ABSTRACT
Animals frequently eat less after a test-article treatment in nonclinical toxicological studies, and it can be difficult to distinguish test article-derived toxicities from secondary changes related to this reduced food intake. Therefore, in this study, we restricted the food intake of cynomolgus monkeys (Cambodian, male, n=2 or 3, 48 ± 3 months old) to 25% of the control for two weeks and evaluated the effects on toxicological parameters (general conditions, body weight, electrocardiography, urinalysis, hematology, blood chemistry, bone marrow analysis, pathological examination). After 2 weeks, the monkeys exhibited decreases in bone marrow erythropoiesis (e.g., decreases in reticulocytes and bone marrow erythrocytes), as well as glycogenesis induction (e.g., increase in aspartate aminotransferase (AST)) and malnutrition (e.g., decrease in triglyceride and systemic adipocytes atrophy). Additionally, histopathological analysis revealed granuloma and inflammatory cell infiltration in coronary fat, which had never been found in previous food restriction studies using other animal species. These findings will enable researchers to more accurately evaluate the toxicological risks of test articles that simultaneously induce food intake reduction.
Subject(s)
Eating , Food , Male , Animals , Macaca fascicularis , Body Weight , ElectrocardiographyABSTRACT
CD3 bispecific constructs show promising therapeutic potential as anti-tumor antibodies, but it has concurrently been difficult to manage cytokine release syndrome (CRS) in clinical use. Currently, the most effective measure for reducing CRS is considered a combination of intra-patient/animal dose escalation and corticosteroid premedication. To examine how effectively an intra-animal ascending dose regimen without premedication would mitigate CRS, we compared plasma cytokine levels in two groups of cynomolgus monkeys; one group was given a single dose, and the other a three-fold daily ascending dose of a CD3 bispecific construct that targets and cross-reacts with both glypican 3 and CD3 (ERY22). Ascending doses up to 1000⯵g/kg of ERY22 dramatically reduced the peak cytokine levels of IL-6, TNF-α, and IFN-γ, IL-2 as well the clinical severity of CRS compared with a single dose of 1000⯵g/kg. Peak cytokine levels following the single and ascending doses were 60,095â¯pg/mL and 1221â¯pg/mL for IL-6; 353â¯pg/mL and 14â¯pg/mL for TNF-α; 123â¯pg/mL and 16â¯pg/mL for IFN-γ; and 2219â¯pg/mL and 42â¯pg/mL for IL-2. The tolerance acquired with daily ascending doses up to 1000⯵g/kg remained in effect for the following weekly doses of 1000⯵g/kg.
Subject(s)
Antibodies, Bispecific/therapeutic use , Antineoplastic Agents, Immunological/therapeutic use , Cytokine Release Syndrome/drug therapy , Immunotherapy/methods , Neoplasms/therapy , T-Lymphocytes/immunology , Animals , Antibodies, Bispecific/administration & dosage , Antibodies, Bispecific/immunology , Antineoplastic Agents, Immunological/administration & dosage , Antineoplastic Agents, Immunological/immunology , CD3 Complex/immunology , Cytokine Release Syndrome/blood , Cytokine Release Syndrome/immunology , Drug Administration Schedule , Interferon-gamma/blood , Interleukin-2/blood , Interleukin-6/blood , Macaca fascicularis , Male , Neoplasms/immunology , Tumor Necrosis Factor-alpha/bloodABSTRACT
γH2AX, the phosphorylated form of a histone variant H2AX at Ser 139, is already widely used as a biomarker to research the fundamental biology of DNA damage and repair and to assess the risk of environmental chemicals, pollutants, radiation, and so on. It is also beginning to be used in the early non-clinical stage of pharmaceutical drug development as an in vitro tool for screening and for mechanistic studies on genotoxicity. Here, we review the available information on γH2AX-based test systems that can be used to develop drugs and present our own experience of practically applying these systems during the non-clinical phase of drug development. Furthermore, the potential application of γH2AX as a tool for in vivo non-clinical safety studies is also discussed.
ABSTRACT
When characterizing the genotoxicity of chemicals that induce micronuclei, it is practical to be able to classify the chemicals as aneugens or clastogens. This classification gives information on the mechanistic properties of chemicals and is indispensable for setting the threshold safety margins for genotoxicity in pharmaceutical development. A widely used method for detecting aneugens is fluorescence in situ hybridization (FISH) but, even though the rat is an experimental animal generally used in preclinical studies in drug development, DNA probes that hybridize to all the centromeres of rat chromosomes have not yet been established. In the present study, in addition to the previously known satellite I sequence, we identified two novel satellite sequences, satellite II and satellite III, from the rat genome database. DNA probes with a mixture of these satellite DNA sequences were used to establish a FISH method for pan-centromeric staining of rat chromosomes. To confirm the feasibility of the method, vinblastine (VBS) and mitomycin C (MMC) were administered to rats as a typical aneugen and clastogen, respectively. Micronucleated polychromatic erythrocytes (MNPCE) from bone marrow were enriched by sorting in flow cytometry and subjected to the FISH method. As a result, the ratio of centromere-positive MNPCE increased in VBS-treated rats but not in MMC-treated ones. Since the FISH method using the novel DNA probes clearly discriminates the aneugens from the clastogens, we suggest this method as a useful tool for providing mechanistic information for micronucleus induction in vivo.