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Janos Szebeni - One of the best experts on this subject based on the ideXlab platform.
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Human Clinical Relevance of the Porcine Model of Pseudoallergic Infusion Reactions
Biomedicines, 2020Co-Authors: Janos Szebeni, Raj BawaAbstract:Pigs provide a highly sensitive animal model for pseudoallergic infusion reactions, which are mild-to-severe hypersensitivity reactions (HSRs) that arise following intravenous administration of certain nanoparticulate drugs (nanomedicines) and other macromolecular structures. This model has been used in research for three decades and was also proposed by regulatory bodies for preclinical assessment of the risk of HSRs in the clinical stages of nano-drug development. However, there are views challenging the human relevance of the model and its utility in preclinical safety evaluation of nanomedicines. The argument challenging the model refers to the “global response” of pulmonary intravascular macrophages (PIM cells) in the lung of pigs, preventing the distinction of reactogenic from non-reactogenic particles, therefore overestimating the risk of HSRs relative to its occurrence in the normal human population. The goal of this review is to present the large body of experimental and clinical evidence negating the “global response” claim, while also showing the concordance of symptoms caused by different reactogenic nanoparticles in pigs and hypersensitive man. Contrary to the model’s demotion, we propose that the above features, together with the high reproducibility of quantifiable physiological endpoints, validate the porcine “complement activation-related Pseudoallergy” (CARPA) model for safety evaluations. However, it needs to be kept in mind that the model is a disease model in the context of hypersensitivity to certain nanomedicines. Rather than toxicity screening, its main purpose is specific identification of HSR hazard, also enabling studies on the mechanism and mitigation of potentially serious HSRs.
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Complement Activation-Related Pathophysiological Changes in Anesthetized Rats: Activator-Dependent Variations of Symptoms and Mediators of Pseudoallergy.
Molecules (Basel Switzerland), 2019Co-Authors: László Dézsi, Janos Szebeni, Tamas Meszaros, Tamas Fulop, László Rosivall, Péter Hamar, Erik Őrfi, Mark Hennies, Gábor SzénásiAbstract:Complement (C) activation can underlie the infusion reactions to liposomes and other nanoparticle-based medicines, a hypersensitivity syndrome that can be partially reproduced in animal models. However, the sensitivities and manifestations substantially differ in different species, and C activation may not be the only cause of pathophysiological changes. In order to map the species variation of C-dependent and -independent Pseudoallergy (CARPA/CIPA), here we used known C activators and C activator liposomes to compare their acute hemodynamic, hematological, and biochemical effects in rats. These C activators were cobra venom factor (CVF), zymosan, AmBisome (at 2 doses), its amphotericin B-free vehicle (AmBisombo), and a PEGylated cholesterol-containing liposome (PEG-2000-chol), all having different powers to activate C in rat blood. The pathophysiological endpoints measured were blood pressure, leukocyte and platelet counts, and plasma thromboxane B2, while C activation was assessed by C3 consumption using the Pan-Specific C3 assay. The results showed strong linear correlation between C activation and systemic hypotension, pointing to a causal role of C activation in the hemodynamic changes. The observed thrombocytopenia and leukopenia followed by leukocytosis also correlated with C3 conversion in case of C activators, but not necessarily with C activation by liposomes. These findings are consistent with the double hit hypothesis of hypersensitivity reactions (HSRs), inasmuch as strong C activation can fully account for all symptoms of HSRs, but in case of no-, or weak C activators, the pathophysiological response, if any, is likely to involve other activation pathways.
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Doxebo (doxorubicin-free Doxil-like liposomes) is safe to use as a pre-treatment to prevent infusion reactions to PEGylated nanodrugs.
Journal of controlled release : official journal of the Controlled Release Society, 2019Co-Authors: Yaelle Bavli, Janos Szebeni, Rivka Cohen, Ilan Winkler, Bing Mae Chen, Steve R. Roffler, Yechezkel BarenholzAbstract:The increasing use in the last decade of PEGylated nanodrugs such as Doxil® has seen a rise in the number of associated occurrences of hypersensitivity reactions (HSRs). These reactions (also called infusion reactions or IR), can range from harmless symptoms to life-threatening reactions. Current means to prevent IR include the prophylactic use of antihistamines and steroids, but they cannot ensure total prevention. We previously showed that an intravenous injection of doxorubicin-free Doxil-like PEGylated nano-liposomes (Doxebo) prior to Doxil treatment suppresses Doxil-induced complement activation-related Pseudoallergy (CARPA) in pigs, a model of human hypersensitivity reactions to Doxil. However, in order to use Doxebo to prevent Doxil-induced IR, we have to prove its safety and that it does not affect Doxil's performance. Here we show that Doxebo itself does not have toxic effects on the host or tumor, and it does not interfere with Doxil's antitumor activity in mice. Blood, microscopic and macroscopic organ evaluation of rats after repeated administration confirm the lack of intrinsic adverse effect of Doxebo. Likewise, the repeated injection of Doxebo before Doxil did not impact Doxil's pharmacokinetics in plasma and therefore does not cause accelerated blood clearance (ABC). Taken together with our previous publications, these data suggest that the injection of Doxebo prior to Doxil administration can help protect against Doxil-induced IR without adversely affecting treatment efficacy and safety.
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involvement of complement activation in the pulmonary vasoactivity of polystyrene nanoparticles in pigs unique surface properties underlying alternative pathway activation and instant opsonization
International Journal of Nanomedicine, 2018Co-Authors: Tamas Meszaros, Gergely Tibor Kozma, Taro Shimizu, Koga Miyahara, Keren Turjeman, Tatsuhiro Ishida, Yechezkel Barenholz, Rudolf Urbanics, Janos SzebeniAbstract:Background It has been proposed that many hypersensitivity reactions to nanopharmaceuticals represent complement (C)-activation-related Pseudoallergy (CARPA), and that pigs provide a sensitive animal model to study the phenomenon. However, a recent study suggested that pulmonary hypertension, the pivotal symptom of porcine CARPA, is not mediated by C in cases of polystyrene nanoparticle (PS-NP)-induced reactions. Goals To characterize PS-NPs and reexamine the contribution of CARPA to their pulmonary reactivity in pigs. Study design C activation by 200, 500, and 750 nm (diameter) PS-NPs and their opsonization were measured in human and pig sera, respectively, and correlated with hemodynamic effects of the same NPs in pigs in vivo. Methods Physicochemical characterization of PS-NPs included size, ζ-potential, cryo-transmission electron microscopy, and hydrophobicity analyses. C activation in human serum was measured by ELISA and opsonization of PS-NPs in pig serum by Western blot and flow cytometry. Pulmonary vasoactivity of PS-NPs was quantified in the porcine CARPA model. Results PS-NPs are monodisperse, highly hydrophobic spheres with strong negative surface charge. In human serum, they caused size-dependent, significant rises in C3a, Bb, and sC5b-9, but not C4d. Exposure to pig serum led within minutes to deposition of C5b-9 and opsonic iC3b on the NPs, and opsonic iC3b fragments (C3dg, C3d) also appeared in serum. PS-NPs caused major hemodynamic changes in pigs, primarily pulmonary hypertension, on the same time scale (minutes) as iC3b fragmentation and opsonization proceeded. There was significant correlation between C activation by different PS-NPs in human serum and pulmonary hypertension in pigs. Conclusion PS-NPs have extreme surface properties with no relevance to clinically used nanomedicines. They can activate C via the alternative pathway, entailing instantaneous opsonization of NPs in pig serum. Therefore, rather than being solely C-independent reactivity, the mechanism of PS-NP-induced hypersensitivity in pigs may involve C activation. These data are consistent with the "double-hit" concept of nanoparticle-induced hypersensitivity reactions involving both CARPA and C-independent Pseudoallergy.
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Understanding the Role of Anti-PEG Antibodies in the Complement Activation by Doxil in Vitro.
Molecules (Basel Switzerland), 2018Co-Authors: Barry W. Neun, Janos Szebeni, Yechezkel Barenholz, Marina A. DobrovolskaiaAbstract:Infusion reactions (IRs) are common immune-mediated side effects in patients treated with a variety of drug products, including, but not limited to, nanotechnology formulations. The mechanism of IRs is not fully understood. One of the best studied mechanisms of IRs to nanomedicines is the complement activation. However, it is largely unknown why some patients develop reactions to nanomedicines while others do not, and why some nanoparticles are more reactogenic than others. One of the theories is that the pre-existing anti-polyethylene glycol (PEG) antibodies initiate the complement activation and IRs in patients. In this study, we investigated this hypothesis in the case of PEGylated liposomal doxorubicin (Doxil), which, when used in a clinical setting, is known to induce IRs; referred to as complement activation-related Pseudoallergy (CARPA) in sensitive individuals. We conducted the study in vitro using plasma derived from C57BL/6 mice and twenty human donor volunteers. We used mouse plasma to test a library of well-characterized mouse monoclonal antibodies with different specificity and affinity to PEG as it relates to the complement activation by Doxil. We determined the levels of pre-existing polyclonal antibodies that bind to PEG, methoxy-PEG, and PEGylated liposomes in human plasma, and we also assessed complement activation by Doxil and concentrations of complement inhibitory factors H and I in these human plasma specimens. The affinity, specificity, and other characteristics of the human polyclonal antibodies are not known at this time. Our data demonstrate that under in vitro conditions, some anti-PEG antibodies contribute to the complement activation by Doxil. Such contribution, however, needs to be considered in the context of other factors, including, but not limited to, antibody class, type, clonality, epitope specificity, affinity, and titer. In addition, our data contribute to the knowledge base used to understand and improve nanomedicine safety.
Rudolf Urbanics - One of the best experts on this subject based on the ideXlab platform.
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involvement of complement activation in the pulmonary vasoactivity of polystyrene nanoparticles in pigs unique surface properties underlying alternative pathway activation and instant opsonization
International Journal of Nanomedicine, 2018Co-Authors: Tamas Meszaros, Gergely Tibor Kozma, Taro Shimizu, Koga Miyahara, Keren Turjeman, Tatsuhiro Ishida, Yechezkel Barenholz, Rudolf Urbanics, Janos SzebeniAbstract:Background It has been proposed that many hypersensitivity reactions to nanopharmaceuticals represent complement (C)-activation-related Pseudoallergy (CARPA), and that pigs provide a sensitive animal model to study the phenomenon. However, a recent study suggested that pulmonary hypertension, the pivotal symptom of porcine CARPA, is not mediated by C in cases of polystyrene nanoparticle (PS-NP)-induced reactions. Goals To characterize PS-NPs and reexamine the contribution of CARPA to their pulmonary reactivity in pigs. Study design C activation by 200, 500, and 750 nm (diameter) PS-NPs and their opsonization were measured in human and pig sera, respectively, and correlated with hemodynamic effects of the same NPs in pigs in vivo. Methods Physicochemical characterization of PS-NPs included size, ζ-potential, cryo-transmission electron microscopy, and hydrophobicity analyses. C activation in human serum was measured by ELISA and opsonization of PS-NPs in pig serum by Western blot and flow cytometry. Pulmonary vasoactivity of PS-NPs was quantified in the porcine CARPA model. Results PS-NPs are monodisperse, highly hydrophobic spheres with strong negative surface charge. In human serum, they caused size-dependent, significant rises in C3a, Bb, and sC5b-9, but not C4d. Exposure to pig serum led within minutes to deposition of C5b-9 and opsonic iC3b on the NPs, and opsonic iC3b fragments (C3dg, C3d) also appeared in serum. PS-NPs caused major hemodynamic changes in pigs, primarily pulmonary hypertension, on the same time scale (minutes) as iC3b fragmentation and opsonization proceeded. There was significant correlation between C activation by different PS-NPs in human serum and pulmonary hypertension in pigs. Conclusion PS-NPs have extreme surface properties with no relevance to clinically used nanomedicines. They can activate C via the alternative pathway, entailing instantaneous opsonization of NPs in pig serum. Therefore, rather than being solely C-independent reactivity, the mechanism of PS-NP-induced hypersensitivity in pigs may involve C activation. These data are consistent with the "double-hit" concept of nanoparticle-induced hypersensitivity reactions involving both CARPA and C-independent Pseudoallergy.
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A porcine model of complement activation-related Pseudoallergy to nano-pharmaceuticals: Pros and cons of translation to a preclinical safety test
Precision Nanomedicine, 2018Co-Authors: Janos Szebeni, László Dézsi, Péter Bedőcs, Rudolf UrbanicsAbstract:Pigs provide a sensitive and quantitative animal model of non-IgE-mediated (pseudoallergic) hypersensitivity reactions (HSRs) caused by liposomes and many other nanoparticulate drugs or drug-carrier nanosystems (nanomedicines). The rapidly arising symptoms, including cardiopulmonary, hemodynamic, hematological, blood chemistry and skin changes, resemble the clinical picture in man undergoing infusion reactions to reactogenic nanoparticles. In addition to summarizing the basic features of the pig CARPA model, the review considers some of the advantages and disadvantages of using the model for preclinical evaluation of nanomedicine safety. [READ THE ARTICLE](https://doi.org/10.29016/180427.1)
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Complement activation in vitro and reactogenicity of low-molecular weight dextran-coated SPIONs in the pig CARPA model: Correlation with physicochemical features and clinical information.
Journal of controlled release : official journal of the Controlled Release Society, 2017Co-Authors: Tamas Fulop, Tamas Meszaros, Rudolf Urbanics, Joshua A. Jackman, Nam-joon Cho, Gert Storm, Reka Nemes, Robbert J. Kok, Janos SzebeniAbstract:The unique magnetic properties of superparamagnetic iron oxide nanoparticles (SPIONs) have led to their increasing use in drug delivery and imaging applications. Some polymer-coated SPIONs, however, share with many other nanoparticles the potential of causing hypersensitivity reactions known as complement (C) activation-related Pseudoallergy (CARPA). In order to explore the roles of iron core composition and particle surface coating in SPION-induced CARPA, we measured C activation by 6 different SPIONs in a human serum that is known to react to nanoparticles (NPs) with strong C activation. Remarkably, only the carboxymethyldextran-coated (ferucarbotran, Resosvist®) and dextran-coated (ferumoxtran-10, Sinerem®) SPIONs caused significant C activation, while the citric acid, phosphatidylcholine, starch and chitosan-coated SPIONs had no such effect. Focusing on Resovist and Sinerem, we found Sinerem to be a stronger activator of C than Resovist, although the individual variation in 15 different human sera was substantial. Further analysis of C activation by Sinerem indicated biphasic dose dependence and significant production of C split product Bb but not C4d, attesting to alternative pathway C activation only at low doses. Consistent with the strong C activation by Sinerem and previous reports of HSRs in man, injection of Sinerem in a pig led to dose-dependent CARPA, while Resovist was reaction-free. Using nanoparticle tracking analysis, it was further determined that Sinerem, but not Resovist, displayed multimodal size distribution and significant fraction of aggregates – factors which are known to promote C activation and CARPA. Taken together, our findings offer physicochemical insight into how key compositional factors and nanoparticle size distribution affect SPION-induced CARPA, a knowledge that could lead to the development of SPIONs with improved safety profiles.
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Non-immunogenic dextran-coated superparamagnetic iron oxide nanoparticles: a biocompatible, size-tunable contrast agent for magnetic resonance imaging.
International journal of nanomedicine, 2017Co-Authors: Harald Unterweger, Rudolf Urbanics, Tamas Fulop, László Dézsi, Erik Őrfi, Christina Janko, Marc Schwarz, Jasmin Matuszak, Tobias Bäuerle, Janos SzebeniAbstract:Iron oxide-based contrast agents have been in clinical use for magnetic resonance imaging (MRI) of lymph nodes, liver, intestines, and the cardiovascular system. Superparamagnetic iron oxide nanoparticles (SPIONs) have high potential as a contrast agent for MRI, but no intravenous iron oxide-containing agents are currently approved for clinical imaging. The aim of our work was to analyze the hemocompatibility and immuno-safety of a new type of dextran-coated SPIONs (SPIONdex) and to characterize these nanoparticles with ultra-high-field MRI. Key parameters related to nanoparticle hemocompatibility and immuno-safety were investigated in vitro and ex vivo. To address concerns associated with hypersensitivity reactions to injectable nanoparticulate agents, we analyzed complement activation-related Pseudoallergy (CARPA) upon intravenous administration of SPIONdex in a pig model. Furthermore, the size-tunability of SPIONdex and the effects of size reduction on their biocompatibility were investigated. In vitro, SPIONdex did not induce hemolysis, complement or platelet activation, plasma coagulation, or leukocyte procoagulant activity, and had no relevant effect on endothelial cell viability or endothelial-monocytic cell interactions. Furthermore, SPIONdex did not induce CARPA even upon intravenous administration of 5 mg Fe/kg in pigs. Upon SPIONdex administration in mice, decreased liver signal intensity was observed after 15 minutes and was still detectable 24 h later. In addition, by changing synthesis parameters, a reduction in particle size
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Comparison of complement activation-related Pseudoallergy in miniature and domestic pigs: foundation of a validatable immune toxicity model
Nanomedicine : nanotechnology biology and medicine, 2016Co-Authors: Joshua A. Jackman, Janos Szebeni, Tamas Meszaros, Rudolf Urbanics, Tamas Fulop, Nam-joon ChoAbstract:Abstract Complement activation-related Pseudoallergy (CARPA) is an acute adverse immune reaction caused by many nanomedicines. There is a regulatory need for a sensitive and standardizable in vivo predictive assay. While domestic pigs are a sensitive animal model, miniature pigs are favored in toxicological studies yet their utility as a CARPA model has not yet been explored. Herein, we used liposomal doxorubicin and amphotericin B (Doxil/Caelyx and AmBisome), Cremophor EL and zymosan as CARPA triggers to induce reactions in miniature and domestic pigs, and compared the hemodynamic, hematological, biochemical, and skin alterations. The changes observed after administration of the test agents were very similar in both pig strains, suggesting that miniature pigs are a sensitive, reproducible, and, hence, validatable animal model for CARPA regulatory testing. From the Clinical Editor With the advances in nanomedicine research, many new agents are now tested for use in clinical setting. Nonetheless, complement activation-related Pseudoallergy (CARPA) is a well known phenomenon which can be caused by nanoparticles. In this study, the authors looked at and compared the use of domestic pigs versus miniature pigs as experimental animals for toxicological studies. Their findings confirmed the possible use of miniature pigs for regulatory testing.
Tamas Meszaros - One of the best experts on this subject based on the ideXlab platform.
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Complement Activation-Related Pathophysiological Changes in Anesthetized Rats: Activator-Dependent Variations of Symptoms and Mediators of Pseudoallergy.
Molecules (Basel Switzerland), 2019Co-Authors: László Dézsi, Janos Szebeni, Tamas Meszaros, Tamas Fulop, László Rosivall, Péter Hamar, Erik Őrfi, Mark Hennies, Gábor SzénásiAbstract:Complement (C) activation can underlie the infusion reactions to liposomes and other nanoparticle-based medicines, a hypersensitivity syndrome that can be partially reproduced in animal models. However, the sensitivities and manifestations substantially differ in different species, and C activation may not be the only cause of pathophysiological changes. In order to map the species variation of C-dependent and -independent Pseudoallergy (CARPA/CIPA), here we used known C activators and C activator liposomes to compare their acute hemodynamic, hematological, and biochemical effects in rats. These C activators were cobra venom factor (CVF), zymosan, AmBisome (at 2 doses), its amphotericin B-free vehicle (AmBisombo), and a PEGylated cholesterol-containing liposome (PEG-2000-chol), all having different powers to activate C in rat blood. The pathophysiological endpoints measured were blood pressure, leukocyte and platelet counts, and plasma thromboxane B2, while C activation was assessed by C3 consumption using the Pan-Specific C3 assay. The results showed strong linear correlation between C activation and systemic hypotension, pointing to a causal role of C activation in the hemodynamic changes. The observed thrombocytopenia and leukopenia followed by leukocytosis also correlated with C3 conversion in case of C activators, but not necessarily with C activation by liposomes. These findings are consistent with the double hit hypothesis of hypersensitivity reactions (HSRs), inasmuch as strong C activation can fully account for all symptoms of HSRs, but in case of no-, or weak C activators, the pathophysiological response, if any, is likely to involve other activation pathways.
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involvement of complement activation in the pulmonary vasoactivity of polystyrene nanoparticles in pigs unique surface properties underlying alternative pathway activation and instant opsonization
International Journal of Nanomedicine, 2018Co-Authors: Tamas Meszaros, Gergely Tibor Kozma, Taro Shimizu, Koga Miyahara, Keren Turjeman, Tatsuhiro Ishida, Yechezkel Barenholz, Rudolf Urbanics, Janos SzebeniAbstract:Background It has been proposed that many hypersensitivity reactions to nanopharmaceuticals represent complement (C)-activation-related Pseudoallergy (CARPA), and that pigs provide a sensitive animal model to study the phenomenon. However, a recent study suggested that pulmonary hypertension, the pivotal symptom of porcine CARPA, is not mediated by C in cases of polystyrene nanoparticle (PS-NP)-induced reactions. Goals To characterize PS-NPs and reexamine the contribution of CARPA to their pulmonary reactivity in pigs. Study design C activation by 200, 500, and 750 nm (diameter) PS-NPs and their opsonization were measured in human and pig sera, respectively, and correlated with hemodynamic effects of the same NPs in pigs in vivo. Methods Physicochemical characterization of PS-NPs included size, ζ-potential, cryo-transmission electron microscopy, and hydrophobicity analyses. C activation in human serum was measured by ELISA and opsonization of PS-NPs in pig serum by Western blot and flow cytometry. Pulmonary vasoactivity of PS-NPs was quantified in the porcine CARPA model. Results PS-NPs are monodisperse, highly hydrophobic spheres with strong negative surface charge. In human serum, they caused size-dependent, significant rises in C3a, Bb, and sC5b-9, but not C4d. Exposure to pig serum led within minutes to deposition of C5b-9 and opsonic iC3b on the NPs, and opsonic iC3b fragments (C3dg, C3d) also appeared in serum. PS-NPs caused major hemodynamic changes in pigs, primarily pulmonary hypertension, on the same time scale (minutes) as iC3b fragmentation and opsonization proceeded. There was significant correlation between C activation by different PS-NPs in human serum and pulmonary hypertension in pigs. Conclusion PS-NPs have extreme surface properties with no relevance to clinically used nanomedicines. They can activate C via the alternative pathway, entailing instantaneous opsonization of NPs in pig serum. Therefore, rather than being solely C-independent reactivity, the mechanism of PS-NP-induced hypersensitivity in pigs may involve C activation. These data are consistent with the "double-hit" concept of nanoparticle-induced hypersensitivity reactions involving both CARPA and C-independent Pseudoallergy.
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Infusion Reactions Associated with the Medical Application of Monoclonal Antibodies: The Role of Complement Activation and Possibility of Inhibition by Factor H.
Antibodies (Basel Switzerland), 2018Co-Authors: Tamas Fulop, Janos Szebeni, Tamas Meszaros, Gergely Tibor Kozma, Mihály JózsiAbstract:Human application of monoclonal antibodies (mAbs), enzymes, as well as contrast media and many other particulate drugs and agents referred to as “nanomedicines”, can initiate pseudoallergic hypersensitivity reactions, also known as infusion reactions. These may in part be mediated by the activation of the complement system, a major humoral defense system of innate immunity. In this review, we provide a brief outline of complement activation-related Pseudoallergy (CARPA) in general, and then focus on the reactions caused by mAb therapy. Because the alternative pathway of complement activation may amplify such adverse reactions, we highlight the potential use of complement factor H as an inhibitor of CARPA.
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Complement activation in vitro and reactogenicity of low-molecular weight dextran-coated SPIONs in the pig CARPA model: Correlation with physicochemical features and clinical information.
Journal of controlled release : official journal of the Controlled Release Society, 2017Co-Authors: Tamas Fulop, Tamas Meszaros, Rudolf Urbanics, Joshua A. Jackman, Nam-joon Cho, Gert Storm, Reka Nemes, Robbert J. Kok, Janos SzebeniAbstract:The unique magnetic properties of superparamagnetic iron oxide nanoparticles (SPIONs) have led to their increasing use in drug delivery and imaging applications. Some polymer-coated SPIONs, however, share with many other nanoparticles the potential of causing hypersensitivity reactions known as complement (C) activation-related Pseudoallergy (CARPA). In order to explore the roles of iron core composition and particle surface coating in SPION-induced CARPA, we measured C activation by 6 different SPIONs in a human serum that is known to react to nanoparticles (NPs) with strong C activation. Remarkably, only the carboxymethyldextran-coated (ferucarbotran, Resosvist®) and dextran-coated (ferumoxtran-10, Sinerem®) SPIONs caused significant C activation, while the citric acid, phosphatidylcholine, starch and chitosan-coated SPIONs had no such effect. Focusing on Resovist and Sinerem, we found Sinerem to be a stronger activator of C than Resovist, although the individual variation in 15 different human sera was substantial. Further analysis of C activation by Sinerem indicated biphasic dose dependence and significant production of C split product Bb but not C4d, attesting to alternative pathway C activation only at low doses. Consistent with the strong C activation by Sinerem and previous reports of HSRs in man, injection of Sinerem in a pig led to dose-dependent CARPA, while Resovist was reaction-free. Using nanoparticle tracking analysis, it was further determined that Sinerem, but not Resovist, displayed multimodal size distribution and significant fraction of aggregates – factors which are known to promote C activation and CARPA. Taken together, our findings offer physicochemical insight into how key compositional factors and nanoparticle size distribution affect SPION-induced CARPA, a knowledge that could lead to the development of SPIONs with improved safety profiles.
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Comparison of complement activation-related Pseudoallergy in miniature and domestic pigs: foundation of a validatable immune toxicity model
Nanomedicine : nanotechnology biology and medicine, 2016Co-Authors: Joshua A. Jackman, Janos Szebeni, Tamas Meszaros, Rudolf Urbanics, Tamas Fulop, Nam-joon ChoAbstract:Abstract Complement activation-related Pseudoallergy (CARPA) is an acute adverse immune reaction caused by many nanomedicines. There is a regulatory need for a sensitive and standardizable in vivo predictive assay. While domestic pigs are a sensitive animal model, miniature pigs are favored in toxicological studies yet their utility as a CARPA model has not yet been explored. Herein, we used liposomal doxorubicin and amphotericin B (Doxil/Caelyx and AmBisome), Cremophor EL and zymosan as CARPA triggers to induce reactions in miniature and domestic pigs, and compared the hemodynamic, hematological, biochemical, and skin alterations. The changes observed after administration of the test agents were very similar in both pig strains, suggesting that miniature pigs are a sensitive, reproducible, and, hence, validatable animal model for CARPA regulatory testing. From the Clinical Editor With the advances in nanomedicine research, many new agents are now tested for use in clinical setting. Nonetheless, complement activation-related Pseudoallergy (CARPA) is a well known phenomenon which can be caused by nanoparticles. In this study, the authors looked at and compared the use of domestic pigs versus miniature pigs as experimental animals for toxicological studies. Their findings confirmed the possible use of miniature pigs for regulatory testing.
Yechezkel Barenholz - One of the best experts on this subject based on the ideXlab platform.
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Doxebo (doxorubicin-free Doxil-like liposomes) is safe to use as a pre-treatment to prevent infusion reactions to PEGylated nanodrugs.
Journal of controlled release : official journal of the Controlled Release Society, 2019Co-Authors: Yaelle Bavli, Janos Szebeni, Rivka Cohen, Ilan Winkler, Bing Mae Chen, Steve R. Roffler, Yechezkel BarenholzAbstract:The increasing use in the last decade of PEGylated nanodrugs such as Doxil® has seen a rise in the number of associated occurrences of hypersensitivity reactions (HSRs). These reactions (also called infusion reactions or IR), can range from harmless symptoms to life-threatening reactions. Current means to prevent IR include the prophylactic use of antihistamines and steroids, but they cannot ensure total prevention. We previously showed that an intravenous injection of doxorubicin-free Doxil-like PEGylated nano-liposomes (Doxebo) prior to Doxil treatment suppresses Doxil-induced complement activation-related Pseudoallergy (CARPA) in pigs, a model of human hypersensitivity reactions to Doxil. However, in order to use Doxebo to prevent Doxil-induced IR, we have to prove its safety and that it does not affect Doxil's performance. Here we show that Doxebo itself does not have toxic effects on the host or tumor, and it does not interfere with Doxil's antitumor activity in mice. Blood, microscopic and macroscopic organ evaluation of rats after repeated administration confirm the lack of intrinsic adverse effect of Doxebo. Likewise, the repeated injection of Doxebo before Doxil did not impact Doxil's pharmacokinetics in plasma and therefore does not cause accelerated blood clearance (ABC). Taken together with our previous publications, these data suggest that the injection of Doxebo prior to Doxil administration can help protect against Doxil-induced IR without adversely affecting treatment efficacy and safety.
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involvement of complement activation in the pulmonary vasoactivity of polystyrene nanoparticles in pigs unique surface properties underlying alternative pathway activation and instant opsonization
International Journal of Nanomedicine, 2018Co-Authors: Tamas Meszaros, Gergely Tibor Kozma, Taro Shimizu, Koga Miyahara, Keren Turjeman, Tatsuhiro Ishida, Yechezkel Barenholz, Rudolf Urbanics, Janos SzebeniAbstract:Background It has been proposed that many hypersensitivity reactions to nanopharmaceuticals represent complement (C)-activation-related Pseudoallergy (CARPA), and that pigs provide a sensitive animal model to study the phenomenon. However, a recent study suggested that pulmonary hypertension, the pivotal symptom of porcine CARPA, is not mediated by C in cases of polystyrene nanoparticle (PS-NP)-induced reactions. Goals To characterize PS-NPs and reexamine the contribution of CARPA to their pulmonary reactivity in pigs. Study design C activation by 200, 500, and 750 nm (diameter) PS-NPs and their opsonization were measured in human and pig sera, respectively, and correlated with hemodynamic effects of the same NPs in pigs in vivo. Methods Physicochemical characterization of PS-NPs included size, ζ-potential, cryo-transmission electron microscopy, and hydrophobicity analyses. C activation in human serum was measured by ELISA and opsonization of PS-NPs in pig serum by Western blot and flow cytometry. Pulmonary vasoactivity of PS-NPs was quantified in the porcine CARPA model. Results PS-NPs are monodisperse, highly hydrophobic spheres with strong negative surface charge. In human serum, they caused size-dependent, significant rises in C3a, Bb, and sC5b-9, but not C4d. Exposure to pig serum led within minutes to deposition of C5b-9 and opsonic iC3b on the NPs, and opsonic iC3b fragments (C3dg, C3d) also appeared in serum. PS-NPs caused major hemodynamic changes in pigs, primarily pulmonary hypertension, on the same time scale (minutes) as iC3b fragmentation and opsonization proceeded. There was significant correlation between C activation by different PS-NPs in human serum and pulmonary hypertension in pigs. Conclusion PS-NPs have extreme surface properties with no relevance to clinically used nanomedicines. They can activate C via the alternative pathway, entailing instantaneous opsonization of NPs in pig serum. Therefore, rather than being solely C-independent reactivity, the mechanism of PS-NP-induced hypersensitivity in pigs may involve C activation. These data are consistent with the "double-hit" concept of nanoparticle-induced hypersensitivity reactions involving both CARPA and C-independent Pseudoallergy.
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Understanding the Role of Anti-PEG Antibodies in the Complement Activation by Doxil in Vitro.
Molecules (Basel Switzerland), 2018Co-Authors: Barry W. Neun, Janos Szebeni, Yechezkel Barenholz, Marina A. DobrovolskaiaAbstract:Infusion reactions (IRs) are common immune-mediated side effects in patients treated with a variety of drug products, including, but not limited to, nanotechnology formulations. The mechanism of IRs is not fully understood. One of the best studied mechanisms of IRs to nanomedicines is the complement activation. However, it is largely unknown why some patients develop reactions to nanomedicines while others do not, and why some nanoparticles are more reactogenic than others. One of the theories is that the pre-existing anti-polyethylene glycol (PEG) antibodies initiate the complement activation and IRs in patients. In this study, we investigated this hypothesis in the case of PEGylated liposomal doxorubicin (Doxil), which, when used in a clinical setting, is known to induce IRs; referred to as complement activation-related Pseudoallergy (CARPA) in sensitive individuals. We conducted the study in vitro using plasma derived from C57BL/6 mice and twenty human donor volunteers. We used mouse plasma to test a library of well-characterized mouse monoclonal antibodies with different specificity and affinity to PEG as it relates to the complement activation by Doxil. We determined the levels of pre-existing polyclonal antibodies that bind to PEG, methoxy-PEG, and PEGylated liposomes in human plasma, and we also assessed complement activation by Doxil and concentrations of complement inhibitory factors H and I in these human plasma specimens. The affinity, specificity, and other characteristics of the human polyclonal antibodies are not known at this time. Our data demonstrate that under in vitro conditions, some anti-PEG antibodies contribute to the complement activation by Doxil. Such contribution, however, needs to be considered in the context of other factors, including, but not limited to, antibody class, type, clonality, epitope specificity, affinity, and titer. In addition, our data contribute to the knowledge base used to understand and improve nanomedicine safety.
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Prevention of infusion reactions to PEGylated liposomal doxorubicin via tachyphylaxis induction by placebo vesicles: a porcine model.
Journal of controlled release : official journal of the Controlled Release Society, 2012Co-Authors: Janos Szebeni, Rudolf Urbanics, László Rosivall, Péter Bedőcs, Miklós Tóth, Rolf Bünger, Yechezkel BarenholzAbstract:PEGylated liposomal doxorubicin (Doxil) has been used in cancer chemotherapy for 16 years. Clinical experience shows that it can cause mild-to-severe hypersensitivity (infusion) reactions, which are manifestations of complement (C) activation-related Pseudoallergy (CARPA). Although in most cases CARPA is inconsequential, a main symptom, cardiopulmonary distress, may be life threatening in hypersensitive individuals. To date, the prevention of Doxil-induced CARPA is based on premedication and a slow infusion protocol. The present study suggests desensitization by Doxil-like empty liposomes, called placebo Doxil (Doxebo), as an alternative strategy, which is based on the tachyphylactic nature of Doxil reactions. Doxebo-induced tolerance to Doxil was shown to develop within minutes and to be specific to Doxil-like PEGylated liposomes. The procedure of desensitization involves slow, low-dose pre-infusion of Doxebo before Doxil treatment which minimizes the ensuing physiological changes or keeps them subclinical. Although the mechanism of tolerance induction is not yet clear, the effector arm of C response is unlikely to be affected, as the vascular reactivity of desensitized pigs to zymosan remains intact. Desensitization with empty vesicles represents a novel approach for reducing the risk of anaphylactic reactions to drug carrier liposomes. The underlying immediate, most likely passive silencing of an innate immune response may represent a novel mechanism of tolerance induction which may work for other reactogenic nanosystems as well.
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Activation of complement by therapeutic liposomes and other lipid excipient-based therapeutic products: prediction and prevention.
Advanced drug delivery reviews, 2011Co-Authors: Janos Szebeni, Franco M. Muggia, Alberto Gabizon, Yechezkel BarenholzAbstract:Some therapeutic liposomes and lipid excipient-based anticancer drugs are recognized by the immune system as foreign, leading to a variety of adverse immune phenomena. One of them is complement (C) activation, the cause, or major contributing factor to a hypersensitivity syndrome called C activation-related Pseudoallergy (CARPA). CARPA represents a novel subcategory of acute (type I) hypersensitivity reactions (HSR), which is mostly mild, transient, and preventable by appropriate precautions. However, in an occasional patient, it can be severe or even lethal. Because a main manifestation of C activation is cardiopulmonary distress, CARPA may be a safety issue primarily in cardiac patients. Along with an overview of the various types of liposome-immune system interactions, this review updates the experimental and clinical information on CARPA to different therapeutic liposomes and lipid excipient-based (micellar) anticancer drugs, including PEGylated liposomal doxorubicin sulfate (PLD, Doxil®) and paclitaxel (Taxol®). The substantial individual variation of in vitro and in vivo findings reflects an extremely complex immune phenomenon involving multiple, redundant pathways of C activation, signal transduction in allergy-mediating cells and vasoactive mediator actions at the effector cell level. The latest advances in this field include the proposal of doxorubicin-induced shape changes and aggregation of liposomes in Doxil as possible contributing factors to CARPA caused by PLD, and the finding that Doxil-induced immune suppression prevents HSR to co-administered carboplatin, a significant benefit of Doxil in combination chemotherapy with carboplatin. The review evaluates the use of in vitro C assays and the porcine liposome-induced cardiopulmonary distress model for predicting CARPA. It is concluded that CARPA may become a frequent safety issue in the upcoming era of nanomedicines, necessitating its prevention at an early stage of nanomedicine R&D.
László Rosivall - One of the best experts on this subject based on the ideXlab platform.
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Complement Activation-Related Pathophysiological Changes in Anesthetized Rats: Activator-Dependent Variations of Symptoms and Mediators of Pseudoallergy.
Molecules (Basel Switzerland), 2019Co-Authors: László Dézsi, Janos Szebeni, Tamas Meszaros, Tamas Fulop, László Rosivall, Péter Hamar, Erik Őrfi, Mark Hennies, Gábor SzénásiAbstract:Complement (C) activation can underlie the infusion reactions to liposomes and other nanoparticle-based medicines, a hypersensitivity syndrome that can be partially reproduced in animal models. However, the sensitivities and manifestations substantially differ in different species, and C activation may not be the only cause of pathophysiological changes. In order to map the species variation of C-dependent and -independent Pseudoallergy (CARPA/CIPA), here we used known C activators and C activator liposomes to compare their acute hemodynamic, hematological, and biochemical effects in rats. These C activators were cobra venom factor (CVF), zymosan, AmBisome (at 2 doses), its amphotericin B-free vehicle (AmBisombo), and a PEGylated cholesterol-containing liposome (PEG-2000-chol), all having different powers to activate C in rat blood. The pathophysiological endpoints measured were blood pressure, leukocyte and platelet counts, and plasma thromboxane B2, while C activation was assessed by C3 consumption using the Pan-Specific C3 assay. The results showed strong linear correlation between C activation and systemic hypotension, pointing to a causal role of C activation in the hemodynamic changes. The observed thrombocytopenia and leukopenia followed by leukocytosis also correlated with C3 conversion in case of C activators, but not necessarily with C activation by liposomes. These findings are consistent with the double hit hypothesis of hypersensitivity reactions (HSRs), inasmuch as strong C activation can fully account for all symptoms of HSRs, but in case of no-, or weak C activators, the pathophysiological response, if any, is likely to involve other activation pathways.
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Rodent Models of Complement Activation-Related Pseudoallergy: Inducers, Symptoms, Inhibitors and Reaction Mechanisms
European Journal of Nanomedicine, 2015Co-Authors: László Dézsi, Janos Szebeni, László Rosivall, Péter Hamar, Gábor SzénásiAbstract:Complement activation-related Pseudoallergy (CARPA) is a hypersensitivity reaction to intravenous administration of nanoparticle-containing medicines (nanomedicines). This review focuses on CARPA in rodent models: rats, mice, guinea pigs and rabbits. Information on all aspects of hypersensitivity reactions caused by known complement activators (zymosan, cobra venom factor) and different nanomedicines (liposomes, other drug carrier nanocarriers) in these species has been compiled and analyzed, trying to highlight the similarities and differences. What is most common in all species’ reactions to i.v. complement activators, liposomes and other nanoparticles is a dose-dependent hemodynamic and cardiopulmonary disturbance manifested in acute, reversible rise or fall of blood pressure and respiratory distress that can lead to shock. Other symptoms include heart rate changes, leukopenia followed by leukocytosis, thrombocytopenia, hemoconcentration due to fluid extravasation (rise of hematocrit) and rise of plasma thromboxane B2. The results of a recent rat study are detailed, which show that rats are 2–3 orders of magnitude less sensitive to liposome-induced CARPA than pigs or hypersensitive humans. It is concluded that CARPA can be studied in rodent models, but they do not necessarily mimic the human reactions in terms of symptom spectrum and sensitivity.
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Pulmonary intravascular macrophages: prime suspects as cellular mediators of porcine CARPA
European Journal of Nanomedicine, 2015Co-Authors: Domokos Csukás, László Rosivall, Rudolf Urbanics, György Wéber, Janos SzebeniAbstract:AbstractPigs provide a highly sensitive and quantitative in vivo model for complement (C) activation-related Pseudoallergy (CARPA), a hypersensitivity reaction caused by some state-of-art nanomedicines. In an effort to understand the mechanism of the pigs’ unique sensitivity for CARPA, this review focuses on pulmonary intravascular macrophages (PIMs), which are abundantly present in the lung of pigs. These cells represent a macrophage subpopulation whose unique qualities explain the characteristic symptoms of CARPA in this species, most importantly the rapidly (within minutes) developing pulmonary vasoconstriction, leading to elevation of pulmonary arterial pressure. The unique qualities of PIM cells include the following; 1) they are strongly adhered to the capillary walls via desmosome-like intercellular adhesion plaques, which secure stable and lasting direct exposition of the bulk of these cells to the blood stream; 2) their ruffled surface engaged in intense phagocytic activity ensures efficient binding and phagocytosis of nanoparticles; 3) PIM cells express anaphylatoxin receptors, this way C activation can trigger these cells, 4) they also express pattern recognition molecules on their surface, whose engagement with certain coated nanoparticles may also activate these cells or act in synergy with anaphylatoxins and, finally 5) their high metabolic activity and capability for immediate secretion of vasoactive mediators upon stimulation explain the circulatory blockage and other robust physiological effects that their stimulation may cause. These qualities taken together with reports on liposome uptake by PIM cells during CARPA and the possible presence of these cells in human lung suggests that PIM cells may be a potential therapeutic target against CARPA.
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Features of complement activation-related Pseudoallergy to liposomes with different surface charge and PEGylation: Comparison of the porcine and rat responses
Journal of controlled release : official journal of the Controlled Release Society, 2014Co-Authors: László Dézsi, Tamas Meszaros, Rudolf Urbanics, Tamas Fulop, Gábor Szénási, László Rosivall, Erik Őrfi, Csenge Vázsonyi, Reka Nemes, Robbert J. KokAbstract:Pigs are known to provide a sensitive model for studying complement (C) activation-related Pseudoallergy (CARPA), a hypersensitivity reaction to liposomal and many other nanomedicines that limits their clinical use. The utility of rats as a CARPA model has, however, not been analyzed to date in detail. The present study compared the two models by inducing CARPA with i.v. bolus injections of two reactogenic liposomes that differed from each other in surface properties: one was AmBisome, a strong anionic, free-surface small unilamellar liposome (SUV), while the other was neutral, polyethylene glycol (PEG)-grafted SUV wherein the 2 kDa-PEG was anchored to the membrane via cholesterol (Chol-PEG). Both in pigs and rats AmBisome caused significant consumption of C3, indicating C activation, along with paralleling massive changes in blood pressure, white blood cell, platelet counts and in plasma thromboxane B2 levels, indicating CARPA. These processes were similar in the two species in terms of kinetics, but significantly differed in the doses that caused major hemodynamic changes (~ 0.01 and ~ 22 mg phospholipid (PL)/kg in pigs and rats, respectively). Pigs responded to AmBisome with pulmonary hypertension and systemic hypotension, and the reaction was not tachyphylactic. The major response of rats was systemic hypotension, leukopenia followed by leukocytosis, and thrombocytopenia. Chol-PEG liposomes caused severe reaction in pigs at 0.1 mg/kg, while the reaction they caused in rats was mild even at 300 mg PL/kg. Importantly, the reaction to Chol-PEG in pigs was partly tachyphylactic. These observations highlight fundamental differences in the immune mechanisms of porcine and rat CARPA, and also show a major impact of liposome surface characteristics, determining the presence or absence of tachyphylaxis. The data suggest that rats are 2–3 orders of magnitude less sensitive to liposomal CARPA than pigs; however, the causes of these differences, the PEG-dependent tachyphylaxis and the massive reactivity of Chol-PEG liposomes remain unclear.
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Cardiopulmonary and hemodynamic changes in complement activation-related Pseudoallergy
Health, 2013Co-Authors: László Dézsi, Rudolf Urbanics, Gábor Szénási, László Rosivall, Janos SzebeniAbstract:Complement activation-related Pseudoallergy (CARPA) is a frequent side effect of intravenous therapies with nanoparticle-containing drugs and biologicals that are recognized by the immune system as foreign. It is an acute infusion reaction dominated by cutaneous and hemodynamic changes, most significantly a cardiopulmonary distress involving major pulmonary hypertension, systemic hypotension and arrhythmias. Because CARPA is unpredictable by conventional allergy tests and it may be life threatening, it can represent a major barrier to the safe therapeutic application of many modern medicines, including liposomal drugs and monoclonal antibodies. This review summarizes and updates the facts and opens questions regarding this phenomenon, with particular focus on its porcine model.
