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Temperatures of 32.5 °C and above impact zebrafish embryonic development
14
Abstracts / Reproductive Toxicology 48 (2014) 12–14
periconceptional period. Several such actions are often sparse and with limited co-ordination at national level. The European recommendations aim at exploiting the integration added value of current actions; so to provide a comprehensive tool for implementing primary prevention into national policies on rare diseases in Europe.
Reference [1] Recommendations on policies to be considered for the primary prevention of congenital anomalies in National Plans and Strategies on Rare Diseases. http://www.eucerd.eu/wp-content/uploads/2013/03/Eurocat Reco Primary Prevention.pdf.
that temperatures at 32.5 ◦ C and above not only elicit malformations in more embryos, but they also cause more malformations in each individual. As such, a higher incidence of tail malformations and oedema of the head was observed at 32.5 ◦ C compared with controls, whereas several other parameters showed an increase at 34.5 ◦ C. At 36.5 ◦ C, almost each parameter was statistically different from control embryos. These results indicate that a temperature of 32.5 ◦ C, which is currently used in a co-incubation system with zebrafish embryos, is too high and affects embryonic development of the zebrafish. http://dx.doi.org/10.1016/j.reprotox.2014.07.006
http://dx.doi.org/10.1016/j.reprotox.2014.07.005
F-6
F-5 Temperatures of 32.5 ◦ C and above impact zebrafish embryonic development
Developmental toxicity assessment of tanezumab, an anti-nerve growth factor monoclonal antibody, in Cynomolgus monkeys (Macaca fascicularis)
Casper Pype 1,∗ , Evy Verbueken 1 , Moayad A. Saad 1 , Els M. Van Peer 1 , Christophe R. Casteleyn 1 , Chris J. Van Ginneken 1 , Dries Knapen 2 , Steven J. Van Cruchten 1
Christopher J. Bowman 1,∗ , Mark Evans 2 , Thomas Cummings 1 , Satoru Oneda 3 , Mark Butt 4 , Susan Hurst 1 , Jessica-lyn Gremminger 1 , David Shelton 5 , Cris Kamperschroer 1 , Mark Zorbas 2
1 University of Antwerp, Applied Veterinary Morphology, Universiteitsplein 1, 2610 Wilrijk, Belgium 2 University of Antwerp, Veterinary Physiology and Biochemistry, Universiteitsplein 1, 2610 Wilrijk, Belgium
1
Zebrafish (Danio rerio) embryos are currently used for screening of teratogenicity during drug development. However, as the embryos develop externally, there is no maternal metabolisation of (pro-)drugs. Consequently, potential (pro-)teratogens may be missed if the biotransformation processes of the embryo are still immature. There are indeed indications that the zebrafish embryo lacks or shows only very low CYP activity. Therefore, several research groups suggest the use of an exogenous metabolic activation system (MAS). Commonly rat liver microsomes (rMAS) are used as a co-incubation system but they are embryotoxic for the zebrafish embryo. The temperature (i.e. 32 ◦ C) that is being used for co-incubation of zebrafish embryos with rMAS may contribute to the observed embryotoxicity. A temperature of 32 ◦ C was arbitrarily chosen as a compromise to still have (1) sufficient biotransformation by rMAS and (2) minimal to no malformations in developing embryos. However, as this temperature is higher than the normal temperature (ca. 28.5 ◦ C) at which zebrafish embryos develop, morphological malformations and even embryonic death might occur. Therefore, the aim of this study was to assess the morphological effect(s) of increased temperature on the embryonic development of zebrafish. Each experiment consisted of 96 control and 96 test embryos divided in 48-well plates and the experiments were repeated (in a different week). Embryos were kept in standardized conditions, implying a 14/10 h light/darkcycle. The control embryos were incubated at 28.5 ◦ C, whereas the test embryos were incubated at 30.5 ◦ C, 32.5 ◦ C, 34.5 ◦ C or 36.5 ◦ C. All embryos were scored for coagulation and several morphological parameters such as hatching, oedema, deviations of the tail and malformations of the cardiovascular system at 6 time points during organogenesis (ranging from 51/4 h post fertilisation (hpf) – 96 hpf). In general, a statistically higher incidence of embryos showing at least one malformation was observed at temperatures from 32.5 ◦ C to 36.5 ◦ C, reaching 100% of affected embryos at 36.5 ◦ C at 96 hpf. Not only the total number of affected embryos increased with temperatures from 32,5 ◦ C onwards, but also the total number of malformations/embryo was higher. This indicates
Pfizer, Inc., Groton, CT, USA Pfizer Inc., San Diego, CA, USA 3 SNBL USA, Ltd., Everett, WA, USA 4 Tox Path Specialists, Frederick, MD, USA 5 Pfizer, Inc., South San Francisco, CA, USA 2
Introduction: Tanezumab, a humanized monoclonal antibody against nerve growth factor, is being investigated for treatment of chronic pain. Methods: Two studies were completed to assess effects of tanezumab on pregnancy and pre- and postnatal development of the offspring. In Study 1, intravenous tanezumab (0, 0.5, 4, or 30 mg/kg; 18 per group) was administered weekly to pregnant cynomolgus monkeys from early organogenesis (gestation day 20) through parturition, with infants examined up to ∼12 months of age. In Study 2, pregnant cynomolgus monkeys received intravenous tanezumab (0, 0.5, or 30 mg/kg; 20–21 per group) weekly from gestation day 20 through the end of organogenesis (gestation day 48), with infants examined up to 2 months of age. Infant evaluations included: clinical signs, external morphology, body weight, heart rate, clinical pathology, immunophenotyping, T-cell dependent antibody response, skeletal development, heart rate, behavior, learning and memory, and gross and microscopic evaluation (including morphologic, stereologic and morphometric evaluation). Plasma/serum and milk were analyzed for exposure and anti-drug antibodies. Results: In maternal animals, no tanezumabrelated changes were identified, including no effect on pregnancy maintenance (abortion/in utero embryo-fetal death), body weight, and gestation length. Notably, maternal tanezumab administration increased stillbirth and post-birth infant mortality/morbidity, and decreased infant growth. In addition, microscopic changes in the peripheral sympathetic and sensory nervous system of the infants at all doses in the absence of any changes in neurobehavior, function, or cognitive ability. Decreased primary antibody responses and increased incidences in skin changes in infants were also observed following tanezumab exposure at ≥4 mg/kg. No other tanezumab-related effects were noted. Conclusions: The noobserved-adverse-effect-level for maternal toxicity was 30 mg/kg and <0.5 mg/kg for developmental toxicity based on increased stillbirth and post-birth infant mortality/morbidity, decreased infant growth, and microscopic changes in the peripheral sympathetic and sensory nervous system of the infants. http://dx.doi.org/10.1016/j.reprotox.2014.07.007
Abstracts / Reproductive Toxicology 48 (2014) 12–14
periconceptional period. Several such actions are often sparse and with limited co-ordination at national level. The European recommendations aim at exploiting the integration added value of current actions; so to provide a comprehensive tool for implementing primary prevention into national policies on rare diseases in Europe.
Reference [1] Recommendations on policies to be considered for the primary prevention of congenital anomalies in National Plans and Strategies on Rare Diseases. http://www.eucerd.eu/wp-content/uploads/2013/03/Eurocat Reco Primary Prevention.pdf.
that temperatures at 32.5 ◦ C and above not only elicit malformations in more embryos, but they also cause more malformations in each individual. As such, a higher incidence of tail malformations and oedema of the head was observed at 32.5 ◦ C compared with controls, whereas several other parameters showed an increase at 34.5 ◦ C. At 36.5 ◦ C, almost each parameter was statistically different from control embryos. These results indicate that a temperature of 32.5 ◦ C, which is currently used in a co-incubation system with zebrafish embryos, is too high and affects embryonic development of the zebrafish. http://dx.doi.org/10.1016/j.reprotox.2014.07.006
http://dx.doi.org/10.1016/j.reprotox.2014.07.005
F-6
F-5 Temperatures of 32.5 ◦ C and above impact zebrafish embryonic development
Developmental toxicity assessment of tanezumab, an anti-nerve growth factor monoclonal antibody, in Cynomolgus monkeys (Macaca fascicularis)
Casper Pype 1,∗ , Evy Verbueken 1 , Moayad A. Saad 1 , Els M. Van Peer 1 , Christophe R. Casteleyn 1 , Chris J. Van Ginneken 1 , Dries Knapen 2 , Steven J. Van Cruchten 1
Christopher J. Bowman 1,∗ , Mark Evans 2 , Thomas Cummings 1 , Satoru Oneda 3 , Mark Butt 4 , Susan Hurst 1 , Jessica-lyn Gremminger 1 , David Shelton 5 , Cris Kamperschroer 1 , Mark Zorbas 2
1 University of Antwerp, Applied Veterinary Morphology, Universiteitsplein 1, 2610 Wilrijk, Belgium 2 University of Antwerp, Veterinary Physiology and Biochemistry, Universiteitsplein 1, 2610 Wilrijk, Belgium
1
Zebrafish (Danio rerio) embryos are currently used for screening of teratogenicity during drug development. However, as the embryos develop externally, there is no maternal metabolisation of (pro-)drugs. Consequently, potential (pro-)teratogens may be missed if the biotransformation processes of the embryo are still immature. There are indeed indications that the zebrafish embryo lacks or shows only very low CYP activity. Therefore, several research groups suggest the use of an exogenous metabolic activation system (MAS). Commonly rat liver microsomes (rMAS) are used as a co-incubation system but they are embryotoxic for the zebrafish embryo. The temperature (i.e. 32 ◦ C) that is being used for co-incubation of zebrafish embryos with rMAS may contribute to the observed embryotoxicity. A temperature of 32 ◦ C was arbitrarily chosen as a compromise to still have (1) sufficient biotransformation by rMAS and (2) minimal to no malformations in developing embryos. However, as this temperature is higher than the normal temperature (ca. 28.5 ◦ C) at which zebrafish embryos develop, morphological malformations and even embryonic death might occur. Therefore, the aim of this study was to assess the morphological effect(s) of increased temperature on the embryonic development of zebrafish. Each experiment consisted of 96 control and 96 test embryos divided in 48-well plates and the experiments were repeated (in a different week). Embryos were kept in standardized conditions, implying a 14/10 h light/darkcycle. The control embryos were incubated at 28.5 ◦ C, whereas the test embryos were incubated at 30.5 ◦ C, 32.5 ◦ C, 34.5 ◦ C or 36.5 ◦ C. All embryos were scored for coagulation and several morphological parameters such as hatching, oedema, deviations of the tail and malformations of the cardiovascular system at 6 time points during organogenesis (ranging from 51/4 h post fertilisation (hpf) – 96 hpf). In general, a statistically higher incidence of embryos showing at least one malformation was observed at temperatures from 32.5 ◦ C to 36.5 ◦ C, reaching 100% of affected embryos at 36.5 ◦ C at 96 hpf. Not only the total number of affected embryos increased with temperatures from 32,5 ◦ C onwards, but also the total number of malformations/embryo was higher. This indicates
Pfizer, Inc., Groton, CT, USA Pfizer Inc., San Diego, CA, USA 3 SNBL USA, Ltd., Everett, WA, USA 4 Tox Path Specialists, Frederick, MD, USA 5 Pfizer, Inc., South San Francisco, CA, USA 2
Introduction: Tanezumab, a humanized monoclonal antibody against nerve growth factor, is being investigated for treatment of chronic pain. Methods: Two studies were completed to assess effects of tanezumab on pregnancy and pre- and postnatal development of the offspring. In Study 1, intravenous tanezumab (0, 0.5, 4, or 30 mg/kg; 18 per group) was administered weekly to pregnant cynomolgus monkeys from early organogenesis (gestation day 20) through parturition, with infants examined up to ∼12 months of age. In Study 2, pregnant cynomolgus monkeys received intravenous tanezumab (0, 0.5, or 30 mg/kg; 20–21 per group) weekly from gestation day 20 through the end of organogenesis (gestation day 48), with infants examined up to 2 months of age. Infant evaluations included: clinical signs, external morphology, body weight, heart rate, clinical pathology, immunophenotyping, T-cell dependent antibody response, skeletal development, heart rate, behavior, learning and memory, and gross and microscopic evaluation (including morphologic, stereologic and morphometric evaluation). Plasma/serum and milk were analyzed for exposure and anti-drug antibodies. Results: In maternal animals, no tanezumabrelated changes were identified, including no effect on pregnancy maintenance (abortion/in utero embryo-fetal death), body weight, and gestation length. Notably, maternal tanezumab administration increased stillbirth and post-birth infant mortality/morbidity, and decreased infant growth. In addition, microscopic changes in the peripheral sympathetic and sensory nervous system of the infants at all doses in the absence of any changes in neurobehavior, function, or cognitive ability. Decreased primary antibody responses and increased incidences in skin changes in infants were also observed following tanezumab exposure at ≥4 mg/kg. No other tanezumab-related effects were noted. Conclusions: The noobserved-adverse-effect-level for maternal toxicity was 30 mg/kg and <0.5 mg/kg for developmental toxicity based on increased stillbirth and post-birth infant mortality/morbidity, decreased infant growth, and microscopic changes in the peripheral sympathetic and sensory nervous system of the infants. http://dx.doi.org/10.1016/j.reprotox.2014.07.007