suppress osteoclastogenesis mediated by MM cells.

Effect of repeated freezing and thawing on 18 clinical chemistry analytes in rat serum.

In a preclinical research laboratory, using serum samples that have been frozen and thawed repeatedly is sometimes unavoidable when needing to confirm previous results or perform additional analysis. Here we determined the effects of multiple cycles of refrigeration or freezing and thawing of rat serum at 3 temperature conditions for different storage times on clinical chemistry analytes. Serum samples obtained from adult Wistar rats were stored at 2 to 8 °C and -10 to -20 °C for as long as 72 h and at -70 °C for as long as 30 d. At different time points (24, 48, and 72 h for samples stored at 2 to 8 °C or -10 to -20 °C and 1, 7, and 30 d for samples stored at -70 °C), the samples were brought to room temperature, analyzed, and then stored again at the designated temperature. The results obtained after each storage cycle were compared with those obtained from the initial analysis of fresh samples. Of the 18 serum analytes evaluated, 14 were stable without significant changes, even after 3 freeze-thaw cycles at the tested temperature ranges. Results from this study will help researchers working with rat serum to interpret the biochemical data obtained from serum samples that have been frozen and thawed repeatedly.

Neuroglial expression of the MHCI pathway and PirB receptor is upregulated in the hippocampus with advanced aging.

The hippocampus undergoes changes with aging that impact neuronal function, such as synapse loss and altered neurotransmitter release. Nearly half of the aged population also develops deficits in spatial learning and memory. To identify age-related hippocampal changes that may contribute to cognitive decline, transcriptomic analysis of synaptosome preparations from adult (12 months) and aged (28 months) Fischer 344-Brown Norway rats assessed for spatial learning and memory was performed. Bioinformatic analysis identified the MHCI pathway as significantly upregulated with aging. Age-related increases in mRNAs encoding the MHCI genes RT1-A1, RT1-A2, and RT1-A3 were confirmed by qPCR in synaptosomes and in CA1 and CA3 dissections. Elevated levels of the MHCI cofactor (B2m), antigen-loading components (Tap1, Tap2, Tapbp), and two known MHCI receptors (PirB, Klra2) were also confirmed. Protein expression of MHCI was elevated with aging in synaptosomes, CA1, and DG, while PirB protein expression was induced in both CA1 and DG. MHCI expression was localized to microglia and neuronal excitatory postsynaptic densities, and PirB was localized to neuronal somata, axons, and dendrites. Induction of the MHCI antigen processing and presentation pathway in hippocampal neurons and glia may contribute to age-related hippocampal dysfunction by increasing neuroimmune signaling or altering synaptic homeostasis.

Effect of fasting duration on clinical pathology results in Wistar rats.

BACKGROUND: Fasting is an important preanalytical factor that may affect the interpretation of hematology and clinical biochemistry data in toxicology or pharmacology studies. Limited information is available on how the results may be affected by different durations of fasting. OBJECTIVE: The purpose of this study was to assess the influence of fasting duration on clinical pathology results in male and female rats and to determine an optimum fasting time for preclinical studies. METHODS: Male and female Wistar rats (10 each per group) were fasted for 0, 4, 8, 16, 24, and 48 hours. Changes in body weight and in the results of routine CBC and clinical chemistry analysis were evaluated by 1-way ANOVA. RESULTS: Body weight was significantly decreased by 4 hours of fasting in all rats, and hemoglobin concentration was significantly increased at 16 hours in male rats. Serum glucose and triglyceride concentrations in both sexes and cholesterol and high-density lipoprotein-C concentrations in female rats were also significantly decreased beginning at 16 hours. The creatinine concentration was increased in females after 16 hours of fasting. Serum alkaline phosphatase and alanine aminotransferase activities were significantly decreased after 8 hours in males and 16 hours in females. CONCLUSIONS: Fasting-induced changes in clinical pathology results were consistent with hemoconcentration and altered nutrition and metabolic function. Most changes occurred at 16 hours, with minimal subsequent changes. Hence, a 16-hour fasting duration may be recommended for preclinical studies involving clinical pathology measurements.