Embryotoxicity of artesunate in animal species related to drug tissue distribution and toxicokinetic profiles

BACKGROUND: Injectable artesunate (AS) can cause fetal death and teratogenic effects in animals at levels below the human therapeutic dose. Similar toxicity has also been found for oral artemisinins in various animal species, but has not been found in humans. METHODS: Studies on tissue distribution (5 mg/kg) and toxicokinetics (TK, 30 mg/kg × 3) were conducted in pregnant (GD11–13) and non‐pregnant rats. RESULTS: TK profiles of AS showed that the two groups of rats were similar after a single AS dose but were significantly different after multiple doses. Following a daily dose for 3 days, no change in AS concentration was found in the pregnant animals, but a significant concentration decline was seen in the non‐pregnant rats on day 3. In addition, a higher conversion rate of AS to dihydroartemisinin (DHA) was detected in the pregnant rats after either single or multiple doses compared to non‐pregnant controls. The ratios of AUC_DHA/AUC_AS were 0.99–1.02 for the pregnant rats and 0.42–0.48 for non‐pregnant animals, resulting in a total AUC_{DHA D1–3} that was about 3.7‐fold higher in pregnant rats (15,049 ng·h/ml) than in non‐pregnant rats (4,015 ng·h/ml). Furthermore, the tissue/blood partition coefficients demonstrated that the level of radiolabeled AS that was distributed into uterus, placenta, and ovary was 2–4‐fold higher than in blood. CONCLUSIONS: TK data showed that unchanged AS and DHA in the blood of pregnant rats were 1.53‐ and 3.74‐fold, respectively, higher than those of non‐pregnant animals, which all likely relate to the severe embryotoxicity of AS, even with a low‐dosage regimen in pregnant animals. Birth Defects Res (Part B), 2008. Published 2008 Wiley‐Liss, Inc.     

Comparison of the developmental toxicity of aspirin in rabbits when administered throughout organogenesis or during sensitive windows of development

BACKGROUND: A review of the nonsteroidal anti‐inflammatory drug (NSAID) literature suggested occurrences of low‐level incidences of cardiovascular and midline defects in rabbit fetuses exposed in utero. Aspirin (acetylsalicylic acid, ASA) is a widely used NSAID that irreversibly inhibits cyclooxygenases (COXs) 1 and 2. ASA has been studied extensively in rats and has consistently increased low‐incidence cardiovascular malformations and defects in midline closure. The objectives of the current study were to comprehensively define the developmental toxicology profile of ASA in rabbits by using a dosing paradigm encompassing the period of organogenesis and to test the hypothesis that maternal gastrointestinal toxicity after repeated dose administrations hampers the detection of low‐incidence malformations with ASA in rabbits by limiting ASA administration to sensitive windows for cardiovascular development and midline closure. METHODS: ASA was administered to pregnant New Zealand White rabbits from gestation days (GDs) 7 to 19 at dose levels of 125, 250, and 350 mg/kg per day and as single doses of 500, 750, or 1000 mg/kg on GD 9, 10, or 11. Cesarean sections were performed on GD 29, and the fetuses were examined for external, visceral, and skeletal development. RESULTS: In the repeated dose study, maternal toxicity was exhibited in the 250‐ and 350‐mg/kg per day groups by mortality and decreased food consumption and body weight gain. In the single dose studies, maternal toxicity was exhibited at all doses by reductions in body weight gain and food consumption for 3 days after treatment. Fetal body weight was significantly reduced in the repeated dose study at 350 mg/kg per day. Fetal weights were not affected by single doses of ASA on GD 9, 10, or 11. There were no treatment‐related external, visceral, or skeletal malformations associated with ASA administration throughout organogenesis or with single doses administered during critical developmental windows. CONCLUSION: These findings supported previous work demonstrating that ASA is not teratogenic in rabbits, as opposed to rats, even when large doses are administered on single days during specific windows of development. Birth Defects Research (Part B) 68:38–46, 2003. © 2003 Wiley‐Liss, Inc.     

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 (≥12 mg/kg/day) were variable and generally minor. CONCLUSIONS: Artesunate was embryolethal at ≥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. Birth Defects Res (Part B) 83:418–434, 2008. © 2008 Wiley‐Liss, Inc.     

Development in the cynomolgus macaque following administration of ustekinumab,a human anti‐il‐12/23p40 monoclonal antibody,during pregnancy and lactation

BACKGROUND: Ustekinumab is a human monoclonal antibody that binds to the p40 subunit of interleukin (IL) 12 and IL‐23 and inhibits their pharmacological activity. To evaluate potential effects of ustekinumab treatment during pregnancy, developmental studies were conducted in cynomolgus macaques. METHODS: Ustekinumab was tested in two embryo/fetal development (EFD) studies and in a combined EFD/pre and postnatal development (PPND) study. In the EFD studies, pregnant macaques (12/group) were dosed with saline or ustekinumab (9 mg/kg IV, 22.5 mg/kg SC, or 45 mg/kg IV or SC during the period of major organogenesis, gestation day [GD] 20–50). Fetuses were harvested on GD100–102 and examined for any effects on development. In the EFD/PPND study, pregnant macaques were injected with saline or ustekinumab (22.5 or 45 mg/kg SC) from GD20 through lactation day 33. Infants were examined from birth through 6 months of age for morphological and functional development. Potential effects on the immune system were evaluated by immunophenotyping of peripheral blood lymphocytes and immunohistopathology of lymphoid tissues in fetuses and infants and by T‐dependent antibody response (TDAR) to KLH and TTX and by DTH response in infants. Ustekinumab concentrations were measured in serum from dams, fetus, and infants and in breast milk. RESULTS: Ustekinumab treatment produced no maternal toxicity and no toxicity in the fetuses or infants, including no effects on the TDAR or DTH responses. Ustekinumab was present in serum from GD100 fetuses and was present in infant serum through day 120 post‐birth. Low levels of ustekinumab were present in breast milk. CONCLUSIONS: Exposure of macaque fetuses and infants to ustekinumab had no adverse effects on pre‐ and postnatal development. Birth Defects Res (Part B) 89:351–363, 2010. © 2010 Wiley‐Liss, Inc.     

Development of a new version of the Liverpool Malaria Model. I. Refining the parameter settings and mathematical formulation of basic processes based on a literature review

Artesunate (AS) is a clinically versatile artemisinin derivative utilized for the treatment of mild to severe malaria infection. Given the therapeutic significance of AS and the necessity of appropriate AS dosing, substantial research has been performed investigating the pharmacokinetics of AS and its active metabolite dihydroartemisinin (DHA). In this article, a comprehensive review is presented of AS clinical pharmacokinetics following administration of AS by the intravenous (IV), intramuscular (IM), oral or rectal routes. Intravenous AS is associated with high initial AS concentrations which subsequently decline rapidly, with typical AS half-life estimates of less than 15 minutes. AS clearance and volume estimates average 2 - 3 L/kg/hr and 0.1 - 0.3 L/kg, respectively. DHA concentrations peak within 25 minutes post-dose, and DHA is eliminated with a half-life of 30 - 60 minutes. DHA clearance and volume average between 0.5 - 1.5 L/kg/hr and 0.5 - 1.0 L/kg, respectively. Compared to IV administration, IM administration produces lower peaks, longer half-life values, and higher volumes of distribution for AS, as well as delayed peaks for DHA; other parameters are generally similar due to the high bioavailability, assessed by exposure to DHA, associated with IM AS administration (> 86%). Similarly high bioavailability of DHA (> 80%) is associated with oral administration. Following oral AS, peak AS concentrations (Cmax) are achieved within one hour, and AS is eliminated with a half-life of 20 - 45 minutes. DHA Cmax values are observed within two hours post-dose; DHA half-life values average 0.5 - 1.5 hours. AUC values reported for AS are often substantially lower than those reported for DHA following oral AS administration. Rectal AS administration yields pharmacokinetic results similar to those obtained from oral administration, with the exceptions of delayed AS Cmax and longer AS half-life. Drug interaction studies conducted with oral AS suggest that AS does not appreciably alter the pharmacokinetics of atovaquone/proguanil, chlorproguanil/dapsone, or sulphadoxine/pyrimethamine, and mefloquine and pyronaridine do not alter the pharmacokinetics of DHA. Finally, there is evidence suggesting that the pharmacokinetics of AS and/or DHA following AS administration may be altered by pregnancy and by acute malaria infection, but further investigation would be required to define those alterations precisely.     

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. Birth Defects Res (Part B) 83:397–406, 2008. © 2008 Wiley‐Liss, Inc.     

Developmental toxicity of Citrus aurantium in rats

BACKGROUND: Ephedra was commonly used in herbal products marketed for weight loss until safety concerns forced its removal from products. Even before the ban, manufacturers had begun to replace ephedra with other compounds, including Citrus aurantium, or bitter orange. The major component in the bitter orange extract is synephrine which is chemically similar to ephedrine. The purpose of this study was to determine if relatively pure synephrine or synephrine present as a constituent of a bitter orange extract produced developmental toxicity in rats. METHOD: Sprague‐Dawley rats were dosed daily by gavage with one of several different doses of synephrine from one of two different extracts. Caffeine was added to some doses. Animals were sacrificed on GD 21, and fetuses were examined for the presence of various developmental toxic endpoints. RESULTS AND CONCLUSION: At doses up to 100 mg synephrine/kg body weight, there were no adverse effects on embryolethality, fetal weight, or incidences of gross, visceral, or skeletal abnormalities. There was a decrease in maternal weight at 50 mg synephrine/kg body weight when given as the 6% synephrine extract with 25 mg caffeine/kg body weight; there was also a decrease in maternal weight in the caffeine only group. This decrease in body weight may have been due to decreased food consumption which was also observed in these two groups. Overall, doses of up to 100 mg synephrine/kg body weight did not produce developmental toxicity in Sprague‐Dawley rats. Birth Defects Res (Part B) 92:216–223, 2011. © 2011 Wiley‐Liss, Inc.     

Embryotoxic and teratogenic effects of aluminum nitrate in rats upon oral administration

In order to determine the embryotoxic and teratogenic potential of aluminum, pregnant Sprague-Dawley rats were treated by gavage with a daily dose of 180, 360, or 720 mg/kg of aluminum nitrate from the sixth through to the fourteenth day of gestation. Fetal examinations were performed on day 20. The number of corpora lutea, total implants, and resorptions as well as the number of live and dead fetuses in the treated animals were not significantly different from the control group. Therefore, embryolethality of aluminum cannot be induced (as a measure of percent dead and resorbed fetuses). However, exposure of rats to aluminum nitrate resulted in decreased fetal body weight and increased the incidence and types of external, visceral, and skeletal malformations and variations in all the treated groups. Consequently, teratogenic effects of aluminum-nitrate administration may result in rats given high oral doses that induce concomitant maternal toxicity.     

Characterization of the pharmacokinetics of dioscin in rat

Dioscin (diosgenyl 2,4-di-O-alpha-l-rhamnopyranosyl-beta-d-glucopyranoside) is an important constituent of some traditional Chinese medicines with several bioactivities. We have investigated the pharmacokinetics of dioscin in rat after intravenous and oral administrations. Compartmental methods were used to perform pharmacokinetic data analysis. The dose-dependent pharmacokinetics of dioscin was characterized after intravenous administrations (0.064, 0.16, 0.4 and 1.0mg/kg) to rats. There was significant decrease in clearance with increasing dose (4.67+/-0.09 ml/min/kg (0.064 mg/kg) versus 3.49+/-0.23 ml/min/kg (1.0 mg/kg), P<0.05), and the plot of reciprocal clearance values versus the doses was linear (r=0.909, P<0.05). After an I.V. dose of 1mg/kg, simultaneous oral gavage of activated charcoal did not change the pharmacokinetic parameters indicating enterohepatic recycling of dioscin is not important in rat. The absolute oral bioavailability was very low (0.2%). In tissue distribution and bile excretion studies after I.V. and oral administrations, dioscin was shown to undergo a prolonged absorption from the intestinal tract and slow elimination from organs, and only a small amount of drug was recovered in bile. The cumulative amounts of dioscin in feces and urine indicated that the parent drug is mainly excreted in the feces.     

Embryofetal Development Study of Vismodegib,a Hedgehog Pathway Inhibitor,in Rats

Vismodegib (Erivedge) is a first‐in‐class small‐molecule hedgehog pathway inhibitor for the treatment of adults with advanced basal‐cell carcinoma. Because this pathway is known to play key roles in patterning and growth during vertebrate development, vismodegib was anticipated to be embryotoxic. To support marketing applications, an embryofetal development study was completed in which a limited number of pregnant rats (n = 6/group) was administered vismodegib by oral gavage on gestation days 6 to 17. When vismodegib was administered at ≥60 mg/kg/day, doses associated with evidence of pharmacologic activity in previous rat toxicity studies, all conceptuses were resorbed at an early embryonic stage in the absence of significant maternal toxicity. When administered at 10 mg/kg/day, corresponding to an exposure (AUC_0–24h) approximately 15% of the median in patients at steady state, a variety of malformations were observed, including absent/fused digits in the hindlimb of multiple fetuses, multiple craniofacial abnormalities in one fetus, and an anorectal defect in one fetus. In addition, the incidence of variations, including dilated renal pelvis or ureter and incompletely or unossified skeletal elements, was significantly greater when compared with the controls. These results confirmed that vismodegib is likely to be embryotoxic at clinically relevant maternal exposures, and doses ≥60 mg/kg/day resulted in a 100% incidence of embryolethality that likely resulted from severe defects in early embryonic development. In contrast, craniofacial defects typically associated with hedgehog pathway inhibition were only observed in one fetus at the low dose of 10 mg/kg/day, which likely reflected minimal or intermittent pathway inhibition at low exposures.     

Ameltolide. I: Developmental toxicology studies of a novel anticonvulsant

Ameltolide, a novel anticonvulsant agent, has been shown in animal models to be effective in controlling seizures. The developmental toxicity of ameltolide was evaluated in two species. Naturally mated rats and rabbits were dosed once daily by gavage on gestation days (GD) 6-17 and 6-18, respectively. Rats were given doses of 0, 10, 25, or 50 mg/kg; rabbits were given 0, 25, 50, or 100 mg/kg. Laparotomy was performed on rats on GD 20 and on rabbits on GD 28. In rats, maternal toxicity was indicated at the 25- and 50-mg/kg dose levels by depressed body weight gain. Fetal body weight was depressed at the 50-mg/kg dose level. Fetal viability and morphology were not affected. The no-observed effect levels (NOEL) for adult and developmental toxicity in the rat were 10 and 25 mg/kg, respectively. In rabbits, maternal toxicity was indicated by a net loss in body weight at the 50- and 100-mg/kg dose levels. Fetal viability and body weight were depressed at the 100 mg/kg dose level. Shortened digits occurred on the right forepaw of one fetus at the 50-mg/kg dose level (in conjunction with severe maternal toxicity) and on the hindpaws of two fetuses from separate litters at the 100-mg/kg dose level. Incomplete ossification of the phalanges occurred on the forepaws of nine fetuses from four litters at the 100-mg/kg dose level. Ameltolide was weakly teratogenic in the rabbit. The NOEL for adult and developmental toxicity in the rabbit was 25 mg/kg.     

Embryotoxicity studies of norfloxacin in cynomolgus monkeys: I. Teratology studies and norfloxacin plasma concentration in pregnant and nonpregnant monkeys

Norfloxacin, a new orally active antibiotic, was investigated in cynomolgus monkeys for potential developmental toxicity. Fifty-seven monkeys were administered a control vehicle or norfloxacin by nasogastric gavage during the major period of organogenesis on gestational days (GD) 21 through 50 at doses of 0, 50, 100, 150, or 200/300 mg/kg/day. There was no evidence of teratogenicity at any dose level. Maternotoxicity and a significant increase in embryolethality occurred following doses of 200/300 mg/kg/day. The maternotoxicity was not expected based on range-finding studies in nonpregnant female monkeys, which showed no signs of toxicity in doses up to 500 mg/kg/day. Additional studies were conducted to determine if norfloxacin caused similar toxicity later in gestation. Forty-six pregnant monkeys were dosed with a control vehicle or 200 mg/kg/day norfloxacin for one of three 10-day periods on GD 36-45, 71-80, or 111-120. There were no maternotoxic, embryotoxic, or fetotoxic effects observed. Plasma concentrations of norfloxacin in five cynomolgus monkeys following 50 and 200 mg/kg oral doses were not dose-proportionate. However, at a given dose, administered in cross-over fashion, plasma concentrations of norfloxacin were higher in nonpregnant females (approximately 20-40%) than during pregnancy when the same subject was compared. At the no-observed-effect dose for maternal and embryotoxicity (50 mg/kg), peak plasma concentrations of norfloxacin in pregnant cynomolgus monkeys are approximately threefold higher than those observed in human volunteers receiving norfloxacin at the maximum recommended therapeutic dose of 400 mg (5.7 mg/kg based on 70 kg body weight) twice per day.     

Developmental toxicity evaluation of sodium thioglycolate administered topically to Sprague-Dawley (CD) rats and New Zealand White rabbits

BACKGROUND: Sodium thioglycolate, which has widespread occupational and consumer exposure to women from cosmetics and hair‐care products, was evaluated for developmental toxicity by topical exposure during the embryonic and fetal periods of pregnancy METHODS: Timed‐mated Sprague–Dawley rats (25/group) and New Zealand White (NZW) rabbits (24/group) were exposed to sodium thioglycolate in vehicle (95% ethanol:distilled water, 1:1) by unoccluded topical application on gestational days (GD) 6–19 (rats) or 6–29 (rabbits) for 6 hr/day, at 0, 50, 100, or 200 mg/kg body weight/day (rats) and 0, 10, 15, 25, or 65 mg/kg/day (rabbits). At termination (GD 20 rats; GD 30 rabbits), fetuses were examined for external, visceral, and skeletal malformations and variations. RESULTS: In rats, maternal topical exposure to sodium thioglycolate, at 200 mg/kg/day (the highest dose tested) on GD 6–19, resulted in maternal toxicity, including reduced body weights and weight gain, increased relative water consumption and one death. Treatment‐related increases in feed consumption and changes at the application site occurred at all doses, in the absence of increased body weights or body weight change. Fetal body weights/litter were decreased at 200 mg/kg/day, with no other embryo/fetal toxicity and no treatment‐related teratogenicity in any group. In rabbits, maternal topical exposure to sodium thioglycolate on GD 6–29 resulted in maternal dose‐related toxicity at the dosing site in all groups; no maternal systemic toxicity, embryo/fetal toxicity, or treatment‐related teratogenicity were observed in any group. CONCLUSIONS: A no observed adverse effect level (NOAEL) was not identified for maternal toxicity in either species with the dosages tested. The developmental toxicity NOAEL was 100 mg/kg/day (rats) and ≥65 mg/kg/day (rabbits; the highest dose tested). The clinical relevance of theses study results is uncertain because no data were available for levels, frequency, or duration of exposures in female workers or end users. Birth Defects Research Part B 68:144–161, 2003. © 2003 Wiley‐Liss, Inc.     

Developmental Toxicity of the HMG‐CoA Reductase Inhibitor (PPD10558) in Rats and Rabbits

PPD10558 is an orally active, lipid‐lowering 3–hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitor (statin) being developed as a treatment for hypercholesterolemia in patients who have not been able to tolerate statins because of statin‐associated myalgia. We have studied the potential developmental toxicity effects of PPD10558 in pregnant rats and rabbits given daily oral doses during the period of organogenesis. Rats were dosed with 0, 20, 80, or 320 mg/kg/day from Gestation Day (GD) 6 to 17 and rabbits received dose levels of 0, 12.5, 25, or 50 mg/kg/day from GD 6 to 18. Additional groups in both studies served as toxicokinetic animals and received the PPD10558 in the same manner as the main study groups at the same dose levels. Blood samples were collected from toxicokinetic animals at designated time points on GD 6 and 17 in rats and GD 6 and 18 in rabbits. Fetal exposure in rats was assessed on GD 20. Maternal and developmental parameters were evaluated in rats and rabbits on GD 20 and GD 29, respectively. No maternal and developmental toxicity was observed at any of the dose levels used in the rat study. Evidence of fetal exposure was determined in fetal plasma with mean fetal concentrations of PPD10558 and the metabolite (PPD11901) found to be between 1 and 6% of the mean maternal concentrations. In rabbits, marked maternal toxicity including mortality (eight deaths; 1 dose at 25 and 7 at 50 mg/kg/day), abortions (2 at 25 mg/kg/day and 6 at 50 mg/kg/day) and reduction in gestation body weight, gestation body weight changes and decreased food consumption were observed. In addition, fetal body weights of the combined sexes were significantly reduced at 50 mg/kg/day in comparison with the controls. Mean peak exposure (Cmax) and total exposure (AUC(0–24)) of PPD11901 in both rats and rabbits were higher than that of PPD10558 on GD 6 and GD 17 at each of the three dose levels.. Based on the results of these studies, the no observed adverse effect level (NOAEL) for maternal and developmental toxicity in rats was considered to be ≥320 mg/kg/day, the highest dose level used in the study. The NOAEL for maternal and developmental toxicity in rabbits was 12.5 mg/kg/day and 25 mg/kg/day, respectively.     

Teratogenicity of all-trans retinoic acid during early embryonic development in the cynomolgus monkey (Macaca fascicularis).

The embryotoxic and teratogenic potential of all-trans retinoic acid was assessed following exposure prior to and during early organogenesis in the cynomolgus monkey (Macaca fascicularis). Sixteen pregnant females were orally administered all-trans retinoic acid (Tretinoin, Hoffmann-La Roche) once daily from GD 10-20 and twice daily from GD 21-24 at three different dosages, 5 (n = 9), 10 (n = 6) and 20 mg/kg (n = 1). Adverse clinical signs resembling hypervitaminosis A were observed in one animal at 5 mg/kg, in three animals at 10 mg/kg, and in the animal treated with 20 mg/kg all-trans retinoic acid. Maternal weight loss was observed in the 10- and 20-mg/kg groups. A dose-dependent increase in embryolethality was observed, with 22% (2/9), 50% (3/6), and 100% (1/1) occurring at 5, 10, and 20 mg/kg, respectively. The majority of embryonic deaths occurred between GD 16 and 20; the incidence of these early losses was higher than in historical and concurrent controls. No malformations, but a single growth-retarded fetus, was observed in the 5-mg/kg group. Craniofacial malformations, consisting of external ear defects, mandibular hypoplasia, cleft palate, and temporal bone abnormalities, were seen in three viable fetuses in the 10-mg/kg group. Skeletal variations were common to the majority (70%, 7/10) of viable fetuses in both dose groups and were increased relative to historical controls (32%, 25/77). Unlike previous studies with 13-cis-retinoic acid during the pre- and early organogenic stages of development (Hummler et al., Teratology 42:263-272, 1990), no thymic hypo- or aplasia or heart anomalies were observed, which may be attributable to the slightly longer 13-cis retinoic acid treatment period, i.e., GD 10-27. However, external ear and temporal bone defects were common to both all-trans and 13-cis retinoic acid. The similarity observed in the malformation syndrome induced by both all-trans and 13-cis retinoic acid in the cynomolgus monkey and 13-cis retinoic acid embryopathy in humans supports this macaque species as a model for further developmental toxicity studies of vitamin A-related compounds.     

Effects of CNTO 530, an erythropoietin mimetic‐IgG4 fusion protein,on embryofetal development in rats and rabbits

BACKGROUND: CNTO 530is a biopharmaceutical consisting of a novel peptide that mimics the actions of erythropoietin, fused to the Fc fragment of human IgG4. Pharmacokinetic and pharmacodynamic studies showed that CNTO 530 produced sustained increases in red blood cell parameters in rats and rabbits and that the serum half life of CNTO 530 was 2 days in rabbits and 3 days in rats. METHODS: For the evaluation of embryofetal development, CNTO 530 was injected at loading doses of 0, 0.9/1, 6, or 60 mg/kg subcutaneously (SC) on gestation day (GD)7 followed by maintenance doses of 0, 0.3, 2, or 20 mg/kg SC every 3 days through GD16 in rats and every 2 days through GD19 in rabbits (GD0 was the day of mating). Rats were Caesarean sectioned on GD21, rabbits on GD29. RESULTS: Administration of CNTO 530 was associated with an increase in hematocrit at all dose levels and a decrease in maternal body weight gains. Fetuses exhibited reduced body weight and delayed ossification. Soft tissue changes were limited to cardiovascular alterations in the high‐dose rabbits only. Rat and rabbit fetuses were exposed to CNTO 530 in all dose groups. CONCLUSIONS: These studies show that the embryo/fetal development effects observed following CNTO 530 treatment during organogenesis are qualitatively similar to those seen with other erythropoietin agonists and are likely a secondary consequence of increased hematocrit in the dams. Unlike other erythropoietin receptor agonists, CNTO 530 was able to cross the placental barrier, which was considered likely the result of FcRn‐mediated transcytosis. Birth Defects Res (Part B) 89:87–96, 2010. © 2010 Wiley‐Liss, Inc.     

Induction of malformations in the cynomolgus monkey with 13-cis retinoic acid

The embryotoxic and teratogenic potential of 13-cis retinoic acid was assessed in the cynomolgus macaque (Macaca fascicularis). A total of 41 animals was orally administered 13-cis retinoic acid in four sequential experiments. In Exp. 1 three dose levels, 2, 10, and 25 mg/kg, were administered on gestational day (GD) 18-28; 5 mg/kg was administered as an equally divided dose twice daily in Exp. 2 and 3 on GD 21-24 and on GD 25-27, respectively; in Exp. 4 the drug was administered at 2.5 mg/kg once daily from GD 10 to 25 and twice daily (2 x 2.5 mg/kg) on GD 26 and 27. Maternal death and toxicity, manifested as reduction in maternal weight and food consumption, and diarrhea, was observed in Exp. 1 in all three dose groups. No significant maternal toxicity was observed in the treatment groups in Exp. 2, 3, and 4 or in the control group. The primary manifestation of developmental toxicity was embryolethality in Exp. 1 and 2. The incidence of embryonic deaths in Exp. 3 was comparable to the historical controls. No malformations in GD 100 fetuses were observed in Exp. 1, 2, and 3. In Exp. 4, five of seven fetuses (71%) had malformations of both external ears, four of seven fetuses (57%) exhibited hypo- or aplasia of the thymus, and two of seven (29%) had malformations (transposition of the great vessels, ventricular septal defect) of the heart. The teratogenic dose for the cynomolgus monkey in the present study was lower than that reported for all other experimental species. Although central nervous system and craniofacial defects were not observed, the incidence of ear and thymus defects was similar to that reported for the human. The cardiovascular defects resembled those reported clinically, but the incidence was lower in the cynomolgus monkey. The similarity in teratogenic sensitivity to humans supports the use of the monkey as a model for developmental toxicity studies of vitamin A-related compounds.     

Postnatal closure of membranous ventricular septal defects in Sprague-Dawley rat pups after maternal exposure with trimethadione

BACKGROUND: Congenital membranous ventricular septal defects (VSD) have been shown to close during postnatal development in rats [Solomon et al., Teratology 55:185-194, 1997]. Although they may differ in size, spontaneous and treatment-related VSD are histologically similar; however, the postnatal fate of treatment-induced VSD is not known. The objective of this study was to determine if treatment-induced VSD persist throughout postnatal development. METHODS: Groups of 40 female rats were given oral doses of trimethadione (TMD) at 400 mg/kg/day (200 b.i.d.) or 600 mg/kg/day (300 b.i.d.) on Gestation Days (GD) 9 and 10. Twenty dams in each group were designated for Cesarean section and 20 were allowed to deliver and rear their offspring to Postnatal Day (PND) 21. The integrity of the ventricular septum was evaluated in fetuses (GD 21) and pups (PND 21). RESULTS: The incidence of membranous VSD was 0.6, 7.6, and 49.8% per litter in the Control, 400, and 600 mg/kg groups, respectively, on GD 21. Both the incidence and severity of VSD increased with dose. The VSD at 400 mg/kg were small in size and initially detected by the presence of blood flowing through the defect from the closed right ventricle. In the 600 mg/kg dose group, the VSD, although still membranous, were larger and more readily detected without the need to examine the blood flow. At 600 mg/kg, not only were the VSD larger than those in the Control or the 400 mg/kg group, 10.1% per litter of the affected fetuses had other vessel anomalies associated with the VSD, which were incompatible with pup survival. On PND 21, VSD was noted in 0.3, 0, and 6.4% per litter evaluated in the Control, 400, and 600 mg/kg groups, respectively. This demonstrates that the small, isolated treatment-related VSD can resolve postnatally; however, the closure of the larger or more severe VSD may be prolonged or may not occur at all. Although TMD exposure reduced group mean fetal weights at both dose levels, there was no difference between the mean weight of fetuses with VSD and those fetuses without VSD in the same group. CONCLUSION: Treatment-induced VSD close postnatally, and appears to be a delay in cardiac development not associated with fetal weight. The timing of closure and survivability during closure is dependent on the severity of the VSD. Further characterization of the two sizes of VSD may provide diagnostic clarity; however, the current data support the smaller VSD as a variation with no significant impact on viability and growth, and the more severe VSD to be a malformation.     

Neuroembryopathic effect of trichloroacetic acid in rats exposed during organogenesis

BACKGROUND: Halogenated hydrocarbons such as trichloroacetic acid (TCA) are among the most common water supply contaminants in the world. This study examines the effect of TCA on the developing brain of the Charles Foster rat. METHODS: Adult pregnant rats were placed in the test group and exposed to various concentration of TCA (i.e., 1000, 1200, 1400, 1600, and 1800 mg/kg body weight [b.w.]) by oral gavage throughout the period of organogenesis from Gestation Day (GD) 6-15 of gestation. Trichloroacetic acid was administered in the form of trichloroacetate, which is reduced to TCA in the body. The control mother rats were administered an equal volume of distilled water. Fetal brains were examined for their external and histological malformation. RESULTS: On GD 19, TCA administration led to an initial increase of brain weight at 1000 mg/kg b.w. and then a weight reduction after TCA doses of 1200 mg/kg b.w. and over. The brain of the formalin-fixed fetuses at 1000 and 1200 mg/kg b.w. showed hydrocephalus with breech of the ependymal lining, altered choroids plexus architecture, and increased apoptosis. At doses of 1400 mg/kg b.w. and above, the brain showed not only enhanced apoptosis of the neuronal cells, but extravasation of erythrocytes within the cortical parenchyma, vacuolation of the neuropil, and multiple cavity formation. CONCLUSION: With an increase in dose of TCA i.e., 1200 mg/kg b.w. and above, there is enhanced apoptosis, leading to increased neuronal death, which consequently led to the reduction in the brain weight as compared to controls. The fetal central nervous system is susceptible to the toxic effect of TCA.     

Inhibiting matrix metalloproteinases with prinomastat produces abnormalities in fetal growth and development in rats

BACKGROUND: Matrix metalloproteinases (MMPs) play key roles in remodeling of the extracellular matrix during embryogenesis and fetal development. The objective of this study was to determine the effects of prinomastat, a potent selective MMP inhibitor, on fetal growth and development. METHODS: Prinomastat (25, 100, 250 mg/kg/day, p.o.) was administered to pregnant female Sprague-Dawley rats on gestational days (GD) 6-17. A Cesarian section was carried out on GD 20 and the fetuses were evaluated for viability and skeletal and soft tissue abnormalities. RESULTS: Prinomastat treatment at the 250 mg/kg/day dose produced a decrease in body weight and food consumption in the dams. A dose-dependent increase in post-implantation loss was observed in the 100 and 250 mg/kg/day-dose groups, resulting in only 22% of the dams having viable litters for evaluation at the 250 mg/kg/day dose. Fetal skeletal tissue variations and malformations were present in all prinomastat treated groups and their frequency increased with dose. Variations and malformation in fetal soft tissue were also increased at the 100 and 250 mg/kg/day doses. Prinomastat also interfered with fetal growth of rat embryo cultures in vitro. CONCLUSIONS: These data confirm that MMP inhibition has a profound effect on fetal growth and development in vivo and in vitro.