The comparative toxicology of narasin in laboratory animals.

The toxicology of narasin has been extensively investigated in several species of laboratory animals. Acute median lethal po doses varied considerably between species (> 10 to 40.8 mg/kg). Animals of various species given acutely toxic doses of narasin manifested similar clinical signs of toxicity, including anorexia, hypoactivity, leg weakness, ataxia, depression and diarrhea. Clinical effects were usually delayed 1 to several days, depending on the dose, and some were reversible even with continued narasin administration. In repeated dose toxicity studies, narasin dosages have been demonstrated at which animals could be exposed daily for long periods of time without producing harmful effects. The no-observed effect levels (NOELs) by the dietary route were 60 ppm in mice and 15 ppm in rats after 3 mo of dosing and 15 ppm in rats after 1 y. In dogs, NOELs were 1 mg/kg body weight after 3 mo and 0.5 mg/kg body weight after 1 y of dosing. In breeding animals, narasin did not affect reproductive performance through 4 generations and was not teratogenic. Two-y chronic bioassays in 2 rodent species showed that narasin did not produce cumulative toxicity or carcinogenicity. In genetic toxicity tests narasin was not mutagenic to bacterial or mammalian cells and did not induce DNA repair or sister chromatid exchange. Narasin neither caused dermal toxicity nor skin sensitization, but was a severe eye irritant in rabbits. In dogs, local irritation and systemic toxicity occurred following repeated inhalation exposure to narasin aerosol concentrations greater than 0.114 mg/M3 of air.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental toxicity of gemcitabine, an antimetabolite oncolytic, administered during gestation to CD-1 mice.

Gemcitabine was given intravenously to female mice on gestation days (GD) 6-15 at doses of 0, 0.05, 0.25, or 1.5 mg/kg/day (0, 0.15, 0.75, or 4.5 mg/m2/day, respectively). Animals assigned to the teratology segment (25/group) were killed on GD 18 for examination of maternal hematologic parameters and organ weights, as well as fetal viability, weights, and morphology. The postnatal segment females (20/group) were allowed to deliver, and offspring physical, behavioral, and reproductive parameters were monitored. After offspring weaning, these dams were killed for hematologic and organ weight evaluations. At necropsy, 3 days after the final dose, the teratology segment dams showed dose-related increases in spleen and thymus weights. These changes were accompanied by a dose-related decrease in leukocytes and modest increases in mean corpuscular volume (MCV) and hemoglobin (MCH) at the two higher doses. On postpartum day (PPD) 21, the dams in the postnatal segment showed no treatment-related effects on these organ weights or hematologic parameters, indicating recovery of these maternal parameters within 3.5 weeks following termination of treatment. The decreases in maternal body weight and food consumption observed during gestation, and in liver and uterine weights at term in the 1.5 mg/kg/day group, were considered to be secondary to a high rate of prenatal mortality, evidenced by increased resorptions in the teratology segment and decreased live litter size in both segments of the study. Additional indications of developmental toxicity in this dose group were an increased incidence of malformations, primarily cleft palate, decreased fetal weights in the teratology segment, and decreased neonatal survival in the postnatal segment.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of metabolism and toxicity to the structure of the anticancer agent sulofenur and related sulfonylureas.

The metabolic formation of p-chloroaniline from the oncolytic agent sulofenur [N-(5-indanesulfonyl)-N'-(4-chlorophenyl)urea, LY186641,] and from similar diaryl-substituted sulfonylureas, and its possible relevance to the compound's toxicity, was studied. In previous studies it was found that significant amounts of metabolites such as 2-amino-5-chlorophenyl sulfate (II), which is also a metabolite of p-chloroaniline, are formed from sulofenur in mice, rats, monkeys, and humans. The metabolism of N-(4-tolyl)-N'-(2-hydroxy-4-chlorophenyl)-urea (V) was studied, and V was not found to be an intermediate in the metabolic formation of II from the sulfonylurea N-(4-tolyl)-N'-(4-chlorophenyl)urea (LY181984, III). The amounts of this p-chloroaniline metabolite (II) formed in C3H mice from a series of diarylsulfonylureas were found to correlate with the compound's propensities to form methemoglobin, one notable toxicity of p-chloroaniline. This metabolism was also found to correlate with the structure of the arylsulfonyl moiety of the sulfonylurea. Other evidence supports the hypothesis that p-chloroaniline is directly formed by metabolism of sulfofenur and similar diarylsulfonylureas as well. Metabolic formation of p-chloroaniline thus appears to be a plausible explanation for the methemoglobinemia and anemia found to be dose-limiting toxicities of sulofenur in Phase I trials.

Genetic alterations in the 61st codon of the H-ras oncogene isolated from archival sections of hepatic hyperplasias, adenomas and carcinomas in control groups of B6C3F1 mouse bioassay studies conducted from 1979 to 1986.

In order to better understand the molecular events in murine hepatocarcinogenesis, the frequency and types of mutations in the murine H-ras proto-oncogene isolated from 184 independent, spontaneously occurring hepatic lesions were determined. Hepatocellular foci, hyperplasias, adenomas and carcinomas were obtained from archival samples of control male (134 samples) and female (50 samples) B6C3F1 mice used in oncogenicity studies that were conducted at Lilly Research Laboratories from 1979 to 1986. The 61st codon region of the H-ras oncogene from these sections was amplified using the polymerase chain reaction. Mutation frequencies were determined by restriction fragment length polymorphism analysis. The types of mutations were characterized by allele-specific oligonucleotide hybridization and confirmed by DNA sequencing. Forty-two per cent of the carcinomas, 44% of the adenomas, 42% of the hyperplasias and 29% of the foci contained mutations at the 61 codon. The mutation spectra for the carcinomas, adenomas and hyperplasias consisted of mostly CAA-AAA transversions, followed by CAA-CGA transitions, followed by CAA-CTA transversions. These results demonstrate that: (i) the frequency of spontaneous mutations in the H-ras 61st codon is equivalent in murine hyperplasias, adenomas and carcinomas, and (ii) sex was not a determining factor in either the mutation frequency or mutation spectrum for the spontaneous lesions. If these lesions represent successive stages in the carcinogenic process, then these results suggest that mutations in the 61st codon of H-ras are early events in spontaneous murine hepatocarcinogenesis.

A 40-week male fertility study of the anticancer agent sulofenur (LY186641) administered orally to rats.

Sulofenur was evaluated for fertility effects on rats. Five-week old male rats (20/group) received 0, 5, 30, or 60 mg Sulofenur/kg/day for 14 weeks. Fertility was evaluated five times. Treated males were mated with untreated females at 10 weeks. Half the males from each group were necropsied after the 14-week treatment period and the remainder were mated four additional times during a 26-week posttreatment period. Following each mating, females were killed on gestation-day 20 and examined for evidence of pregnancy. Six weeks after mating trial 5, the remaining males were necropsied. Testes and epididymides were collected, weighed, and examined microscopically. All rats mated in each mating trial, and all control and low-dose animals were fertile. A significant reduction in fertility occurred in the middle- and high-dose males. This was consistent with testicular hypospermatogenesis in these groups. Fertility recovered in the high-dose group following cessation of treatment, but remained reduced in the middle-dose group. Preimplantation and postimplantation loss in mating trial 1 were higher in the middle- and high-dose groups. Abnormal fetuses were present in the high-dose group in mating trial 3, but not in mating trials 4 or 5. In conclusion, male rats given doses of 30 and 60 mg/kg/day of Sulofenur showed hypospermatogenesis and decreased fertility. Spermatogenesis and fertility recovered in the high-dose group. A dose of 5 mg/kg/day did not produce any effect on testes or fertility.

Target organ toxicity and leukopenia in Fischer 344 rats given intravenous doses of vinblastine and desacetyl vinblastine.

The toxicity and leukopenia produced by vinblastine or desacetyl vinblastine were established in 1-month studies in rats. Groups of male Fischer 344 rats were given weekly intravenous doses of vinblastine or desacetyl vinblastine at doses of 0.08, 0.16, 0.32, 0.64, or 1.28 mg/kg. The target organ toxicity was similar for both compounds. Decreased cell production in the thymus, testes, and bone marrow was produced in the animals of the two highest dose groups for both compounds with the degree of severity greater in the highest dose group. All high dose rats (1.28 mg/kg) given desacetyl vinblastine and three rats given vinblastine died prior to study termination. Body weight loss was more pronounced in high dose rats given desacetyl vinblastine, but at lower doses there were no significant differences in body weight reduction for rats receiving either compound. Leukopenia occurred at all dose levels of 0.32 mg/kg and higher. During the first 2 weeks of the study, rats given 1.28 mg/kg of desacetyl vinblastine had a greater leukopenic response than rats given 1.28 mg/kg of vinblastine. It is concluded that both compounds produced similar target organ toxicity and leukopenia without deaths at doses of 0.32 and 0.64 mg/kg given once a week for 4 weeks. At the high dose (1.28 mg/kg) desacetyl vinblastine was more toxic than vinblastine resulting in greater mortality and body weight reduction.

Evaluation of the antitumor activity of gemcitabine (2',2'-difluoro-2'-deoxycytidine).

A new pyrimidine antimetabolite, 2',2'-difluorodeoxycytidine, Gemcitabine (LY188011, dFdCyd) has been synthesized and evaluated in experimental tumor models. dFdCyd is a very potent and specific deoxycytidine analogue. The concentration required for 50% inhibition of growth is 1 ng/ml in the CCRF-CEM human leukemia cell culture assay. Concurrent addition of deoxycytidine to the cell culture system provides about a 1000-fold decrease in biological activity. The inhibition of growth of human leukemia cells in culture led to the in vivo evaluation of this compound as a potential oncolytic agent. Maximal activity in vivo was seen with dFdCyd when administered on an every third day schedule. 1-beta-D-Arabinofuranosylcytosine, administered on a daily for 10-day schedule, was directly compared to dFdCyd in this evaluation. dFdCyd demonstrated good to excellent antitumor activity in eight of the eight murine tumor models evaluated. 1-beta-D-Arabinofuranosylcytosine was substantially less active or had no activity in these same tumor models. This in vivo activity against murine solid tumors supports the conclusion that dFdCyd is an excellent candidate for clinical trials in the treatment of cancer.

Comparative cardiovascular toxicity in dogs given inotropic agents by continuous intravenous infusion.

A comparative study of the toxicity of the inotropic amines isoproterenol hydrochloride (IP), 1-norepinephrine bitartrate (NE), dopamine hydrochloride (DP), and dobutamine hydrochloride (DB) was conducted in beagle dogs (2/sex/dose group). All drugs were administered at doses that produced maximal contractile tension in dog myocardium. Doses, continuously infused for 96 hr, were 0.625, 1.25, and 2.5 micrograms/kg/min IP, 2.5 and 5 micrograms/kg/min NE, and 25, 50, and 100 micrograms/kg/min DP and DB. Three of 4 dogs that received 5 micrograms/kg/min NE and one of 4 given 100 micrograms/kg/min DP died. Pronounced tachycardia (mean peak rate increases from baseline of 88-104 beats/min) was observed at all doses of IP. DB produced a transient moderate tachycardia (mean peak rate increases from baseline of 25-27 beats/min) at 25 and 50 micrograms/kg/min and pronounced tachycardia (mean peak rate increases from baseline of 74 beats/min) at 100 micrograms/kg/min. Moderate bradycardia occurred at both doses of NE and at 25 and 50 micrograms/kg/min DP (mean peak rate decreases from baseline of 42-46 and 22-38 beats/min, respectively). At high doses the 4 inotropes produced focal to multifocal myocardial necrosis located mainly in left ventricle and segmental medial necrosis of the coronary arteries, mainly in small intramural muscular branches. Segmental medial hemorrhage was also seen following administration of high doses of NE and DP. An additional intramural coronary arterial lesion produced by all of the inotropes consisted of a mild periadventitial cellular infiltrate and fibroplasia. The results indicated that NE and DP produced the most severe cardiovascular lesions, followed by IP which produced lesions of more moderate severity. DB produced only slight lesions in comparison to the other 3 inotropic amines.

An immunocytochemical study of pituitary adenomas and focal hyperplasia in old Sprague-Dawley and Fischer 344 rats.

Spontaneous pituitary tumors occurring in groups of 100 Sprague-Dawley (SD) and 100 Fischer 344 (F344) rats of each sex on a 2-year aging study were characterized by immunocytochemistry. The SD strain had a total of 75 tumors with 10% in males and 65% in females. Tumors immunoreactive for prolactin (PRL) alone were the most common tumor (64%) with the immunonegative tumor being the second most common (17.3%). F344 rats had a total of 62 tumors with 26% in males and 36% in females. The majority of the tumors were reactive for prolactin alone (56.5%) and tumors reactive for both growth hormone (GH) and PRL were the second most common (21%). Most tumors were immunoreactive for only 1 hormone; however, both strains had tumors that expressed multiple hormones in unusual combinations.

Induction and reversibility of thyroid proliferative changes in rats given an antithyroid compound.

Sixty Fischer 344 rats were fed a diet containing 90 ppm methimazole, a known antithyroid compound. Following exposure to the test compound, groups of ten animals were terminated at 1, 3, and 6 months. Similar groups of ten treated animals were given control diet for a reversibility period of 2, 3, and 6 months, respectively. Groups of ten control rats were terminated after 1, 3, 6, and 12 months. Except for the expected effect on body weight, the treated animals had no physical signs of toxicity. The weight of thyroids increased with the duration of exposure, becoming in males after 6 months about ten times the weight of thyroids from control rats. Thyroids of treated rats after 1 and 3 months had a diffuse homogeneous hypertrophy and hyperplasia of follicular cells, decreased colloid, and increased vascularity. After 6 months' exposure to the antithyroid compound, there was diffuse hyperplasia but also a heterogeneity in the size and morphology of follicles, protrusion of follicular tissue through the gland capsule and into vascular spaces, and the development of follicular nodules. Treated rats placed on control diet, allowing for reversibility, had thyroids which were decreased in size with large follicles and flattened epithelium, and a complete remodeling of most nodules with no evidence of progression. Although the nodules produced after prolonged administration of the antithyroid compound had many of the characteristics of neoplasia, the biologic behavior supports the diagnosis of nodular hyperplasia.

Comparative toxicology of monensin sodium in laboratory animals.

The toxicology of monensin has been studied in several laboratory animal species. There was considerable species variation in acute oral LD50 values. The consistent signs of acute toxicity were: anorexia, hypoactivity, skeletal muscle weakness, ataxia, diarrhea, decreased weight gain and delayed deaths. The 3-mo study in rats fed diets containing 0, 50, 150 or 500 ppm monensin resulted in no effects at the lowest dose level, slight reduction of body weight gain in the middle-dose group and severe depression in body weight gain, skeletal and cardiac lesions, and deaths in the highest dose group. The 3-mo study in dogs given daily oral doses of 0, 5, 15 or 50 mg/kg monensin resulted in no effects at the lowest dose level. Dogs in the 15 and 50 mg/kg groups developed, during test wk 1 to 4, anorexia, weakness, ataxia, labored respiration, body weight loss, increased serum muscle enzyme values, severe skeletal muscle degeneration and necrosis with less severe heart lesions and deaths. Mice fed diets containing 0, 37.5, 75, 150 or 300 ppm monensin for 3 mo had reduced body weight gain in all test groups but no other physical signs. Serum creatine phosphokinase (CPK) values were increased in mice in the two highest dose groups and minimal heart lesions were found in the highest dose group. Dogs given daily oral doses of 0, 1.25, 2.5, 5 or 7.5 mg/kg monensin for 1 yr survived with no evidence of toxicity in the two lowest dose groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat lymphocytic thyroiditis associated with ingestion of an immunosuppressive compound.

Lymphocytic thyroiditis was induced in young Wistar rats by feeding them the immunosuppressive compound frentizole [1-(6-methoxy-2-benzothiazolyl)-3-phenyl urea] for 1 year. About half of the rats given 0.060 and 0.150% frentizole in the diet had lymphocytic thyroiditis. The incidence of thyroiditis was low in the group given the high dose because of severe anemia and hepatic disease which resulted in increased mortality. Reversibility of the thyroid lesion was indicated by reduced incidence rates at 15 and 18 months after treatment was stopped at 1 year. The thyroiditis was characterized by interstitial infiltrates of many lymphocytes and plasma cells and fewer macrophages with mild degenerative changes in the follicular epithelium. This inflammatory cell infiltrate was generally diffuse but occasionally was multifocal, particularly in thyroid glands of rats late in the reversibility phase of the study. The inflammatory cell infiltrate caused the thyroid glands to be several times normal size. Sera from rats with lymphocytic thyroiditis contained hemagglutinating antibody against rat perfect correlation between the presence of anti-thyroglobulin antibody and enlarged thyroid glands. Fine granular deposits of IgG and complement were identified in some areas of the follicular basement membrane. We concluded that the lymphocytic thyroiditis was immunologically mediated, at least in part, by anti-thyroglobulin antibody.

Animal models for the comparative assessment of neurotoxicity following repeated administration of vinca alkaloids.

Neurotoxicity is the major side effect occurring during the clinical use of vincristine (VCR). Animal models predictive of potential neurotoxicity would be very useful in the preclinical development of new vinca compounds. To simulate conditions in which neurotoxicity is produced during the clinical use of VCR, experimental animals (except the guinea pig) were given the test compounds by the iv route over a prolonged time period. Doses were selected based on the production of leukopenia. Vindesine (VDS), a chemically modified vinblastine (VBL) product, was compared with VCR and VBL in animal studies. Definite neurotoxic manifestations developed when VCR was given to chickens, cats, and monkeys. The administration of VDS or VBL did not produce neurotoxic signs in these species. The mouse, rat, dog, and guinea pig were not found to be useful models. Thus, it would appear the chicken, cat, and monkey would be appropriate animal models for the preclinical testing of new vinca compounds.

Structure-activity relationships of dimeric Catharanthus alkaloids. 1. Deacetylvinblastine amide (vindesine) sulfate.

Exploration of the effects of "minor" structural differences on the antitumor activity and toxicity of dimeric Catharanthus alkaloids resulted in the preparation of deacetylvinblastine amide (vindesine, VDS) from either vinblastine (VLB) or deacetylvinblastine. Adequate amounts of vindesine for biological testing were prepared by preferential hydrazinolysis of the C23-ester in the vindoline moiety of VLB, followed by hydrogenolysis of the resulting deacetylvinblastine hydrazide. Vindesine in its activity spectrum against rodent tumor systems resembles vincristine (VCR) rather than its parent VLB, while its neurotoxic potential appears to be less than that of VCR. The experimental models developed to estimate this potential include in vitro measurements of axoplasmic transport effects in the cat sciatic nerve and the estimation of neuromuscular disturbances in chickens and monkeys by vindesine in comparison with VCR. A radioimmunoassay for VLB, VCR, and VDS, developed by means of deacetylvinblastine acid azide, has been used to study the pharmacokinetics of vindesine in man. The clinical investigation of vindesine is in progress. Deacetylvinblastine, in contrast to earlier reports, showed activity against several murine tumor systems.

Toxicology of vindesine (desacetyl vinblastine amide) in mice, rats, and dogs.

Comparative acute intravenous toxicity studies of vinblastine sulfate (VLB), vincristine sulfate (VCR), and vindesine in mice and rats indicated that vindesine was more toxic than VLB and less toxic than VCR. Rats were able to tolerate larger repeated doses of vindesine than dogs. Rats given intravenous doses totaling 0.15 mg/kg-wk vindesine for 3 months developed no remarkable signs of toxicity. Doses of 0.3 mg/kg-wk or greater produced anorexia, depressed blood cell counts, atrophic intestinal mucosa, inhibition of spermatogenesis, extramedullary hematopoiesis, and infections. Dogs were given total weekly intravenous doses of 0.04, 0.08, 0.1, or 0.16 mg/kg vindesine for 3 months. The only observed effect in the two lower dose groups was inhibition of spermatogenesis. Groups receiving 0.1 or 0.16 mg/kg developed leukopenia, slight erythropenia, inhibition of spermatogenesis, focal skeletal muscle degeneration, elevated lactic dehydrogenase, and an increase in bone marrow myeloid: erythroid ratio. No evidence of functional or structural changes in neural tissues was found. The above effects are common to animals given VCR at lower doses and for a shorter test period. It is therefore concluded that vindesine is less toxic in animals than VCR.