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Budapest, Hungary
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Orfi E.,Semmelweis University | Szebeni J.,Semmelweis University | Szebeni J.,University of Miskolc | Szebeni J.,SeroScience Ltd.
Advanced Drug Delivery Reviews | Year: 2016

There is substantial effort in modern pharmacotherapy to use nanoparticle-based drug delivery systems (nDDS) for improving the oral absorption of drugs. An often neglected circumstance regarding this approach is that the gut is a major part of the immune system that may be vulnerable for immune-cell toxicity, or mediate humoral immune response against various components of nDDS, recognized as foreign. This review recapitulates the structure and function of gut-associated lymphoid tissue (GALT), i.e., the enteral section of mucosa-associated lymphoid tissue (MALT) and reminds how virus-like nDDS may potentially induce immunogenicity just as attenuated or killed viruses do in oral vaccines. Furthermore, we present examples for immune toxicities of emulsifiers and polymer-containing micelles, manifested in complement activation-related pseudoallergy (CARPA). A major message of the review is that early testing of immunogenicity or other adverse immune effects of nDDS in appropriate test systems or models may be prudent to recognize the risk of rare immune problems that may surface in late-stage clinical trials or after marketing of nDDS. © 2016 Elsevier B.V.


Weiszhar Z.,Seroscience Ltd. | Weiszhar Z.,Semmelweis University | Czucz J.,Seroscience Ltd. | Revesz C.,Seroscience Ltd. | And 6 more authors.
European Journal of Pharmaceutical Sciences | Year: 2012

Immunosafety analysis of pharmaceutical surfactants is an important step in understanding the complex mechanisms by which they induce side effects in susceptible patients. This paper provides experimental evidences that polyethoxylated surfactants, Cremophor-EL and Tween-80, also known as Polysorbate-80, activate the complement system in vitro, in normal human serum and plasma. They appeared to be more efficient reactogens than their structural homolog, Tween-20. Cremophor-EL and Tween-80 promoted the generation of biologically active complement products, C3a, C5a and C5b-9. Consistently, Paclitaxel and Taxotere (Docetaxel), pharmaceuticals formulated in Cremophor-EL and Tween-80, activated the complement system in similar extent. Moreover, comparison of serum reactivity against the drug-loaded and drug-free formulations exhibited a significant linear correlation. Taken together, these results are consistent with the hypothesis that therapeutic side effects, such as acute hypersensitivity and systemic immunostimulation, caused by intravenous nanomedicines containing polyethoxylated detergents such as Cremophor-EL and Tween-80, can be attributed to complement activation-derived inflammatory mediators. © 2011 Elsevier B.V. All rights reserved.


Epstein-Barash H.,Hebrew University of Jerusalem | Gutman D.,Hebrew University of Jerusalem | Markovsky E.,Hebrew University of Jerusalem | Mishan-Eisenberg G.,Hebrew University of Jerusalem | And 3 more authors.
Journal of Controlled Release | Year: 2010

Partial inactivation and transient depletion of monocytes/macrophages by liposomal bisphosphonates (LIP-BPs) is widely experimented in various inflammatory disorders including restenosis. Previous studies on activation of cytokines by LIP-BPs are limited to certain cell lines. Moreover, the correlation between in vitro and in vivo studies and complement (C) activation has not been reported. We report here a comprehensive study on the bioactivity of LIP-BPs on various cells' internalization and proliferation, mechanism of cell death, cytokines (in vitro and in vivo) and C activation (in the rat, rabbit and pig). The role of the following parameters has been determined i) drug type (clodronate/alendronate); ii) vesicles size (60-800. nm); iii) charge (neutral/negative/ positive); and iv) cell culture type (various cell lines and primary cultures). It was found that monocyte/macrophage inhibition and cytokine activation depend on the cell type, with a limited correlation to the bioactivity obtained in the rat and rabbit models of restenosis. Negatively charged liposomes (85 ± 20 nm) effectively depleted rabbit's monocytes (67% depletion), with a minor activation of cytokines and no C activation. It is concluded that cell culture studies are insufficient for assessing cytokine activation, and that by controlling LIP-BP properties (size, charge and drug type) optimal bioactivity could be achieved. © 2010 Elsevier B.V.


Szebeni J.,Semmelweis University | Szebeni J.,Seroscience Ltd. | Szebeni J.,University of Miskolc | Bedocs P.,Semmelweis University | And 7 more authors.
Journal of Controlled Release | Year: 2012

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. © 2012 Elsevier B.V.


Dezsi L.,Semmelweis University | Fulop T.,Semmelweis University | Meszaros T.,Semmelweis University | Szenasi G.,Semmelweis University | And 13 more authors.
Journal of Controlled Release | Year: 2014

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. © 2014 Elsevier B.V. All rights reserved.


Szebeni J.,Semmelweis University | Szebeni J.,Bay Zoltan Applied Research Nonprofit Ltd | Szebeni J.,University of Miskolc | Bedocs P.,Semmelweis University | And 7 more authors.
Advanced Drug Delivery Reviews | Year: 2012

Intravenous administration of low (milligram) doses of nanoparticulate materials in pigs can lead to acute cardiopulmonary, hemodynamic, hematological, biochemical and dermatological changes within minutes, mimicking the human infusion (or anaphylactoid) reactions to many state-of-the-art (nano)medicines and biologicals. Because of the causal role of complement (C) activation, the phenomenon was called C activation-related pseudoallergy (CARPA). This review summarizes the available information on porcine CARPA caused by different liposomes and polymers. It provides methodical details of the model and addresses the quantitation, sensitivity, specificity, reproducibility and variability of symptoms caused by different reactogenic drugs. We describe a unique feature of the model: the rise of tachyphylaxis (self-induced tolerance) as a function of structural properties of reactogenic agents. For drugs that cause tachyphylactic CARPA, such as liposomal doxorubicin (Doxil), the review recapitulates a recently reported method of desensitization, which may prevent this, as well as many similar hypersensitivity reactions. In explaining the underlying mechanism of tachyphylactic CARPA, a new theory on "double hit" is outlined, wherein the pulmonary intravascular macrophages (PIM cells) of pigs give aggravated response to simultaneous stimulation of their anaphylatoxin and other surface receptors (e.g., toll-like, PAMP, DAMP or mannose) that recognize vesicle surface molecular patterns. The porcine CARPA model might provide unique advantages in studying the mechanism of severe hypersensitivity reactions in man to i.v. drugs, as well as in identifying drugs and drug carriers that may cause such reactions. © 2012 Elsevier B.V.


PubMed | University of Twente, Semmelweis University, Medical University of Graz, nanoPET Pharma GmbH and 5 more.
Type: Journal Article | Journal: Nanomedicine (London, England) | Year: 2016

We report the physicochemical analysis of nanosystems intended for cardiovascular applications and their toxicological characterization in static and dynamic cell culture conditions.Size, polydispersity and -potential were determined in 10 nanoparticle systems including liposomes, lipid nanoparticles, polymeric and iron oxide nanoparticles. Nanoparticle effects on primary human endothelial cell viability were monitored using real-time cell analysis and live-cell microscopy in static conditions, and in a flow model of arterial bifurcations.The majority of tested nanosystems were well tolerated by endothelial cells up to the concentration of 100 g/ml in static, and up to 400 g/ml in dynamic conditions. Pilot experiments in a pig model showed that intravenous administration of liposomal nanoparticles did not evoke the hypersensitivity reaction. These findings are of importance for future clinical use of nanosystems intended for intravascular applications.


Merkel O.M.,University of Marburg | Urbanics R.,Seroscience Ltd | Bedocs P.,Seroscience Ltd | Bedocs P.,Semmelweis University | And 8 more authors.
Biomaterials | Year: 2011

Complement activation by polymeric gene and drug delivery systems has been overlooked in the past. As more reports appear in the literature concerning immunogenicity of polymers and their impact on gene expression patterns, it is important to address possible immune side effects of polymers, namely complement activation. Therefore, in this study the activity of low and high molecular weight poly(ethylene imine) and two PEGylated derivatives to induce complement activation were investigated in human serum. These in vitro results revealed that PEI 25 kDa caused significant and concentration dependent complement activation, whereas none of the other polymers induced such effects at their IC50 concentrations determined by MTT-assays. To verify these in vitro results, additionally, studies were carried out in a swine model after intravenous administration, showing complement activation-related pseudoallergy (CARPA), reflected in symptoms of transient cardiopulmonary distress. Injections of PEI 25 kDa or PEI(25k)-PEG(2k)10 at a dose of 0.05 and 0.1 mg/kg caused strong reactivity, while PEI 5 kDa and with PEI(25k)-PEG(20k)1 were also reactogenic at 0.1 mg/kg. It was found that PEI 25 kDa caused both self- and cross-tolerance, whereas the PEG-PEIs were neither self- nor cross-reactively tachyphylactic. As a result of this study, it was shown that PEGylation of polycations with PEG of 20 kDa or higher molecular weight may be favorable. However, potential safety concerns in the development of PEI-based polymeric carriers for drugs and nucleic acids and their translation from bench to bedside need to be taken into consideration for human application. © 2011 Elsevier Ltd.


Szebeni J.,University of Miskolc | Szebeni J.,Seroscience Ltd. | Bedocs P.,Semmelweis University | Bedocs P.,Uniformed Services University of the Health Sciences | And 10 more authors.
Nanomedicine: Nanotechnology, Biology, and Medicine | Year: 2012

Hypersensitivity reactions to liposomal drugs, often observed with Doxil and AmBisome, can arise from activation of the complement (C) system by phospholipid bilayers. To understand the mechanism of this adverse immune reaction called C activation-related pseudoallergy (CARPA), we analyzed the relationship among liposome features, C activation in human serum in vitro, and liposome-induced cardiovascular distress in pigs, a model for human CARPA. Among the structural variables (surface charge, presence of saturated, unsaturated, and PEGylated phospholipids, and cisplatin vs. doxorubicin inside liposomes), high negative surface charge and the presence of doxorubicin were significant contributors to reactogenicity both in vitro and in vivo. Morphological analysis suggested that the effect of doxorubicin might be indirect, via distorting the sphericity of liposomes and, if leaked, causing aggregation. The parallelism among C activation, cardiopulmonary reactions in pigs, and high rate of hypersensitivity reactions to Doxil and AmBisome in humans strengthens the utility of the applied tests in predicting the risk of CARPA. From the Clinical Editor: The authors studied complement activation-related pseudoallergy (CARPA) in a porcine model and demonstrate that high negative surface charge and drug effects leading to distortion of liposome sphericity might be the most critical factors leading to CARPA. The applied tests might be used to predict CARPA in humans. © 2012 Elsevier Inc.


Bedocs P.,Defense and Veterans Center for Integrative Pain Management | Capacchione J.,Uniformed Services University of the Health Sciences | Potts L.,U.S. National Institutes of Health | Chugani R.,Washington University in St. Louis | And 4 more authors.
Anesthesia and Analgesia | Year: 2014

BACKGROUND: Reports in the recent experimental literature have provided contradicting results in different animal species regarding the efficacy of IV lipid emulsion (ILE) in the reversal of cardiovascular and central nervous system symptoms of local anesthetic and other lipophilic drug overdoses. In particular, ILE seemed to be effective in rats, rabbits, dogs, and humans, but not in swine, for which it not only failed to reverse the adverse effects of anesthetics, but the animals also developed a generalized cutaneous mottling or a dusky appearance immediately after ILE, suggestive of another type of toxicity. The latter symptoms arise in complement (C) activation-related pseudoallergy, a hypersensitivity reaction to particulate drugs and agents. METHODS: Ten Yorkshire swine (15-20 kg) were sedated with ketamine and anesthetized with isoflurane. ILE 1.5 and 5 mL/kg 20% was administered via the ear vein while pulmonary arterial pressure, systemic arterial blood pressure, electrocardiogram, and end-tidal CO2 were recorded continuously. Thromboxane was measured in blood collected at baseline and 2 and 10 minutes after injections. Complement activation by lipid emulsion was also assessed in vitro with soluble terminal complement complex (SC5b-9) and sheep red blood cell assays. RESULTS: Significant increases were observed in the pulmonary pressure (median [interquartile range]) within minutes after the administration of ILE, both at doses 1.5 and 5 mL/kg (15 [12-16.5] to 18.5 [16-20] mm Hg, P = 0.0058 and 15.5 [13-17.25] to 39.5 [30.5-48.5], respectively). The systemic arterial blood pressure increased, and the heart rate decreased after both injections. Thromboxane B2 concentration (median [interquartile range]) in the blood plasma increased from a baseline of 617.3 [412.4-920] to 1132 [597.9-1417] pg/mL (P = 0.0055) and from 1276 [1200-2581] to 4046 [2946-8442] pg/mL (P = 0.0017) after the administration of 1.5 and 5 mL/kg ILE, respectively. Intralipid did not cause in vitro complement activation in human serum. CONCLUSIONS: ILE causes clinically significant hemodynamic changes in pigs, in concert with significant increases in the plasma thromboxane concentration. However, the in vitro tests did not confirm involvement of the complement system in human sera, leaving the underlying mechanism of these findings in doubt. Nonetheless, the observed hemodynamic and biochemical effects of ILE serve as a caveat that the pig is not an ideal model for the study of interventions involving ILE. Copyright © 2014 International Anesthesia Research Society.

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