Effects of LRRK2 Inhibitors in Nonhuman Primates.

Toxicology studies in nonhuman primates were conducted to evaluate selective, brain penetrant inhibitors of LRRK2. GNE 7915 was limited to 7-day administration in cynomolgus monkeys at 65 mg/kg/day or limited to 14 days in rhesus at 22.5 mg/kg b.i.d. due to physical signs. Compound 25 demonstrated acceptable tolerability at 50 and 225 mg/kg b.i.d. for 7 days in rhesus monkeys. MK-1468 was tolerated during 7-day administration at 100, 200 or 800 mg/kg/day or for 30-day administration at 30, 100, or 500 mg/kg b.i.d. in rhesus monkeys. The lungs revealed hypertrophy of type 2 pneumocytes, with accumulation of intra-alveolar macrophages. Transmission electron microscopy confirmed increased lamellar structures within hypertrophic type 2 pneumocytes. Hypertrophy and hyperplasia of type 2 pneumocytes with accumulation of intra-alveolar macrophages admixed with neutrophils were prominent at peripheral lungs of animals receiving compound 25 or MK-1468. Affected type 2 pneumocytes were immuno-positive for pro-surfactant C, but negative for CD11c, a marker for intra-alveolar macrophages. Accumulation of collagen within alveolar walls, confirmed by histochemical trichrome stain, accompanied changes described for compound 25 and MK-1468. Following a 12-week treatment-free interval, animals previously receiving MK-1468 for 30 days exhibited remodeling of alveolar structure and interstitial components that did not demonstrate reversibility.

The effect of methylmercury exposure on behavior and cerebellar granule cell physiology in aged mice.

Epidemiology studies have clearly documented that the central nervous system is highly susceptible to methylmercury toxicity, and exposure to this neurotoxicant in humans primarily results from consumption of contaminated fish. While the effects of methylmercury exposure have been studied in great detail, comparatively little is known about the effects of moderate to low dose methylmercury toxicity in the aging central nervous system. We examined the toxic effects of a moderate dose of methylmercury on the aging mouse cerebellum. Male and female C57BL/6 mice at 16-20 months of age were exposed to methylmercury by feeding a total dose of 5.0 mg kg(-1) body weight and assessed using four behavioral tests. Methylmercury-treated aged mice performed significantly worse in open field, footprint analysis and the vertical pole test compared with age-matched control mice. Isolated cerebellar granule cells from methylmercury-treated aged mice exhibited higher levels of reactive oxygen species and reduced mitochondrial membrane potentials, but no differences in basal intracellular calcium ion levels compared with age-matched control mice. When aged mice were exposed to a moderate dose of methylmercury, they exhibited a similar degree of impairment when compared with young adult mice exposed to the same moderate dose of methylmercury, as reported in earlier studies from this laboratory. Thus, at least in mice, exposure of the aged brain to moderate concentrations methylmercury does not pose greater risk compared with the young adult brain exposed to similar concentrations of methylmercury.

Early toxicology signal generation in the mouse.

The rat has been the preferred rodent toxicology species since before regulatory requirements have been in place, and there exists in the pharmaceutical industry and the regulatory agencies a significant amount of historical data for the rat. The resulting experience base with the rat makes the possibility of replacing it with the mouse for regulated toxicology studies untenable for all but the most extreme circumstances. However, toxicologists are very familiar with the mouse as a model for chronic carcinogenicity studies, and there exist multiple preclinical mouse models of disease. The authors evaluated the use of the mouse for early in vivo toxicology signal generation and prioritization of small molecule lead compounds prior to nomination of a development candidate. In five-day oral gavage studies with three test agents in the mouse, the authors were able to identify the same dose-limiting toxicities as those identified in the rat, including examples of compound-mediated hemolysis as well as microscopic lesions in the alimentary canal, kidney, and pancreas. Performing early signal generation studies in the mouse allows for earlier assessment of the safety liabilities of small molecules, requires significantly less compound, and allows evaluation of more compounds earlier in the project's life cycle.

Changes in biochemical processes in cerebellar granule cells of mice exposed to methylmercury.

At postnatal day 34, male and female C57BL/6J mice were exposed orally once a day to a total of five doses totaling 1.0 or 5.0 mg/kg of methylmercuric chloride or sterile deionized water in moistened rodent chow. Eleven days after the last dose cerebellar granule cells were acutely isolated to measure reactive oxygen species (ROS) levels and mitochondrial membrane potential using CM-H(2)DCFDA and TMRM dyes, respectively. For visualizing intracellular calcium ion distribution using transmission electron microscopy, mice were perfused 11 days after the last dose of methylmercury (MeHg) using the oxalate-pyroantimonate method. Cytosolic and mitochondrial protein fractions from acutely isolated granule cells were analyzed for cytochrome c content using Western blot analysis. Histochemistry (Fluoro-Jade dye) and immunohistochemistry (activated caspase 3) was performed on frozen serial cerebellar sections to label granule cell death and activation of caspase 3, respectively. Granule cells isolated from MeHg-treated mice showed elevated ROS levels and decreased mitochondrial membrane potential when compared to granule cells from control mice. Electron photomicrographs of MeHg-treated granule cells showed altered intracellular calcium ion homeostasis ([Ca(2+)](i)) when compared to control granule cells. However, in spite of these subcellular changes and moderate relocalization of cytochrome c into the cytosol, the concentrations of MeHg used in this study did not produce significant neuronal cell death/apoptosis at the time point examined, as evidenced by Fluoro-Jade and activated caspase 3 immunostaining, respectively. These results demonstrate that short-term in vivo exposure to total doses of 1.0 and 5.0 mg/kg MeHg through the most common exposure route (oral) can result in significant subcellular changes that are not accompanied by overt neuronal cell death.

Coordination deficits induced in young adult mice treated with methylmercury.

Male and female C57BL/6J mice starting at postnatal (P) day 34 were exposed orally to five divided doses totaling 1.0 or 5.0 mg/kg of methylmercury (MeHg; given as methylmercuric chloride) or sterile deionized water in moistened rodent chow. After a 5-day waiting period, control and MeHg-treated mice were subjected to a standard battery of behavior tests for balance and motor coordination. Latency to falling on the accelerating rota-rod was significantly decreased in 5.0 mg/kg MeHg-exposed mice when compared to control mice. In the open field, horizontal exploration with respect to total distance traveled during the first 5 min on the first test day was significantly reduced in 1.0 mg/kg MeHg-exposed mice when compared to control mice. Rearing activity was not affected by MeHg treatment. In the footprint analysis, angle of foot placement measured in 1.0 mg/kg MeHg-treated mice was significantly greater compared to control mice. Base stance and stride length were unaffected by MeHg treatment. On the vertical pole test, 10 mice from each treatment group fell off the pole during the time the pole was shifted from a horizontal position to a vertical position, whereas none of the control mice fell. These results indicate that short-term, low to moderate doses of MeHg in young adult mice can be detrimental to motor coordination and balance.

Assessment of mercury concentrations in mouse brain using different routes of administration and different tissue preparations.

ABSTRACT The objective of this study was to determine the best method for preparing brain tissue for mercury analysis from mice exposed to methylmercury either through subcutaneous (SC) injection or through ingestion. C57BL/6 J mice at postnatal day 29 were exposed to 0.0 or 5.0 mg/kg methylmercuric chloride (MMC) given SC or through food containing MMC. Eighteen mice received vehicle (sodium bicarbonate; SC) and 18 additional mice received 5.0 mg/kg MMC (SC). Whole brain tissue was prepared using one of four tissue preparation methods: rapid freezing, saline perfusion, 4% paraformaldehyde perfusion fixation, or 4% paraformaldehyde immersion fixation. Brains from vehicle-treated mice exhibited minimal levels of mercury (0.0007 to 0.0018 ppm) in all preparation methods. Mercury content in rapidly frozen control brains differed statistically from immersion-fixed control brain tissue. There was no significant difference in mercury content from mice given 5.0 mg/kg MMC (SC) in all preparation methods (0.2660 to 0.3650 ppm). Additional mice were divided into groups of six mice each: single oral dose of 5.0 mg/kg MMC; total oral dose of 5.0 mg/kg MMC divided into five doses; and vehicle only. Forebrain (0.3243 ppm) and hindbrain (0.1908 ppm) mercury content in MMC-treated mice given multiple doses was 10 times higher than in brain tissue from mice given a single 5.0 mg/kg dose. Brain mercury content following administration of 5.0 mg/kg MMC via the oral route (0.5354 ppm) differed statistically from the SC route (0.3430 ppm). In conclusion, different tissue preparation methods do not significantly affect brain mercury content, but route of administration and dosing regimen can influence total brain mercury content.