Introduction: CARPA as an immune-mediated stress reaction in blood triggered by nanomedicines
Nanotechnology has achieved remarkable success in improving the therapeutic index of numerous drugs and agents by the use drug carrier nanosystems (nanocarriers) that carry and target the active pharmaceutical ingredient (API) to its site of action and/or control its ADME properties. However, the resulting increase of efficacy or decrease of toxicity is in some cases not without a price, as many of such nanodrugs or agents can cause adverse effects that the API alone would not do. We refer to them as reactogenic nanomedicines (RNMs). One of such adverse effects is complement (C) activation, which can lead to a syndrome called C activation-related pseudoallergy (CARPA). It is an acute and reversible immune reaction, also known as infusion reaction (or anaphylactic/anaphylactoid or idiosyncratic reaction), or non-immune allergy, whose symptoms, clinical significance, mechanism and many other properties were detailed previously in numerous reviews (1–8). An apparently trivial observation 20 years ago, that C activation underlies the major hemodynamic disturbance in rats following the infusion of liposomes (9) launched the progress of the CARPA concept, whose latest milestone is a broad claim that CARPA is a manifestation of a chemical stress on blood (8). More explicitely, it is a universal defense process against the “threat” of nanomedicines, that can occur in any organisms that has blood and C proteins therein, which falsely perceive the nanoparticles as pathogenic viruses. It is a stress reaction because just like in classical stress, an external harm (“pseudovirus”) triggers a nonspecific, standard battery of physiological changes via a multiorgan “axis”, namely the “immuno-cardiovascular” axis, which corresponds to the “hypothalamo-pituitary-adrenal” axis involved in classical stress (8).
Depending on the features of RNMs, their administration speed and the use of anti-allergic premedication, the prevalence of CARPA in man can reach 30%–40% with mostly transient and mild symptoms. The reaction usually occurs unpredictably at the first use of the drug, as there is no known laboratory or any clinical test that could estimate the risk of a reaction. Because in rare cases CARPA may be severe, even fatal, the phenomenon is getting increasing regulatory attention. For example, the European Medicines Agency’s latest guidance on generic liposome development recommends CARPA assays for preclinical safety testing (10). However, it has not solidified to date which CARPA tests are appropriate, or best for regulatory purposes, which parameters need to be measured and under what conditions? In general, the use and utility of CARPA tests are in an early stage of scientific evaluation.
In vitro tests for CARPA
In vitro testing of CARPA can be non-cellular and cellular. The non-cellular testing is based on the measurement of C activation by the RNM in serum, plasma or whole blood, using C split product ELISAs. There are many C split products that serve as activation marker and have commercially available pathway specific ELISAs, the best known being C3a, C5a, iC3b, C4d, Bb and SC5b-9. One issue in this regard is the inter-laboratory variation of test results, which problem has been addressed recently by efforts to produce international C standards (11–13). The information that these in vitro assays provide on the risk of CARPA is limited, since they report only on the activity of afferent arm of CARPA, the extent of anaphylatoxin formation. The efferent arm, the body’s response to anaphylatoxins, remains unknown. The most relevant cellular assays that can measure anaphylatoxin sensitivity are various basophil assays, which quantitate basophil leukocyte activation and/or secretion to model the mast cells’ response to allergens or other RNMs (14–16). We reported some preliminary, promising results with a basophil assay measuring CD203c upregulation as a predictor of liposomal CARPA (6), however, a study dedicated for the evaluation of the predictive value of the basophil test in pseudoallergy remains to be done.
Animal models of immune toxicity: which is good for CARPA evaluation?
Animal models represent a major tool for the study of mechanisms in virtually all biomedical research. Hypersensitivity reactions result from a complex combination of genetic, environmental and temporal factors as well as complex interactions between the immune and other organ systems and the drug, making these reactions uniquely diverse. As delineated above, in vitro systems are unlikely able to mimic such complexity. There is a consensus in the field that HSRs can only be tested in whole animal models (17–19). The critical question in this regard whether the standard, already accepted tests are appropriate for CARPA evaluation, or not.
Among the established animal models of immune toxicity, some can a priori be ruled out as CARPA test, based on their mode of operation. These include the mouse popliteal lymph node assay (PLNA) (20–22), the mouse ear-swelling test (23–25), the guinea pig (GP) maximization test (GPMT), the GP occluded patch test (Buehler’s test) (26, 27) and the murine local lymph node assay (MLLA) (26). In fact, the standard guinea pig assays were shown to be useless in predicting systemic hypersensitivity (28). The measurement of blood lymphocyte counts and lymph node weight or assaying B, T or other immune cell proliferation or function evaluate long-term immune toxicity, and not CARPA, which is a short term toxicity. Taken together, to our best knowledge, none of the regulatory standard animal tests may be appropriate to assess CARPA.
Non-standard immunotoxicity tests of CARPA in different animals
As pointed out, CARPA may be considered as a universal stress reaction, which implies that it may be present in most, if not all levels of mammalian evolution. Accordingly, there is a great number of reports in the literature on C activation and its consequences in various animals, including rats (9, 29–33), mice (33), dogs (34–37), rabbits (38, 39) nonhuman primates (40–49) and pigs (50–55). Among these experimental systems, it is the pigs’ response to C activator liposomes that best mimics the human infusion reactions to liposomes in terms of kinetics and spectrum of symptoms and the conditions of reaction induction (Table 1). For Doxil, for example, it was calculated that the drug dose that triggers CARPA in pigs corresponds to the dose that triggers infusion reactions in hypersensitive man (56). These facts, taken together with the favorable ethical and financial aspects of working with pigs rather than dogs or primates, rationalizes the use of pigs as CARPA model. Thus, CARPA adds to the list of diseases that are successfully studied in pigs, as discussed below.
The use of pigs as disease models
Pigs are widely used as large animal models in biomedical research (57–61), particularly in studies on cardiovascular diseases (62–67), trauma (68–71), sepsis (72–76), drug intoxications (54, 55), and, since 1999 (53), CARPA. As described in many previous experimental studies (50–53, 77) and a recent review (7), the porcine CARPA model represents a highly sensitive and reproducible model for the most serious, life threatening HSRs in man caused by RNM.
Technical details of the porcine CARPA model
Figure 1 illustrates the setup and instruments used in the model. In brief, domestic pigs (usually 2–3 months old) or miniature pigs are initially sedated (with Calypsol/Xilazine) and, after tracheal intubation, anesthesized with isoflurane while breathing spontaneously. The animals thereafter undergo surgery to place multiple catheters into their circulation for the measurement of different hemodynamic parameters, administration of test drugs and blood sampling. Namely, a Swan-Ganz catheter is placed to the pulmonary artery wedge, for the measurement of pulmonary arterial pressure (PAP), and (optionally) central venous pressure (CVP) and cardiac output (CO). Additional catheters are placed into the femoral artery to record the systemic arterial pressure (SAP) and, (optionally), left ventricular end-diastolic pressure (LVEDP). The left femoral vein is canulated for blood sampling, and the external jugular vein for the administration of test articles and to maintain a slow drop infusion of saline (∼3 mL/kg/h). For more sophisticated hemodynamic analysis to measure systemic vascular resistance (SVR) and pulmonary vascular resistance (PVR), additional catheters are placed and measurements and calculations are carried out. The hemodynamic, EKG and respiratory parameters are measured continuously, while blood cell counts, O2 saturation, blood analytes (inflammatory and vasoactive mediators) and temperature are measured at predetermined times, usually in 10–20 min intervals. EKG leads I-III are placed at the standard Einthoven positions.
The symptom tetrad
Figure 2 illustrates the four types of symptoms observed during CARPA in pigs: hemodynamic (A), hematological (B); laboratory (C) and skin (D) changes. Among the hemodynamic symptoms, the rise of PAP is the most prominent and reproducible measure of CARPA in the porcine model, which is invariably present with all RNMs. The transient, massive pulmonary hypertension is most likely due to the presence of pulmonary intravascular macrophages (PIM cells) in the lung of pigs, a theory based on the speed (seconds) and prominence (maximal possible) of pulmonary changes in this species taken together with the known properties and functions of PIM cells (78). Namely, PIM cells are directly exposed to blood and their function is to screen out from blood particulate pathogens. They can be activated both by anaphylatoxins and via particle binding to their surface receptors, and they respond to activation with massive secretion of vasoactive mediators (78). These features are necessary and at the same time enough to explain the characteristic changes of PAP during CARPA in pigs.
The changes of SAP are more variable; it can drop, rise, display no change or undulate. The hematological changes typically include initial leukopenia followed by protracted leukocytosis and thrombocytopenia: among these the leukopenic effect is the most frequent. Among the laboratory changes the rise of TXB2 is measured most often as it was found to show massive alterations in CARPA (53). The exploration of changes of further allergy and inflammatory mediators (e.g. tryptase, leukotrienes, PAF, chemokines, cytokines) represents an unmet need in this field. Finally, the skin changes are rare and variable, they are seen only in case of very strong reactions.
Uniqueness of the porcine CARPA model
The uniqueness of the pig CARPA model lies in the identity, or close similarities of symptoms to the human CARPA reactions (Table 1) and the quantitative nature and reproducibility of measured endpoints, most significantly the rise of PAP. It should be stressed here again that the high reproducibility applies to the reactions to the same RNMs in different pigs, and NOT to the reactions to different RNMs.
Invariable parameters of porcine CARPA
As mentioned, the rise of PAP to i.v. bolus injection of certain RNMs is remarkably constant and reproducible if the dose and administration schedule is the same in all animals. This statement is demonstrated in Table 2, which summarizes the rise of PAP to 3 different RNMs in numerous independent experiments performed over many years.
Yet another remarkable constancy of the model is the rise of PAP in the same animal, upon repetitive administration of a non-tachyphylactic RNM, such as large multilamellar liposomes (MLV) consisting of dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol and cholesterol (50:5:45 mole ratios). Figure 3 illustrates this constancy: repeated injection of 5-mg lipid containing MLV in a pig at 30- to 60-min intervals eight times raised the PAP with negligible (6%) coefficient of variation (53).
Variable parameters of porcine CARPA
The standard pulmonary reaction in porcine CARPA, observed immediately after bolus injection of RNMs consists of a rapid rise of PAP within 2–5 min after the bolus, a plateau reached in 5–15 min and decline to baseline or near baseline within 15–60 min (Figures 2A and 5). However, there are unpublished examples for delayed start and/or protracted reactions as well, which we obtained with bolus administration of a surface conjugated liposomal nanomedicine (Figure 4A) and upon stepwise infusion of a PEGylated liposomal drug candidate (Figure 4B).
In the former experiment, testing the reactogenicity of a surface-conjugated SUV (Figure 4A), the rise of PAP started only 40 min after bolus administration of the sample and the curve showed 3 progressively diminishing peaks over ∼1.5 h. This observation remains to be understood, just as the late start of a protracted pulmonary hypertension observed during stepwise infusion of a PEGylated SUV drug candidate (Figure 4B). After the initial, usual sharp and rapid reaction another reaction started at 30 min, the PAP peaked at 110 min and returned to normal after 180 min. These complex curves reflect complex interactions underlying the reactions, whose understanding will require a lot more work. It should be noted though that in clinical practice reactions can also start late, even hours after the infusion, and may also extend over hours.
Blood pressure wave forms
Another feature of porcine CARPA is that the relative intensities and waveforms of PAP and SAP show substantial variation among different reaction inducers. Figure 5 shows a selection of different PAP/SAP/heart rate (HR) curves that were registered following bolus injection of different RNMs specified in the legend. The combination of different changes appears random at this time, as no known particle property can be associated with any particular time course or kinetic variables of PAP or SAP or HR. Nevertheless one observation can be explained: the initial splicing of PAP arises most likely from the coincidence of pulmonary hypertension with systemic hypotension, best discernible in Figure 5A. The example shown for extended reaction is in Panel E, wherein a highly negative SUV formulation of an anticancer lipophilic (pro)drug was tested. The roller-coaster swings of PAP extended over 1.5 h, which may be attributed to metabolism or short-term physiological effects of the liposomes or the released drug leading to varying blood levels of different vasoactive mediators.
Yet another property of CARPA showing significant variation depending on the RNM is the presence/absence, and duration of tachyphylaxis, i.e. self-induced tolerance. The importance of tachyphylactic CARPA lies in the capability it offers for tolerance induction, i.e. to develop safe administration protocols for RNMs. In tachyphylactic CARPA the first dose of the RNM induces tolerance to the second or later administration of the same RNM, and if the first tolerogenic dose is made harmless, the hypersensitivity reactions (HSRs) can be entirely prevented. The utilization of tachyphylaxis for the prevention of HSR was first described for Doxil, wherein the HSR could be prevented by prior slow infusion with Doxebo (82) (Figure 6A), a doxorubicine-free (placebo) Doxil that retains the tachyphylaxis-causing, self- tolerogenic effect of Doxil without causing major HSR (82). However, not all RNMs can cause full tachyphylaxis, as it is a variable and structure-dependent property of RNMs; and it can also be absent (like in Figure 3), partial (Figure 6B) or biphasic (Figure 6C).
To date, the absence of tachyphylaxis was observed with MLV (Figure 3), zymosan (Figure 6A), Ambisome (50), some PEI polymers (80) and, in general, highly charged nanoparticles (unpublished observations). Partial tachyphylaxis occurs more frequently with liposomes and it is a dose dependent phenomenon. The biphasic tachyphylaxis shown in Figure 6C, wherein a section of nontachyphylactic responses switches to another nontachyphylatic section with smaller, but equal peaks, was observed for SAP after repetitive administration of the same dose of zymosan (7).
The mechanism of full or partial tachyphylaxis is not understood; based on its rapid appearance (minutes), it is most likely a passive phenomenon rather than an immune learning-based, cell-mediated active process. One theory, referred to as “double hit” hypothesis, points to allergy-mediating cells (mast cells/basophils/PIM cells as anatomic sites of tachyphylaxis, and suggests that tachyphylactogenic RNMs trigger CARPA both via C activation and via direct binding to these allergy mediating cells, and the two signals together trigger these cells to release vasoactive mediators. If one of these mechanisms is not working for some reason at the time of repeated treatments, because a mediator gets consumed or a signal transduction pathway gets exhausted, tachyphylaxis will ensue (7, 78).
Summary and future directions
This review provided background information for placing the porcine CARPA model on the map of preclinical immunotoxicological tests for CARPA. It also focused, for the first time, on the constant and variable features of CARPA symptoms in pigs, particularly those seen in the hemodynamic response. The inter-animal and inter-experimental stability of PAP changes in response to a certain type of RNM, and switch to another type of response with another type of RNM represent remarkable, yet ill-understood features of the model that reflect the involvement of highly regulated, complex immunological and physiological processes.
Figure 7 illustrates this complexity by providing an overall scheme of molecular and cellular interactions in CARPA (8). It shows the involvement of several organ systems, numerous cells and highly active biomolecules and mediators and multiple redundant pathways in both the afferent (triggering of allergy-mediating cells) and efferent arms (mediation of trigger signal to the effector cells) of CARPA.
Figure 8 provides further glimpses into the complex picture of CARPA pathogenesis, by highlighting the vicious cycle of hemodynamic and other organ derangements that may lead to the worst outcome of this immune toxicity; anaphylactic shock with circulatory collapse.
In summary, the constant and, at the same time highly variable symptoms of CARPA in pigs reflect the simplicity and at the same time the complexity of a “stress reaction in blood” (8), a novel immune phenomenon brought to light by the introduction of nanotechnology in pharmacotherapy. Further studies on the porcine CARPA model will hopefully reveal more details of the phenomenon and more understanding of its clinical use in the safety evaluation of novel nanomedicines.
JS and UR acknowledge the financial support to the Nanomedicine Research and Education Center at Semmelweis University from Gedeon Richter NyRT and EU FP7 projects No: 309820 (NanoAthero) and 310337 (CosmoPhos). We also acknowledge the grants to SeroScience Ltd from EU FP7 projects No: 602923 (TheraGlio) and 281035 (TransInt).
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About the article
János Szebeni, MD, PhD, DSc, MedHabil, immunologist, director of the Nanomedicine Research and Education Center at Semmelweis University, Budapest, Hungary. He is also founder and CEO of a contract research SME “SeroScience”, and full Professor of (immune) biology at Miskolc University, Hungary. He has held various guest professor and scientific positions in Hungary and abroad, mostly in the USA where he lived for 22 years. His research on various themes in hematology, membrane biology and immunology resulted in more than 120 scientific papers (citations: >4550, H index: 35), 14 book chapters, two granted patents, a book entitled “The Complement System: Novel Roles in Health and Disease” (Kluwer, 2004). Three fields stand out where he has been most active: artificial blood, liposomes and the complement system. His original works led to the “CARPA” concept, i.e. that complement activation underlies numerous drug-induced (pseudo)allergic (anaphylactoid) reactions.
Péter Bedőcs, MD, PhD graduated cum laude as a doctor of Medicine from Semmelweis University in 2004. He completed his PhD training at the same institution, focusing on pathophysiological effects and immunotoxicity of drug delivery systems utilizing nanotechnology. In collaboration with Semmelweis University, the Bay Zoltan Foundation in Hungary, and the Uniformed Services University of the Health Sciences (USUHS) in the United States, he participated in the development of an animal model for the testing of complement activation by intravenous therapeutic and diagnostic agents, which became an FDA-recommended preclinical test for nanopharmaceuticals. This also led to the establishment of an effective desensitization procedure to prevent immunotoxic reactions provoked by certain nanomedicines.
Currently he works at the Defense and Veterans Center for Integrative Pain Management as a senior scientist and as an Assistant Professor at the Department of Military and Emergency Medicine at USUHS in Bethesda, MD, overseeing multiple clinical and animal studies in the area of patient safety in anesthesia, developing new effective modalities for pain management. Additionally, he continues to serve as a collaborator and consultant for the Malignant Hyperthermia Diagnostic Center at USUHS.
Rudolf Urbanics, MD, PhD, is Head of the in vivo laboratory of Nanomedicine Research and Education Center of Semmelweis University, and SeroScience Ltd., an immunotoxicity CRO, since 2008 in Budapest, Hungary.
He obtained his MD diploma and the PhD degree at Semmelweis Medical School, Budapest, Hungary. He had various research/collaboration positions at MaxPlanck Institute of Systemphysiology, at University of Pennsylvania, Cerebrovascular Research Center, at Pennsylvania Muscle Institute, working in the field of CNS regulation of blood flow/metabolism, ischemic/hypoxic disorders, stroke and chronic neurodegenerative disease animal models.
He was the Deputy R&D Director and Head of CNS Pharmacology Department at Biorex R&D Co., and worked at IVAX/Drug Research Institute Budapest, as a leading researcher in safety and CNS pharmacology and later at the IVAX/Drug Research Institute, a subsidiary of TEVA as Head of the In Vivo Pharmacology Group.
Currently, he is working with in vivo models of nano drug-nano carrier induced, complement activation related pseudoallergic reactions (CARPA), clarifying their immune-toxicological and safety hazards.
Published Online: 2015-06-06
Published in Print: 2015-06-01