Results from two-year rodent oral carcinogenicity studies of cizolirtine, a substance-P and calcitonin gene-related peptide release modulator.

Cizolirtine is a substance-P and calcitonin gene-related peptide release modulator developed for the treatment of pain and urinary incontinence. To assess its carcinogenic potential, cizolirtine was administered by oral route once daily for up to 104 weeks to CD-1 mice at doses of 40, 90, or 200 mg/kg/day, and to Han Wistar rats at doses of 40, 90 or 200 mg/kg/day to males and 40, 110 or 160 mg/kg/day to females. There were treatment-related neoplastic findings both in mice and rats. In mice, administration of cizolirtine was associated to an increase in skin fibrosarcomas and sarcomas among high-dose males, considered secondary to increased aggression and specific to the animal model. In rats, there was an increased incidence of liver adenomas in males and females, and carcinomas in males, in association with an increased incidence of hepatocyte hypertrophy, vacuolation and clear cell foci, and considered related to sustained long-term enzyme induction resulting in increased liver metabolism and associated hypertrophic changes. The observed neoplastic findings in mouse skin and rat liver after life-time oral administration of cizolirtine are considered related to rodent-specific non-genotoxic mechanisms of questionable relevance to man.

Twenty six-week repeat dose oral rat toxicity study of cizolirtine, a substance-P and calcitonin gene-related peptide release modulator.

Cizolirtine, a substance-P and calcitonin gene-related peptide release modulator developed for the treatment of pain and urinary incontinence, was orally administered for 26-weeks to rats at dosages of 20, 60 and 200 mg/kg/day. Clinical signs were limited to post-dosing salivation and brown staining on head and muzzle. There were slight decreases in bodyweight gain and slight increases in water consumption among cizolirtine-treated animals. Slight increases in plasma alkaline phosphatase activity, and cholesterol and phospholipid concentrations were observed in mid- and/or high-dose animals. Low urinary volume, pH and sodium and potassium outputs were observed after 12-weeks, and low urinary pH, low sodium and high potassium outputs at end of treatment. Increased relative (to bodyweight) liver weight was observed in high-dose animals. Treated males and high-dose females showed a dose-related increase in the incidence and severity of periacinar hepatocytic hypertrophy and midzonal/periacinar hepatocytic fat vacuolization. Increased incidences of hepatic clear cell foci were observed in all cizolirtine-treated male groups and, to a lesser extent, in treated females. Ovaries of treated females showed a dose-dependent increased incidence of absent corpora lutea and, occasionally, follicular cysts. The dosages of 20 and 60 mg/kg/day were considered as the No-Observed-Adverse-Effect Levels for males and females, respectively.

Assessment of the Genotoxic Potential of Cizolirtine a Substance-P and Calcitonin Gene-Related Peptide Release Modulator.

The analysis of the genotoxic potential of cizolirtine, a compound being developed as a drug for analgesia and for urinary incontinence, was carried out using a battery of in vitro and in vivo assays as recommended in the guidelines for medicinal products. Negative results were obtained in an Ames test (up to 5000 µg/plate), in a Mouse Lymphoma assay (up to 2000 µg/ml) and in a single dose mouse bone marrow micronucleus assay (up to 300 mg/kg). In a human lymphocyte chromosome aberration assay, a slight statistical increase in the frequency of cells with chromosome aberrations including gaps was reported for the concentrations of 200 and 1600 µg/ml at the 24-h sampling time. This minor increase in chromosome aberrations was considered of questionable biological relevance since it was moderate, was within the laboratory historical control values, did no show a dose-dependent effect and was not observed at similar concentrations in a repeat assay. Taking into considerations the results obtained in the different in vitro and in vivo assays and a weight-of-evidence analysis, it suggests that cizolirtine would not pose a genotoxic risk when administered to humans.

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 (sigma(1)) 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 sigma(1) 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 sigma(1) receptor knockout (sigma(1)R-KO) and wild type (WT) mice. After administration, animal's 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 sigma(1)R-KO and WT mice. Maximum decreases from preadministration temperature were 12.2 and 13.5 degrees C in sigma(1)R-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 sigma(1)R-KO and WT animals. Examination of peripheral blood samples showed a slight, although nonstatistical significant, increase in MNRET frequency in sigma(1)R-KO mice. No similar effect was observed among WT animals. The results obtained after E-5842 administration to sigma(1)R-KO mice indicate that induction of hypothermic conditions associated with increased MNPCE formation is not mediated by compound interaction with sigma(1) receptor, ruling out concern for a possible class effect of similar high affinity sigma(1) receptor ligands in the mouse MN test.

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.

Assessment of the genotoxic potential of the antipsychotic sigma receptor ligand E-5842.

The genotoxic potential of E-5842, a sigma ligand compound being developed as an antipsychotic drug, was evaluated by means of an extensive battery of in vitro and in vivo assays. Negative results were obtained in an Ames test (up to 5000 µg/plate), a mouse lymphoma assay (up to 535.1 µg/ml (-S9) and 891.8 µg/ml (+S9)), an in vivo rat hepatocyte micronucleus assay (up to 100 mg/kg/day on 2 days), and a two-dose mouse micronucleus assay (up to 40 mg/kg/day on 2 days). In a single-dose mouse bone-marrow micronucleus assay (up to 400 mg/kg; 24, 48 and 72 h sampling) a slight and non-statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (MNPCE) was observed 48 h after administration of a 200 mg/kg dose, in the absence of bone-marrow toxicity. This minor increase in MNPCE frequency was considered of questionable biological relevance, because it was observed under conditions of marked animal toxicity including mortality. In addition, it occurred in association with a strong hypothermic effect produced by administration of E-5842. A clear increase in the frequency of structural chromosomal aberrations was observed in human lymphocytes at concentrations ≥350.6 and 1685.4 µg/ml in the presence and absence of S9, respectively. Mitotic accumulation was observed at those concentrations at which clastogenic effects were observed, a condition that may have masked toxicity. Concentrations lacking clastogenic effects in this chromosome aberration assay (300.7 and 173.2 µg/ml in the presence and absence of S9, respectively) were well in excess of maximum human plasma concentrations attained in clinical studies at the maximum tolerated dose (19.1 ng/ml). A weight-of-evidence analysis, taking into consideration the results obtained in the different in vitro and in vivo assays and the conditions of clinical use, suggest that E-5842 would not pose a genotoxic risk under clinical conditions.

Induction of micronuclei in mouse bone-marrow erythrocytes in association with hypothermia after administration of the sigma receptor ligand E-5842.

Oral administration of E-5842, a new sigma1 receptor ligand being developed as an antipsychotic drug, to male mice at single doses of 50, 100, 200 and 400 mg/kg produced marked and sustained decreases in rectal temperature. Both the intensity and the duration of the hypothermic effect increased with dose. Maximum decreases from the mean pre-administration temperature (36.2 degrees C) ranged from 7.5 to 12.9 degrees C for animals receiving 50 and 400 mg/kg doses, respectively. Examination of bone-marrow smears obtained 24, 48 and 72 h after administration revealed a slight but statistically significant (p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (MNPCE) at the 48 h sampling for animals receiving the 200 mg/kg dose. These animals showed decreases from pre-administration temperature of approximately 12 degrees C, with recovery being observed 24 h after administration. When the hypothermic effect of E-5842 administration was avoided by housing treated animals under conditions of increased environmental temperature (30 degrees C) for 24 h, MNPCE frequency reverted to vehicle control values. Further, in E-5842-treated animals with an increased MNPCE frequency there was a shift in the distribution of the relative areas of micronuclei in MNPCE towards higher values. In addition, there was a statistically significant increase (p < 0.001) in the number of relatively large micronuclei (micronucleus diameter > or = 1/4 cytoplasm diameter) similar to that produced by administration of the mitotic spindle inhibitor colchicine (1 mg/kg), suggesting disturbance of mitotic apparatus as the possible underlying mechanism. The results suggest that the slight increase in MNPCE frequency observed 48 h after administration of a 200 mg/kg dose of E-5842 is due to a hypothermic effect and not to a direct effect of E-5842 on DNA.