Antonio Guzmán

Formation of micronucleated erythrocytes in mouse bone-marrow under conditions of hypothermia is not associated with stimulation of erythropoiesis

Conditions of marked and long-lasting hypothermia have been shown to increase the formation of micronucleated polychromatic erythrocyte (MNPCE) in mouse bone-marrow. Stimulation of erythropoiesis as a consequence of anoxic conditions associated with decreased body temperature has been suggested as a possible mechanism for hypothermia-induced micronucleus formation. We examined whether chemically induced hypothermic conditions that produced increased MNPCE formation were associated with stimulation of erythropoiesis by measuring erythropoietin (EPO) concentrations in blood. Marked and long-lasting hypothermia was induced in male mice by oral administration of the antipsychotic compounds E-5842 (200 mg/kg) or chlorpromazine (100 mg/kg). Maximum decreases from the basal temperature, achieved 8 h after treatment, were 14.8 and 12.8 degrees C, respectively. A statistically significant increase in bone-marrow MNPCE frequency was observed 48 h after administration of E-5842 (p<0.01) or chlorpromazine (p<0.05). Mice made anaemic by retro-orbital bleeding (0.5 ml), which acted as positive control for stimulation of erythropoiesis, showed no relevant variation in mean rectal temperature and a slight non-statistically significant increase in MNPCE frequency after 48 h. Blood samples for determination of EPO levels were obtained 4 (bleed-control animals only), 8, 16 and 24 h after treatment. In spite of the induced hypothermia, no significant variation in EPO blood levels was observed after administration of E-5842 or chlorpromazine. Bleed-induced anaemic mice showed a clear increase in EPO blood levels at all sampled time points, differences from baseline values being statistically significant (p<0.001) at the 8-h samplings and beyond. These results indicate that induction of MNPCE secondary to chemically induced hypothermia is not mediated by stimulation of erythropoiesis.

Evaluation of the genotoxic potential of three phenyltetrahydropyridinyl butylazole-derived sigma-receptor ligand drug candidates.

Three structurally related phenyltetrahydropyridinyl butylazole (PTHPB)-derived drug candidates with sigma receptor-binding properties were evaluated for genotoxic potential in the ICH standard battery of genetic toxicology assays. These comprised an Ames test, a mouse-lymphoma assay, and a mouse bone-marrow micronucleus test. The maximum test concentrations in the in vitro assays were determined by the solubility and/or the cytotoxicity of the compounds. In the mouse micronucleus assay, the compounds were administered orally at three levels up to the maximum tolerated dose (MTD). Negative results were obtained for all three drug candidates in the Ames test and in the mouse-lymphoma assay, both in the absence or presence of metabolic activation. In the mouse micronucleus test, there was no effect on the frequency of micronucleated polychromatic erythrocytes (MNPCE) in bone marrow after oral administration of any of the three test compounds, at any dose level or sampling time (24 and 48h). Administration of all three compounds at the MTD induced a clear decrease in mouse body-temperature of 3.1-4.8 degrees C below normal; the temperature returned to normal within 8h of dose administration. The produced mild hypothermia and absence of micronucleus induction was in contrast to the induction of MNPCE secondary to marked hypothermia reported for a structurally similar PTHPB-derived sigma-receptor ligand, the antipsychotic compound E-5842. The results obtained in the current series of studies suggest that exposure to the three tested PTHPB-derived drug candidates would not pose a genotoxic risk under clinical conditions.

Induction of hypothermic conditions associated with increased micronuclei formation in sigma-1 receptor knockout mice after administration of the antipsychotic compound E-5842

The antipsychotic sigma‐1 (σ1) receptor ligand E‐5842 has been shown to increase micronucleated polychromatic erythrocyte (MNPCE) frequency in mouse bone marrow secondary to compound‐induced hypothermia. Interaction with σ1 receptor has been considered a plausible contributing factor for E‐5842‐induced hypothermia, raising concern for a possible class effect of sigma receptor ligands in the mouse micronucleus (MN) test. We assessed the potential of E‐5842 (200 mg/kg, oral) to produce hypothermic conditions associated with increased micronuclei formation in σ1 receptor knockout (σ1R‐KO) and wild type (WT) mice. After administration, animals rectal temperature was recorded and peripheral blood and bone marrow samples were obtained (48 hr) and assessed for induction of micronucleated reticulocytes (MNRET) and MNPCE, respectively. E‐5842 administration produced marked hypothermia both in σ1R‐KO and WT mice. Maximum decreases from preadministration temperature were 12.2 and 13.5°C in σ1R‐KO and WT mice, respectively. Temperature returned to normal approximately 32 hr after administration. Bone marrow examination revealed a statistical significant increase (P < 0.05) in MNPCE frequency both in σ1R‐KO and WT animals. Examination of peripheral blood samples showed a slight, although nonstatistical significant, increase in MNRET frequency in σ1R‐KO mice. No similar effect was observed among WT animals. The results obtained after E‐5842 administration to σ1R‐KO mice indicate that induction of hypothermic conditions associated with increased MNPCE formation is not mediated by compound interaction with σ1 receptor, ruling out concern for a possible class effect of similar high affinity σ1 receptor ligands in the mouse MN test. Environ. Mol. Mutagen., 2008.

Four-week oral toxicity studies of the new quinolone antibacterial agent cetefloxacin tosylate in rats and marmoset monkeys

Four-week oral toxicity studies with cetefloxacin tosylate ((-)-7[3-(R)-amino-2-(S)-methyl-1-azetidinyl]-1-(2,4- difluorophenyl)-1,4-dihydro-6-fluoro-4-oxo-3-quinolinecarboxylic acid tosylate, CAS 141725-88-4 (base), E-4868.Ts) a new quinolone antibacterial agent, were performed in Sprague-Dawley rats and marmoset monkeys at doses of 100, 450, 2000 mg/kg/d and 25, 50, 125, 300 mg/kg/d, respectively. In rats, due to its toxicity the high dose was decreased to 1000 mg/kg/d after 3 days of treatment. Mortality was recorded among high dose rats receiving 2000 or 1000 mg/kg/d. Rats receiving dosages of 450 or 2000/1000 mg/kg/d showed less activated mandibular lymph nodes, cortical lymphocyte depletion of mandibular and/or mesenteric lymph nodes, atrophy of the white pulp of the spleen, cortical atrophy of thymus and thymic apoptosis. Enlarged caeca, increased water consumption and variations in plasma electrolyte levels were observed in animals receiving these dosages and in male rats receiving 100 mg/kg/d. Low neutrophil counts were observed in rats receiving dosages of 100 or 450 mg/kg/d, and increased alkaline phosphatase and alanine transaminase plasma levels and slightly decreased plasma protein levels in females receiving 450 or 2000/1000 mg/kg/d. Marmosets receiving dosages of 50 mg/kg/d and above displayed several clinical signs which included emesis, diarrhoea, ptosis, occasional episodes of under- and overactivity, and excessive scratching activity. Skin reddening was observed during the first week of treatment in marmosets receiving 300 mg/kg/d. On the basis of the results obtained it can be concluded that the non-toxic doses of E-4868. Ts after 4-week oral administration in rats and marmoset monkeys were 100 and 25 mg/kg/d, respectively.