Developmental toxicity associated with receptor tyrosine kinase Ret inhibition in reproductive toxicity testing.

BACKGROUND: Urogenital abnormalities are among the most common of all human birth defects. In developmental toxicity studies with the Syk kinase inhibitor R788, a spectrum of findings, including renal agenesis, were observed. R788 has also been found to inhibit the receptor tyrosine kinase Ret. Ret kinase is known to be an essential component in the signaling pathway required for renal organogenesis and ureteric duct formation. Previously known is that mutant mice without the c-ret gene, develop urogenital malformations including renal agenesis. METHODS: In GLP developmental toxicity studies, gravid rabbits were treated orally with R788 at doses of 0, 10, 22, and 50 mg/kg/day (gestation days 7-19) and gravid rats received 0, 5, 12.5, and 25 mg/kg/day (gestation days 6-17) by the same route. The activity of R406 against Ret kinase was assessed in biochemical and cell-based assays. RESULTS: A dose-dependent increase in malformations, including renal and ureteric agenesis and a specific major vessel anomaly, retroesophageal right subclavian artery, was observed in both the rat and rabbit. R788 proved to be a potent inhibitor of Ret kinase. CONCLUSIONS: R788 promoted a spectrum of developmental toxicity, including renal and ureteric agenesis and a specific major vessel abnormality, retroesophageal right subclavian artery, in two different species. These effects are likely the result of inhibition of Ret kinase given its importance in the normal ontogeny of the urogenital and cardiovascular systems across species.

Immunotoxicity assessment for the novel Spleen tyrosine kinase inhibitor R406.

Spleen tyrosine kinase (Syk) is a novel pharmaceutical target for treatment of allergic, autoimmune, and neoplastic disorders. Previous studies have indicated that Syk signaling plays critical roles in regulating the lymphohematopoietic system. These observations prompted us to investigate whether inhibition of Syk would promote immunotoxicity. In a series of studies, rats were treated orally with R406, at dose levels up to and including 100 mg/kg/day (or its prodrug R788 at dose levels up to and including 100 mg/kg/day, reduced to 50 mg/kg/day for females as MTD was exceeded), a potent Syk inhibitor, twice daily for 28 days. In addition to standard toxicological assessments, immunophenotyping by flow cytometric analysis, and a study of humoral immune response measuring anti-KLH IgM and IgG levels, were undertaken. Other immunotoxicity studies included three host resistance models in female Balb/c mice to further ascertain effects of R406 on innate and acquired immunity. Following R406 treatment, expected immunomodulating effects (e.g., decreased thymic and spleen weight, hypocellularity of bone marrow, and reduced lymphocyte counts, including T and B cells) were observed in the rat studies. These changes essentially resolved during a 14-day treatment-free recovery period. A KLH challenge in rats demonstrated no adverse effects on IgG or IgM response. R788/406, administered orally at dose levels up to and including 80 mg/kg/day for 28 days, did not affect bacterial or viral clearance in the Listeria, Streptococcal, or Influenza host resistance mouse models, respectively. This correlated with previous in vitro macrophage and neutrophil function assays (assessing migration, phagocytosis, oxidative burst and microbicidal activity), which revealed that R406 did not adversely affect macrophage or neutrophil function in innate immune responses. Collectively, these results demonstrate that R406 has minimal functional immunotoxicity notwithstanding its lymphocytopenic effect, suggesting that inhibition of Syk might not lead to unacceptable mechanism-based adverse effects.

Safety Evaluation of Glucose Isomerase Derived from Flavobacterium arborescens and Used in Production of High Fructose Corn Syrup.

Flavobacterium arborescens is a common rod-shaped, gram-negative bacterium which, when cultivated in a nutrient medium, is an efficient source of glucose isomerase (GI). GI is then used in the production of high fructose corn syrup. Studies were conducted to assure product safety and establish GRAS status for GI derived from F. arborescens . A viable cell suspension of F. arborescens and the cell-free medium in which the organism was cultured were administered i.v. to rats and rabbits. For feeding studies, the cells were immobilized using polycationic polymers and a crosslinking agent (i.e., chitosan, polyethylenimine and glutaraldehyde). GI, in the whole cell immobilized form, was offered at concentrations of 0, 1.5, 3.0 or 5.0% (wt/wt) of the diet to dogs for a minimum of 90 consecutive days and to rats over three generations. Animals were observed daily for signs of toxicosis; body weight and food consumption were monitored; biochemical tests, hematologic determinations, and urinalyses were done on blood and urine samples; and thorough gross and microscopic tissue examinations were performed at terminations. There were no signs of infection or toxicosis following i.v. administration of F. arborescens or the cell-free supernatant fluid. This, and the lack of toxicity in dogs and rats which received daily dietary concentrations of GI many times above the projected highest possible human exposure level, suggest that there should be virtually no risk of toxicity associated with the consumption of food and beverages containing high fructose syrup produced by GI derived from F. arborescens .