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Safety consideration of nanomaterials for biomedical applications
Abstracts / Toxicology Letters 221S (2013) S4–S30
with respect to supra-pharmacological or target related side effects and may lead in the discovery phase to better decision making processes. On the other hand, data generated with those models need to be interpreted carefully in the context of translation into man and due to the low number of historical control data. In 2003, Boelsterli suggested that tailor-made and simplified models of human disease could be conducted in satellite toxicity studies to facilitate candidate selection, help predict rare and unexpected toxicity, and identify new biomarkers. Approximately 10 years later, Morgan et al. (2013) published several examples and a proposal how to use carefully those models. In line with this publication, it is suggested that testing in an animal model of human disease should only be taken after adequate consideration of relevance along with validities, benefits and limitations of the proposed models.
References Boelsterli, U.A., 2003. Animal models of human disease in drug safety assessment. Journal of Toxicological Sciences 28, 109–121. Morgan, S.J., Elangbam, C.S., Berens, S., Janovitz, E., Vitsky, A., Zabka, T., Conour, L., 2013. Use of animal models of human disease for non-clinical safety assessment of novel pharmaceuticals. Toxicologic Pathology 41 (3), 508–518.
http://dx.doi.org/10.1016/j.toxlet.2013.06.017
S01-5 Early safety assessment of biologicals Jennifer Sims Integrated Biologix GmbH, Basel, Switzerland Although industry’s large molecule success rates in Development continue to be significantly higher than that of small molecules, the success rates for large molecules have recently declined. About 17% of large molecules in Phase I are calculated to reach market, compared to about 4% of small molecules (KMR Pharmaceutical Benchmarking Forum, 2012). The issues that impact attrition in discovery and early development will be discussed in relation to protein therapeutics, using illustrative case examples. In recent years an increased emphasis has been placed on optimal molecular design for appropriate PK-PD characteristics to maximize the probability of engaging the target(s),and improving developability assessments aimed at delivering a high quality drug product. More emphasis still needs to be placed on a better understanding of target biology (e.g.target expression, turnover, receptor internalization, shed receptor, endogenous binding proteins), signaling pathways impacted, mechanism of action in normal and disease states, and comparative biology across animal species used for PK-PD and safety assessment to human volunteers and patients. Some biologics such as monoclonal antibodies and related products show very high target binding selectivity. This high target binding selectivity leads to high species specificity with respect to binding of the drug candidate to the target, even to the point that sequence polymorphisms between different strains of cynomolgus macaques may render one strain as not relevant for safety assessment due to lack of target binding by the drug candidate. Other differences between humans and animal species such as differences in Fc? receptors may also impact on the predictive value for humans of safety evaluation in animals. It is important to have a good understanding of the comparative biology issues which may result in limited predictive value of animal data and, where possible, to address any gaps with in vitro data in human systems e.g. assessment of potential for cytokine release in human blood/PBMCs.
S5
Another issue to address early in development is an immunogenicity risk assessment strategy, particularly the potential for any serious safety issues which may result from the development of neutralizing anti-drug-antibodies cross-reactive to endogenous proteins. Predictive immunogenicity approaches are being used more frequently as more novel engineered “less human” constructs and protein scaffolds enter development. Examples and limitations of predictive immunogenicity approaches will be discussed. http://dx.doi.org/10.1016/j.toxlet.2013.06.221 Symposium 2: Recent developments in risk assessment of nanomaterials and nano safety science
S02-1 Nanomaterials as a potential cause of lung disease James C. Bonner North Carolina State University, Raleigh, NC, USA The nanotechnology revolution offers enormous societal and economic benefits for innovation in the fields of engineering, electronics, and medicine. Nevertheless, evidence from rodent inhalation studies show that biopersistent engineered nanomaterials, including carbon nanotubes and metal nanoparticles, have the potential to stimulate immune, inflammatory, or fibroproliferative responses in the lung and pleura. These data suggest possible risks for pulmonary fibrosis or the development of pleural disease as a consequence of occupational or consumer exposure. Some engineered nanomaterials also exacerbate pre-existing allergeninduced inflammation by altering the balance of distinct T-helper cell phenotypes, suggesting that they could serve as sensitizers or adjuvants to alter the innate immune response. These findings suggest that individuals with asthma or other pre-existing respiratory diseases would be particularly susceptible to the adverse health effects of nanomaterials. Due to their nanoscale dimensions and increased surface area per unit mass, engineered nanomaterials have a much greater potential to reach the distal regions of the lung, generate reactive oxygen species, and alter cell signaling pathways linked to disease pathogenesis. The goal of this presentation will be to discuss mechanisms through which engineered nanomaterials cause lung, airway, and pleural disease, especially in the context of susceptible individuals with pre-existing disease. Functionalization of nanomaterials through processes such as atomic layer deposition will also be discussed as a means of altering the pathogenicity of nanomaterials. http://dx.doi.org/10.1016/j.toxlet.2013.06.019
S02-2 Safety consideration of nanomaterials for biomedical applications Chunying Chen 1,∗ , Ying Liu 1 , Liming Wang 2 , Yufeng Li 2 1 CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing China, 2 Institute of High Energy Physics, Beijing China
Many nanomaterials are promising in biological detection, diagnosis, and therapy for diseases and have shown great potential for biomedical applications. Therefore, the toxicity of nanomaterials becomes an increasing concern. Both in vitro and in vivo studies were applied to evaluate biological consequence of nanomaterials.
S6
Abstracts / Toxicology Letters 221S (2013) S4–S30
The underlying mechanisms were investigated including induction of oxidative stress, inflammation and autophagy (Li et al., 2012; Liu et al., 2013; Zhao et al., 2008). The intrinsic physicochemical properties of nanomaterials have decisive influence on their biological consequences and toxicity. These properties include size, shape, surface charge, chemical composition, surface modification, metal impurities, agglomeration and dispersion, degradation, as well as the formation of “protein corona” (Qiu et al., 2010; Wang et al., 2011; Zhao et al., 2011). It is important to obtain a better understanding of the uptake, trafficking, pharmacokinetics, clearance, and role of nanomaterials in biological systems, so that their possible undesirable effects can be avoided. Chemical speciation, dynamics and kinetics of nanomaterials in biological systems are extremely necessary since we have very limited knowledge. The state-of-the art analytical techniques are playing important roles in the study of nanotoxicology and nanobiology by taking advantages of absolute quantification, high sensitivity, excellent accuracy and precision, low matrix effects and non-destructiveness (Li and Chen, 2011). For example, the combination of -SRXRF and microbeam X-ray absorbance near edge structure (-XANES) can simultaneously provide information about the subcellular distribution and chemical species of metalcontaining nanomaterials of interest (Qu et al., 2011).
antioxidant balance, and quantitative morphometry (including collagen) in wild-type and genetically manipulated mice, we were able to reveal the major pathways through which CNT – in doses relevant to potential occupational exposures – exert their toxic effects in the lung/distant organs of exposed animals. Overall, found an unusual and robust inflammatory and fibrogenic response is closely associated with the progression of oxidative stress in the lung. Because realistic exposures to SWCNT are likely to occur in conjunction with other pathogenic influences, e.g., microbial infections, our finding of compromised bacterial clearance in the lungs of CNTexposed mice are of great practical importance. The talk will also address important issues of comparative respiratory outcomes of CNTs and asbestos, particularly with regards to pulmonary injury and potential carcinogenicity. Finally, the mechanisms of toxicity will be discussed in the context of current regulations of protection and their sufficiency in environmental and occupational settings. Supported by NIH008282, NORA700Y, 7ZKCY.
References
Wolfgang G. Kreyling
Li, Y., Chen, C., 2011. Fate and toxicity of metallic and metal-containing nanoparticles for biomedical applications. Small 7 (21), 2965–2980. Li, Y., Liu, Y., Fu, Y., Wei, T., Le Guyader, L., Gao, G., Liu, R.S., Chang, Y.Z., Chen, C., 2012. The triggering of apoptosis in macrophages by pristine graphene through the MAPK and TGF-beta signaling pathways. Biomaterials 33, 402–411. Liu, Y., Zhao, Y., Sun, B., Chen, C., 2013. Understanding the toxicity of carbon nanotubes. Accounts of Chemical Research 46 (3), 702–713. Qiu, Y., Liu, Y., Wang, L.M., Xu, L.G., Bai, R., Ji, Y.L., Wu, X.C., Zhao, Y.L., Li, Y.F., Chen, C.Y., 2010. Surface chemistry and aspect ratio mediated cellular uptake of Au nanorods. Biomaterials 31, 7606–7619. Qu, Y., Li, W., Zhou, Y.L., Liu, X.F., Zhang, L.L., Wang, L.M., Li, Y.F., Iida, A., Tang, Z.Y., Zhao, Y.L., Chai, Z.F., Chen, C.Y., 2011. Full assessment of fate and physiological behavior of quantum dots utilizing Caenorhabditis elegans as a model organism. Nano Letters 11 (8), 3174–3183. Wang, L.M., Liu, Y., Li, W., Jiang, X., Ji, Y.L., Wu, X.C., Xu, L.G., Qiu, Y., Zhao, K., Wei, T.T., Li, Y.F., Zhao, Y., Chen, C.Y., 2011. Selective targeting of gold nanorods at the mitochondria of cancer cells: implications for cancer therapy. Nano Letters 11, 772–780. Zhao, F., Zhao, Y., Liu, Y., Chang, X., Chen, C., 2011. Cellular uptake, intracellular trafficking, and cytotoxicity of nanomaterials. Small 7, 1322–1337. Zhao, Y.L., Xing, G.M., Chai, Z.F., 2008. Nanotoxicology: are carbon nanotubes safe? Nature Nanotechnology 3, 191–192.
Helmholtz Center Munich, Institute of Lung Biology and Disease, Neuherberg/Munich, Germany
http://dx.doi.org/10.1016/j.toxlet.2013.06.020
S02-3 Nanoparticles as an emerging environmental and occupational hazard Anna A. Shvedova Pathology & Physiology Research Branch/NIOSH/CDC, Department of Physiology and Pharmacology, School of Medicine, WVU, Morgantown, WV, USA Advancements in nanotechnology and broad applications of nanomaterials raise the issue of their potential adverse health effects. Among different nanomaterials, carbonaceous nanotubes (CNT) including single/multi-walled carbon nanotubes and nanofibers – with their distinctive physico-chemical, electronic and mechanical properties – are emerging as important objects of toxicological studies. However, toxic effects of CNT have not been well characterized, especially with respect to pulmonary and immune outcomes. By employing proteomics and lipidomics analyses, in vivo ESR spin-trapping technology, redox assessments of
http://dx.doi.org/10.1016/j.toxlet.2013.06.021
S02-4 Dosimetry of nanomaterials after different routes of exposure
Nanoparticles (NP) are increasingly used in science, technology and medicine. Besides NP inhalation in the respiratory tract oral uptake via ingestion is the other direct route of intake into the organism. In fact there is evidence that nanoparticles can cross body membranes – such as the air–blood–barrier in lungs or the gut walls – reaching blood circulation and accumulating in secondary target organs. Therefore, the comparison of inhaled NP versus direct intravenously administered NP into circulation versus NP administered orally provides important insights on the various interactions of crossing body membranes. To quantitatively determine accumulated NP fractions in such organs the ultimate aim is to balance the NP fractions in all interesting organs, skeleton and tissues including the remaining body and total excretion. Based on quantitative biokinetics after inhalation, intratracheal instillation, intravenous injection and gavage in rats we found small NP fractions (iridium, carbon, titanium dioxide, gold) in all secondary organs studied including brain, heart and in the skeleton. Fractions per secondary organ were usually below 0.1% of the administered dose but depended strongly on particle size, material and surface modifications and the route of intake. Interestingly NP which had crossed the ABB showed different patterns of organ distribution compared to intravenously injected NP supporting the hypothesis that the formation and the dynamics of protein conjugation mediates the fate of NP in the organism. http://dx.doi.org/10.1016/j.toxlet.2013.06.022
with respect to supra-pharmacological or target related side effects and may lead in the discovery phase to better decision making processes. On the other hand, data generated with those models need to be interpreted carefully in the context of translation into man and due to the low number of historical control data. In 2003, Boelsterli suggested that tailor-made and simplified models of human disease could be conducted in satellite toxicity studies to facilitate candidate selection, help predict rare and unexpected toxicity, and identify new biomarkers. Approximately 10 years later, Morgan et al. (2013) published several examples and a proposal how to use carefully those models. In line with this publication, it is suggested that testing in an animal model of human disease should only be taken after adequate consideration of relevance along with validities, benefits and limitations of the proposed models.
References Boelsterli, U.A., 2003. Animal models of human disease in drug safety assessment. Journal of Toxicological Sciences 28, 109–121. Morgan, S.J., Elangbam, C.S., Berens, S., Janovitz, E., Vitsky, A., Zabka, T., Conour, L., 2013. Use of animal models of human disease for non-clinical safety assessment of novel pharmaceuticals. Toxicologic Pathology 41 (3), 508–518.
http://dx.doi.org/10.1016/j.toxlet.2013.06.017
S01-5 Early safety assessment of biologicals Jennifer Sims Integrated Biologix GmbH, Basel, Switzerland Although industry’s large molecule success rates in Development continue to be significantly higher than that of small molecules, the success rates for large molecules have recently declined. About 17% of large molecules in Phase I are calculated to reach market, compared to about 4% of small molecules (KMR Pharmaceutical Benchmarking Forum, 2012). The issues that impact attrition in discovery and early development will be discussed in relation to protein therapeutics, using illustrative case examples. In recent years an increased emphasis has been placed on optimal molecular design for appropriate PK-PD characteristics to maximize the probability of engaging the target(s),and improving developability assessments aimed at delivering a high quality drug product. More emphasis still needs to be placed on a better understanding of target biology (e.g.target expression, turnover, receptor internalization, shed receptor, endogenous binding proteins), signaling pathways impacted, mechanism of action in normal and disease states, and comparative biology across animal species used for PK-PD and safety assessment to human volunteers and patients. Some biologics such as monoclonal antibodies and related products show very high target binding selectivity. This high target binding selectivity leads to high species specificity with respect to binding of the drug candidate to the target, even to the point that sequence polymorphisms between different strains of cynomolgus macaques may render one strain as not relevant for safety assessment due to lack of target binding by the drug candidate. Other differences between humans and animal species such as differences in Fc? receptors may also impact on the predictive value for humans of safety evaluation in animals. It is important to have a good understanding of the comparative biology issues which may result in limited predictive value of animal data and, where possible, to address any gaps with in vitro data in human systems e.g. assessment of potential for cytokine release in human blood/PBMCs.
S5
Another issue to address early in development is an immunogenicity risk assessment strategy, particularly the potential for any serious safety issues which may result from the development of neutralizing anti-drug-antibodies cross-reactive to endogenous proteins. Predictive immunogenicity approaches are being used more frequently as more novel engineered “less human” constructs and protein scaffolds enter development. Examples and limitations of predictive immunogenicity approaches will be discussed. http://dx.doi.org/10.1016/j.toxlet.2013.06.221 Symposium 2: Recent developments in risk assessment of nanomaterials and nano safety science
S02-1 Nanomaterials as a potential cause of lung disease James C. Bonner North Carolina State University, Raleigh, NC, USA The nanotechnology revolution offers enormous societal and economic benefits for innovation in the fields of engineering, electronics, and medicine. Nevertheless, evidence from rodent inhalation studies show that biopersistent engineered nanomaterials, including carbon nanotubes and metal nanoparticles, have the potential to stimulate immune, inflammatory, or fibroproliferative responses in the lung and pleura. These data suggest possible risks for pulmonary fibrosis or the development of pleural disease as a consequence of occupational or consumer exposure. Some engineered nanomaterials also exacerbate pre-existing allergeninduced inflammation by altering the balance of distinct T-helper cell phenotypes, suggesting that they could serve as sensitizers or adjuvants to alter the innate immune response. These findings suggest that individuals with asthma or other pre-existing respiratory diseases would be particularly susceptible to the adverse health effects of nanomaterials. Due to their nanoscale dimensions and increased surface area per unit mass, engineered nanomaterials have a much greater potential to reach the distal regions of the lung, generate reactive oxygen species, and alter cell signaling pathways linked to disease pathogenesis. The goal of this presentation will be to discuss mechanisms through which engineered nanomaterials cause lung, airway, and pleural disease, especially in the context of susceptible individuals with pre-existing disease. Functionalization of nanomaterials through processes such as atomic layer deposition will also be discussed as a means of altering the pathogenicity of nanomaterials. http://dx.doi.org/10.1016/j.toxlet.2013.06.019
S02-2 Safety consideration of nanomaterials for biomedical applications Chunying Chen 1,∗ , Ying Liu 1 , Liming Wang 2 , Yufeng Li 2 1 CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing China, 2 Institute of High Energy Physics, Beijing China
Many nanomaterials are promising in biological detection, diagnosis, and therapy for diseases and have shown great potential for biomedical applications. Therefore, the toxicity of nanomaterials becomes an increasing concern. Both in vitro and in vivo studies were applied to evaluate biological consequence of nanomaterials.
S6
Abstracts / Toxicology Letters 221S (2013) S4–S30
The underlying mechanisms were investigated including induction of oxidative stress, inflammation and autophagy (Li et al., 2012; Liu et al., 2013; Zhao et al., 2008). The intrinsic physicochemical properties of nanomaterials have decisive influence on their biological consequences and toxicity. These properties include size, shape, surface charge, chemical composition, surface modification, metal impurities, agglomeration and dispersion, degradation, as well as the formation of “protein corona” (Qiu et al., 2010; Wang et al., 2011; Zhao et al., 2011). It is important to obtain a better understanding of the uptake, trafficking, pharmacokinetics, clearance, and role of nanomaterials in biological systems, so that their possible undesirable effects can be avoided. Chemical speciation, dynamics and kinetics of nanomaterials in biological systems are extremely necessary since we have very limited knowledge. The state-of-the art analytical techniques are playing important roles in the study of nanotoxicology and nanobiology by taking advantages of absolute quantification, high sensitivity, excellent accuracy and precision, low matrix effects and non-destructiveness (Li and Chen, 2011). For example, the combination of -SRXRF and microbeam X-ray absorbance near edge structure (-XANES) can simultaneously provide information about the subcellular distribution and chemical species of metalcontaining nanomaterials of interest (Qu et al., 2011).
antioxidant balance, and quantitative morphometry (including collagen) in wild-type and genetically manipulated mice, we were able to reveal the major pathways through which CNT – in doses relevant to potential occupational exposures – exert their toxic effects in the lung/distant organs of exposed animals. Overall, found an unusual and robust inflammatory and fibrogenic response is closely associated with the progression of oxidative stress in the lung. Because realistic exposures to SWCNT are likely to occur in conjunction with other pathogenic influences, e.g., microbial infections, our finding of compromised bacterial clearance in the lungs of CNTexposed mice are of great practical importance. The talk will also address important issues of comparative respiratory outcomes of CNTs and asbestos, particularly with regards to pulmonary injury and potential carcinogenicity. Finally, the mechanisms of toxicity will be discussed in the context of current regulations of protection and their sufficiency in environmental and occupational settings. Supported by NIH008282, NORA700Y, 7ZKCY.
References
Wolfgang G. Kreyling
Li, Y., Chen, C., 2011. Fate and toxicity of metallic and metal-containing nanoparticles for biomedical applications. Small 7 (21), 2965–2980. Li, Y., Liu, Y., Fu, Y., Wei, T., Le Guyader, L., Gao, G., Liu, R.S., Chang, Y.Z., Chen, C., 2012. The triggering of apoptosis in macrophages by pristine graphene through the MAPK and TGF-beta signaling pathways. Biomaterials 33, 402–411. Liu, Y., Zhao, Y., Sun, B., Chen, C., 2013. Understanding the toxicity of carbon nanotubes. Accounts of Chemical Research 46 (3), 702–713. Qiu, Y., Liu, Y., Wang, L.M., Xu, L.G., Bai, R., Ji, Y.L., Wu, X.C., Zhao, Y.L., Li, Y.F., Chen, C.Y., 2010. Surface chemistry and aspect ratio mediated cellular uptake of Au nanorods. Biomaterials 31, 7606–7619. Qu, Y., Li, W., Zhou, Y.L., Liu, X.F., Zhang, L.L., Wang, L.M., Li, Y.F., Iida, A., Tang, Z.Y., Zhao, Y.L., Chai, Z.F., Chen, C.Y., 2011. Full assessment of fate and physiological behavior of quantum dots utilizing Caenorhabditis elegans as a model organism. Nano Letters 11 (8), 3174–3183. Wang, L.M., Liu, Y., Li, W., Jiang, X., Ji, Y.L., Wu, X.C., Xu, L.G., Qiu, Y., Zhao, K., Wei, T.T., Li, Y.F., Zhao, Y., Chen, C.Y., 2011. Selective targeting of gold nanorods at the mitochondria of cancer cells: implications for cancer therapy. Nano Letters 11, 772–780. Zhao, F., Zhao, Y., Liu, Y., Chang, X., Chen, C., 2011. Cellular uptake, intracellular trafficking, and cytotoxicity of nanomaterials. Small 7, 1322–1337. Zhao, Y.L., Xing, G.M., Chai, Z.F., 2008. Nanotoxicology: are carbon nanotubes safe? Nature Nanotechnology 3, 191–192.
Helmholtz Center Munich, Institute of Lung Biology and Disease, Neuherberg/Munich, Germany
http://dx.doi.org/10.1016/j.toxlet.2013.06.020
S02-3 Nanoparticles as an emerging environmental and occupational hazard Anna A. Shvedova Pathology & Physiology Research Branch/NIOSH/CDC, Department of Physiology and Pharmacology, School of Medicine, WVU, Morgantown, WV, USA Advancements in nanotechnology and broad applications of nanomaterials raise the issue of their potential adverse health effects. Among different nanomaterials, carbonaceous nanotubes (CNT) including single/multi-walled carbon nanotubes and nanofibers – with their distinctive physico-chemical, electronic and mechanical properties – are emerging as important objects of toxicological studies. However, toxic effects of CNT have not been well characterized, especially with respect to pulmonary and immune outcomes. By employing proteomics and lipidomics analyses, in vivo ESR spin-trapping technology, redox assessments of
http://dx.doi.org/10.1016/j.toxlet.2013.06.021
S02-4 Dosimetry of nanomaterials after different routes of exposure
Nanoparticles (NP) are increasingly used in science, technology and medicine. Besides NP inhalation in the respiratory tract oral uptake via ingestion is the other direct route of intake into the organism. In fact there is evidence that nanoparticles can cross body membranes – such as the air–blood–barrier in lungs or the gut walls – reaching blood circulation and accumulating in secondary target organs. Therefore, the comparison of inhaled NP versus direct intravenously administered NP into circulation versus NP administered orally provides important insights on the various interactions of crossing body membranes. To quantitatively determine accumulated NP fractions in such organs the ultimate aim is to balance the NP fractions in all interesting organs, skeleton and tissues including the remaining body and total excretion. Based on quantitative biokinetics after inhalation, intratracheal instillation, intravenous injection and gavage in rats we found small NP fractions (iridium, carbon, titanium dioxide, gold) in all secondary organs studied including brain, heart and in the skeleton. Fractions per secondary organ were usually below 0.1% of the administered dose but depended strongly on particle size, material and surface modifications and the route of intake. Interestingly NP which had crossed the ABB showed different patterns of organ distribution compared to intravenously injected NP supporting the hypothesis that the formation and the dynamics of protein conjugation mediates the fate of NP in the organism. http://dx.doi.org/10.1016/j.toxlet.2013.06.022