Reproductive toxicology studies supporting the safety of molindone, a dopamine receptor antagonist.

BACKGROUND: An extended-release molindone (a dopamine D2 and serotonin antagonist) is currently being developed as a novel treatment for impulsive aggression (IA) in patients optimally treated for ADHD. Oral Good Laboratory Practice reproductive toxicology studies (fertility and early embryonic [FEE], prenatal/postnatal [PPN], embryo-fetal development [EFD]) were conducted with molindone HCl using International Conference on Harmonisation (ICH) S5(R2)-compliant protocols. METHODS: In the FEE study, 0, 5, 15, or 30 mg kg-1 day-1 was administered to female (2 weeks premating through implantation) and male (4 weeks premating for 57 days) rats, and fertility parameters were evaluated. In the EFD studies, rats received 0, 5, 20, or 40 mg kg-1 day-1 on gestational days (GDs) 6-17; rabbits received 0, 5, 10, or 15 mg kg-1 day-1 on GDs 6-18. Ovarian/uterine and fetal parameters were evaluated at term. In the PPN study, F0 rats received 0, 5, 20, or 40 mg kg-1 day-1 (GD6-LD21); behavior and reproduction were evaluated in F1 offspring. RESULTS: Parental hypoactivity and reduced body weight gain occurred in all studies. In the FEE, prolonged estrous cycles and delayed mating occurred at ≥15 mg kg-1 day-1 , without effects on fertility or embryonic development. No developmental toxicity occurred in F1 fetuses. In F1 pups, reduced preweaning growth was observed at 40 mg kg-1 day-1 , but there were no effects on postweaning growth, behavior, or reproduction. CONCLUSIONS: Molindone was not developmentally toxic in rats or rabbits at 69X and 6X clinical exposures, confirming the reproductive safety of molindone. Changes in estrous cyclicity were related to species-specific pharmacological effects of molindone in rodents and are not considered relevant to human risk.

Multigeneration reproduction and male developmental toxicity studies on atrazine in rats.

BACKGROUND: Reproductive toxicity of Atrazine (ATR) was evaluated in two rat multigenerational studies. Development of male reproductive parameters was evaluated in separate studies after prenatal or postnatal exposure. METHODS: In multigenerational studies, rats received dietary concentrations of 0, 10, 50, 100 or 500 ppm ATR. In separate studies in female rats, ATR was administered by gavage at 0, 1, 5, 25 or 125 mg/kg/day during pregnancy (GD6-21) or lactation (LD2-21). Plasma testosterone concentration, testicular and epididymal weights, and sperm counts were measured in male offspring on PND70 and 170. RESULTS: In the multigenerational studies, parental systemic toxicity occurred at 500 ppm (38.7 mg/kg/day), but reproductive endpoints were unaffected. In the prenatal study, maternal toxicity and embryo-fetal mortality occurred at 125 mg/kg/day. In male offspring, testosterone levels and sperm counts were unaffected, although the percentage of abnormal sperm increased at 125 mg/kg/day (PND 70) and 25 mg/kg/day (PND170). In the postnatal study, maternal toxicity and reduced body weights of male offspring occurred at 125 mg/kg/day. Additionally, reduced testicular (PND70, PND170) and epididymal (PND70) weights and increased numbers of abnormal sperm (PND70, PND170) were seen, but no changes in plasma testosterone or sperm counts. CONCLUSIONS: Dietary administration of ATR did not affect rat reproduction up to a parentally toxic dose of 38.7 mg/kg/day. Some effects on male reproductive system development occurred after high dose, bolus administration to dams, but doses were much higher than expected under normal use conditions. Thus, oral RfDs for ATR would be protective for reproductive effects.

Dihydroartemisinin (DHA) treatment causes an arrest of cell division and apoptosis in rat embryonic erythroblasts in whole embryo culture.

Within 24 hr after oral administration of the antimalarial artesunate to rats on Day 10 or 11 postcoitum (pc), there is depletion of embryonic erythroblasts (EEbs), leading to embryo malformation and death. The proximate agent is dihydroartemisinin (DHA), the primary metabolite. We investigated the causes of EEb depletion by evaluating effects of DHA on EEbs in whole embryo culture (WEC). Rat embryos cultured starting on Day 9 pc were treated with 1 or 7 µM DHA for 24 hr starting after 19 hr of culture (∼Day 10 pc) and for 2 to 12 hr starting after 43 hr of culture (∼Day 11 pc). DHA effects indicating the depletion of EEbs were paling of the visceral yolk sac and reductions in visible blood cells, H&E-stained normal (Type II or III) EEbs, and dividing (BrdU-stained) EEbs. DHA-induced abnormal cell division was indicated by increases in symmetric and asymmetric binuclear cells. DHA-induced apoptosis was indicated by increases in TUNEL- and Caspase-3-positive cells and EEbs with fragmented nuclei. In addition, although the overall number of EEbs was decreasing, DHA caused increases in the numbers of circulating early-stage (Type I or earlier) EEbs that could not be accounted for by cell division, suggesting the release of new, less sensitive erythroblasts from the yolk sac. In summary, treatment of Day 10 or 11 pc rat embryos with DHA in WEC resulted in defective and arrested cell division in EEbs followed by apoptosis, suggesting a mechanism for their depletion after artesunate treatment in vivo.

The potential role for corticosterone in the induction of cleft palate in mice after treatment with a selective NK-1 receptor antagonist, casopitant (GW679769B).

BACKGROUND: Casopitant is a potent and selective NK-1 receptor antagonist that has shown clinical efficacy in the prevention of chemotherapy-induced and postoperative-induced nausea and vomiting. METHODS: In an embryo-fetal development study, pregnant mice were given vehicle (sterile water) or doses of 30, 100, or 300 mg/kg/day casopitant on Gestation Day (GD) 6 to 15. Fetuses were evaluated for external, visceral, and skeletal abnormalities on GD 18. In a follow-on study to investigate casopitant-induced hormonal changes during the developmental period for palate formation, pregnant mice were given vehicle (sterile water) or 300 mg/kg/day casopitant once daily on GD 6 to 13. Blood was collected on GD 13 at various time-points for measurement of plasma adrenocorticotropic hormone and corticosterone (CRT) concentrations. RESULTS: There was no evidence of developmental toxicity in mice at 30 or 100 mg/kg/day but 9% of fetuses at 300 mg/kg/day had cleft palate. Mice are sensitive to glucocorticoid-induced cleft palates, and NK-1 antagonists are known to modulate the hypothalamic-pituitary-adrenal axis leading to increases in corticosterone. On GD 13, mean plasma adrenocorticotropic hormone levels at 300 mg/kg/day were elevated by approximately twofold from vehicle-treated levels at 1 hr post-dose and mean plasma CRT levels were elevated by 3, 5, and 10-fold at 0.5, 1, and 2 hr post-dose, respectively. CONCLUSIONS: The increased level of CRT was in the range previously shown in the literature to cause cleft palates in mice and was likely the underlying mechanism behind casopitant-induced cleft palate in mice.

Artesunate: developmental toxicity and toxicokinetics in monkeys.

BACKGROUND: The developmental toxicity, toxicokinetics, and hematological effects of the antimalarial drug, artesunate, were previously studied in rats and rabbits and have now been studied in cynomolgus monkeys. METHODS: Groups of up to 15 pregnant females were dosed on Gestation Days (GD) 20-50 or for 3-7-day intervals. RESULTS: At 30 mg/kg/day, 6 embryos died between GD30 and GD40. Histologic examination of 3 live embryos (GD26-GD36) revealed a marked reduction in embryonic erythroblasts and cardiomyopathy. At 12 mg/kg/day, 6 embryos died between GD30 and GD45. Four surviving fetuses examined on GD100 had no malformations, but long bone lengths were slightly decreased. At the developmental no-adverse-effect-level (4 mg/kg/day), maternal plasma AUC was 3.68 ng.h/mL for artesunate and 6.93 ng.h/ml for its active metabolite, dihydroartemisinin (DHA). No developmental toxicity occurred with administration of 12 mg/kg/day for 3 or 7 days, GD29-31 or GD27-33 (maternal plasma AUC of 9.84 ng.h/mL artesunate and 16.4 ng.h/mL DHA). Exposures at embryotoxic doses were substantially lower than human therapeutic exposures. However, differences in monkey and human Vss for artesunate (0.5 L/kg vs. 0.18 L/kg) confound relying solely on AUC for assessing human risk. Decreases in reticulocyte count occur at therapeutic doses in humans. Changes to reticulocyte counts at embryotoxic doses in monkeys (> or =12 mg/kg/day) were variable and generally minor. CONCLUSIONS: Artesunate was embryolethal at > or =12 mg/kg/day when dosed for at least 12 days at the beginning of organogenesis, but not when dosed for 3 or 7 days, indicating that developmental toxicity of artesunate is dependent upon duration of dosing in cynomologus monkeys.

Developmental toxicity of artesunate in the rat: comparison to other artemisinins, comparison of embryotoxicity and kinetics by oral and intravenous routes, and relationship to maternal reticulocyte count.

BACKGROUND: The antimalarial, artesunate, is teratogenic and embryolethal in rats, with peak sensitivity on Days 10 and 11 postcoitum (pc). METHODS: We compared the developmental toxicity of structurally related artemisinins, dihdyroartemisinin (DHA), artemether (ARTM), and arteether (ARTE) to that of artesunate after oral administration to rats on Day 10 pc. In separate studies, embryolethality was characterized after single intravenous (IV) administration of artesunate on Day 11 pc, and toxicokinetic parameters following oral and IV administration were compared. Lastly, to determine whether maternal hematologic effects occurred at doses that affect embryonic erythroblasts, artesunate was orally administered on Day 11 pc at a dose that caused 100% embryolethality. RESULTS: All artemisinins caused the same pattern of embryolethality and fetal cardiovascular and skeletal abnormalities as previously shown for artesunate. In the IV study, marked postimplantation loss occurred at 1.5 and 3 mg/kg artesunate, but not at 0.75 mg/kg. Among the toxicokinetic parameters evaluated, only the DHA AUC(0-t) was similar at embryolethal oral and IV doses of artesunate. An embryolethal dose of artesunate caused a 15% decrease in maternal reticulocyte counts and no other hematologic effects. CONCLUSIONS: Several structurally related artemisinins cause similar developmental toxicity, suggesting an artemisinin class effect. Equally embryotoxic oral and IV treatments of one artemisinin compound (artesunate) produced similar systemic exposure to the artesunate metabolite, DHA, suggesting that DHA may be the proximate developmental toxicant. Embryolethal doses of artesunate only caused minor changes in maternal reticulocyte counts indicating that adult hematology parameters are not as sensitive as embryonic erythroblasts.

Sensitive periods for developmental toxicity of orally administered artesunate in the rat.

BACKGROUND: Artesunate has been reported to cause embryolethality and malformations when administered orally to rats during organogenesis. The purpose of this study was to determine the most sensitive period(s) for the induction of these effects in order to provide clues about possible mechanisms and to identify a short treatment regimen for further studies. METHODS: Pregnant rats were orally administered artesunate (10, 17 or 30 mg/kg/day) on single or multiple days of gestation. Cesarean sections and fetal evaluations were conducted on Day 21 postcoitum (pc). RESULTS: Embryolethality, cardiovascular malformations and a syndrome of skeletal defects were observed after single doses on days 10 to 14 pc, while no developmental effects were observed before (day 9 pc) or after (days 16 or 17 pc) that period. The most sensitive day for embryo lethality was day 11 pc, where lethality occurred with a very steep dose response (postimplantation loss was approximately 15% at 10 mg/kg and 100% at 17 mg/kg/day). The most sensitive day for the induction of malformations was day 10 pc. Malformations tended to occur in partially resorbed litters and included cardiovascular defects and bent and misshapen long bones and scapulae. CONCLUSIONS: The sensitive window for developmental toxicity of artesunate in the rat was identified as days 10 to 14 pc. Single oral doses produced embryolethality and similar cardiovascular and skeletal malformations as previously reported in longer term dosing experiments. These single dose treatment regimens could be useful to further investigate the mechanistic basis for artesunate-induced developmental toxicity.

Effects of food restriction on testis and accessory sex glands in maturing rats.

Reduced food consumption and associated lower body weights may occur in subacute toxicity studies. The short-term effects of food restriction (FR) on body and reproductive organ weights, hormones, and testis histology were assessed in Sprague-Dawley rats fed 20% to 36% less (21 g feed/day) than rats fed ad libitum (AL) starting at six, eight, ten, or twelve weeks of age for two or six weeks. Body weight and relative seminal vesicle, ventral prostate, and/or epididymis weights were reduced in rats FR for two or six weeks. Degeneration of stage VII pachytene spermatocytes was seen in rats FR for six weeks when initiated at eight, ten, and twelve weeks of age. Plasma testosterone concentrations were lower in rats FR at ages six to eight weeks, eight to ten weeks, six to twelve weeks, and eight to fourteen weeks. Luteinizing hormone was not statistically different in FR rats compared with AL counterparts. Therefore, duration of lower food intake had a greater impact on spermatogenesis, whereas a younger initial age of lower food intake was more influential on testosterone levels. These interactions are important in the interpretation of subacute toxicology studies employing FR or when test articles lower food consumption relative to AL-fed rats.

Artesunate-induced depletion of embryonic erythroblasts precedes embryolethality and teratogenicity in vivo.

BACKGROUND: Artesunate (ART), an artemisinin antimalarial, is embryolethal and teratogenic in rats, with the most sensitive days being 10 and 11 postcoitum (pc), respectively (Clark et al.: Birth Defects Res B 71:380-394, 2004; White et al.: Birth Defects Res A 70:265, 2004). METHODS: In this study, pregnant rats were administered a single oral dose of 17 mg/kg ART on Days 10-11 pc and conceptuses were evaluated through Day 14 pc. RESULTS: Paling of visceral yolk sacs was observed within 3-6 hr after treatment. Within 24 hr, marked paling and embryonic erythroblast depletion were observed macroscopically, which preceded malformations and embryo death, and persisted through Day 14 pc. Histologically, embryonic erythroblasts were reduced and cells showed signs of necrosis within 24 hr, were maximally depleted by 48 hr, and had partially rebounded within 3-4 days after treatment (Days 13 and 14 pc). Iron accumulation was evident in treated erythroblasts as early as 6 hr after treatment, suggesting impairment of heme synthesis. Heart abnormalities (swollen or collapsed chambers) were observed within 24 hr in approximately 25-60% of embryos and within 48 hr in 100% of embryos, correlating with histologic signs of cardiac myopathy (thinned and underdeveloped heart walls and enlarged chambers). Delays in limb and tail development occurred by Day 13 pc. Embryos were viable through Day 13 pc, but approximately 77% of embryos had died by Day 14 pc, presumably due to hypoxia and/or cardiac abnormalities. CONCLUSIONS: In summary, embryonic erythroblasts are the primary target of ART toxicity in the rat embryo after in vivo treatment, preceding embryolethality and malformations.

Developmental toxicity of artesunate and an artesunate combination in the rat and rabbit.

The artemisinins are playing an increasingly important role in treating multidrug-resistant malaria. The artemisinin, artesunate, is currently in use in Southeast Asia and is advocated for use in Africa. In these areas, more than one million people die of malaria each year, with the highest mortality occurring in children and pregnant women. To test the developmental toxicity in ICH-compliant animal studies, embryofetal development studies were conducted in rats and rabbits treated with artesunate alone or a three-drug combination (CDA) consisting of chlorproguanil hydrochloride, Dapsone, and artesunate in the ratio 1.00:1.25:2.00. Developmental toxicity seen with CDA could be attributed to the administered dose of artesunate. The hallmark effect of artesunate exposure was a dramatic induction of embryo loss, apparent as abortions in rabbits and resorptions in both rats and rabbits. In addition, low incidences of cardiovascular malformations and a syndrome of skeletal defects were induced at or close to embryolethal doses of artesunate in both rats and rabbits. The cardiovascular malformations consisted of ventricular septal and vessel defects. The skeletal syndrome consisted of shortened and/or bent long bones and scapulae, misshapen ribs, cleft sternebrae, and incompletely ossified pelvic bones. These developmental effects were observed largely in the absence of any apparent maternal toxicity. The no or low adverse effect levels were in the range of 5 to 7 mg/kg/day artesunate. Encouragingly, no adverse drug-related developmental effects have been observed in a limited number of pregnant women (more than 100 first trimester and 600 second and third trimester) treated with artemisinins, primarily artesunate. Investigations of the mechanism of developmental toxicity are ongoing to attempt to determine whether rats and rabbits are more sensitive to artemisinins than humans.