Hypersensitivity to intravenous iron: classification, terminology, mechanisms and management.

Intravenous (IV) iron therapy is widely used in iron deficiency anaemias when oral iron is not tolerated or ineffective. Administration of IV-iron is considered a safe procedure, but severe hypersensitivity reactions (HSRs) can occur at a very low frequency. Recently, new guidelines have been published by the European Medicines Agency with the intention of making IV-iron therapy safer; however, the current protocols are still non-specific, non-evidence-based empirical measures which neglect the fact that the majority of IV-iron reactions are not IgE-mediated anaphylactic reactions. The field would benefit from new specific and effective methods for the prevention and treatment of these HSRs, and the main goal of this review was to highlight a possible new approach based on the assumption that IV-iron reactions represent complement activation-related pseudo-allergy (CARPA), at least in part. The review compares the features of IV-iron reactions to those of immune and non-immune HSRs caused by a variety of other infused drugs and thus make indirect inferences on IV-iron reactions. The process of comparison highlights many unresolved issues in allergy research, such as the unsettled terminology, multiple redundant classifications and a lack of validated animal models and lege artis clinical studies. Facts and arguments are listed in support of the involvement of CARPA in IV-iron reactions, and the review addresses the mechanism of low reactogenic administration protocols (LRPs) based on slow infusion. It is suggested that consideration of CARPA and the use of LRPs might lead to useful new additions to the management of high-risk IV-iron patients.

Complement activation cascade triggered by PEG-PL engineered nanomedicines and carbon nanotubes: the challenges ahead.

Since their introduction, poly(ethylene glycol)-phospholipid (PEG-PL) conjugates have found many applications in design and engineering of nanosized delivery systems for controlled delivery of pharmaceuticals especially to non-macrophage targets. However, there are reports of idiosyncratic reactions to certain PEG-PL engineered nanomedicines in both experimental animals and man. These reactions are classified as pseudoallergy and may be associated with cardiopulmonary disturbance and other related symptoms of anaphylaxis. Recent studies suggest that complement activation may be a contributing, but not a rate limiting factor, in eliciting hypersensitivity reactions to such nanomedicines in sensitive individuals. This is rather surprising since PEGylated structures are generally assumed to suppress protein adsorption and blood opsonization events including complement. Here, we examine the molecular basis of complement activation by PEG-PL engineered nanomedicines and carbon nanotubes and discuss the challenges ahead.

Sugar-free, glycine-stabilized intravenous immunoglobulin prevents skin but not renal disease in the MRL/lpr mouse model of systemic lupus.

Intravenous immunoglobulin (IVIG) has a therapeutic potential in many autoimmune diseases. Based on its immune modulating and complement inhibiting effects, IVIG has been tested in systemic lupus erythematosus (SLE), but due to osmotic tubular injury caused by immunoglobulin-stabilizing sugar components, lupus nephritis had been accelerated in some patients, thus IVIG use in SLE has been abandoned. The availability of non-sugar-stabilized IVIG raised the possible re-evaluation of IVIG for SLE. We investigated high-dose, long-term non-sugar-stabilized IVIG treatment on skin and renal SLE manifestations in the MRL/lpr mouse model. Animals were treated once a week with glycine-stabilized IVIG or saline (0.2 ml/ 10 g BW) from 6 weeks until they were humanely killed at 5 months of age. IVIG diminished macroscopic cutaneous lupus compared with saline treated mice. Histology and complement-3 immunostaining also demonstrated a significant reduction of skin disease after IVIG treatment. However, renal histology and function were similar in both groups. Compared with typical osmotic tubular damage induced by 5% sucrose and 10% maltose (used for IVIG stabilization), we did not observe any osmotic tubular injury in the glycine-stabilized IVIG treated mice. Our data demonstrate a beneficial effect of IVIG on skin lupus without renal side-effects. Deeper understanding of the organ-specific pathomechanism may aid an individualized SLE therapy.

Poly(ethylene glycol)s generate complement activation products in human serum through increased alternative pathway turnover and a MASP-2-dependent process.

Poly(ethylene glycol) (PEG) is receiving increasing attention as an intravenous therapeutic agent per se in a variety of experimental therapeutics and veterinary settings, such as spinal cord injury and traumatic axonal brain injury. PEG is often perceived to be immunologically safe, but here we demonstrate that near-monodisperse endotoxin-free PEGs, at concentrations relevant to above-mentioned settings, can generate complement activation products in human serum on a time scale of minutes (reflected in significant rises in serum levels of C4d, Bb, C3a-desArg and SC5b-9). With the aid of sera depleted from either C2 or C1q, and devoid of anti-PEG antibodies, we further demonstrate that, depending on PEG concentration and M(wt), generation of complement activation products occur either exclusively through the lectin pathway activation or through both the lectin pathway and increased fluid phase turnover of the alternative pathway. Inhibition of PEG-mediated C4d elevation in C1q-depleted serum by the broad serine protease inhibitor Futhan and anti-MASP-2 antibodies as well as competitive studies with d-mannose and N-acetylglucosamine indicated a likely role for ficolins/MASP-2 in PEG-mediated triggering of the lectin pathway and independent of calcium. PEG-mediated amplification of the alternative pathway is a complex process related to protein partitioning and exclusion effect, but factor H depletion/exclusion seems to play a minor role. Our results are relevant to the proposed potential therapeutic applications of intravenous PEG and warn about possible acute PEG infusion-related reactions in sensitive individuals and animals. PEG-mediated generation of complement activation products further provides a plausible explanation to the previously reported unexplained anaphylaxis or the referred cardiovascular collapse in sensitive animals that have received medicines containing high levels of PEG as solubilizer/carrier.

Stealth liposomes and long circulating nanoparticles: critical issues in pharmacokinetics, opsonization and protein-binding properties.

This article critically examines and evaluates the likely mechanisms that contribute to prolonged circulation times of sterically protected nanoparticles and liposomes. It is generally assumed that the macrophage-resistant property of sterically protected particles is due to suppression in surface opsonization and protein adsorption. However, recent evidence shows that sterically stabilized particles are prone to opsonization particularly by the opsonic components of the complement system. We have evaluated these phenomena and discussed theories that reconcile complement activation and opsonization with prolonged circulation times. With respect to particle longevity, the physiological state of macrophages also plays a critical role. For example, stimulated or newly recruited macrophages can recognize and rapidly internalize sterically protected nanoparticles by opsonic-independent mechanisms. These concepts are also examined.

Complement activation following first exposure to pegylated liposomal doxorubicin (Doxil): possible role in hypersensitivity reactions.

BACKGROUND: Pegylated liposomal doxorubicin (Doxil) has been reported to cause immediate hypersensitivity reactions (HSRs) that cannot be explained as IgE-mediated (type I) allergy. Previous in vitro and animal studies indicated that activation of the complement (C) system might play a causal role in the process, a proposal that has not been tested in humans to date. PATIENTS AND METHODS: Patients with solid tumors (n = 29) treated for the first time with Doxil were evaluated for HSRs and concurrent C activation. HSRs were classified from mild to severe, while C activation was estimated by serial measurement of plasma C terminal complex (SC5b-9) levels. Increases in SC5b-9 were compared in patients with or without reactions, and were correlated with Doxil dose rate. RESULTS: Moderate to severe HSRs occurred in 45% of patients. Plasma SC5b-9 at 10 min after infusion was significantly elevated in 92% of reactor patients versus 56% in the non-reactor group, and the rise was greater in reactors than in non-reactors. We found significant association between C activation and HSRs, both showing direct correlation with the initial Doxil dose rate. CONCLUSIONS: C activation may play a key role in HSRs to Doxil. However, low-level C activation does not necessarily entail clinical symptoms, highlighting the probable involvement of further, as yet unidentified, amplification factors.

Role of complement activation in hypersensitivity reactions to doxil and hynic PEG liposomes: experimental and clinical studies.

Pegylated liposomal doxorubicin (Doxil) and 99mTc-HYNIC PEG liposomes (HPL) were reported earlier to cause hypersensitivity reactions (HSRs) in a substantial percentage of patients treated i.v. with these formulations. Here we report that (1) Doxil, HPL, pegylated phosphatidylethanolamine (PEG-PE)-containing empty liposomes matched with Doxil and HPL in size and lipid composition, and phosphatidylglycerol (PG)-containing negatively charged vesicles were potent C activators in human serum in vitro, whereas small neutral liposomes caused no C activation. (2) Doxil and other size-matched PEG-PE and/or PG-containing liposomes also caused massive cardiopulmonary distress with anaphylactoid shock in pigs via C activation, whereas equivalent neutral liposomes caused no hemodynamic changes. (3) A clinical study showed more frequent and greater C activation in patients displaying HSR than in non-reactive patients. These data suggest that liposome-induced HSRs in susceptible individuals may be due to C activation, which, in turn, is due to the presence of negatively charged PEG-PE in these vesicles.

Formation of complement-activating particles in aqueous solutions of Taxol: possible role in hypersensitivity reactions.

We reported earlier that the anticancer drug paclitaxel (Taxol) activated the complement (C) system in human serum in vitro, raising the possibility that C activation might play a role in the ill-understood hypersensitivity reactions (HSRs) to this drug [J. Natl. Cancer Inst. 90 (1998) 300]. In pursuing the mechanism of C activation by Taxol, the present study provided evidence that dilution of the injection concentrate in aqueous solvents led to the formation of micelles and needle-like structures, both of which caused C activation in vitro. Micelles were formed mainly from Cremophor EL (CrEL), the nonionic emulsifier vehicle of paclitaxel, whose level in Taxol infusion exceeded its critical micelle concentration by at least 400-fold. CrEL micelles were shown by quasi-elastic light scattering and cryo-transmission electron microscopy (cryo-TEM) to be spherical with diameters in the 8-22 nm range; however, de novo formation of 50-300 nm microdroplets following incubation with human plasma suggested further fundamental structural transformation in blood. The needle-like structures extended to the multimicron range and were shown by electron diffraction to be crystalline paclitaxel. Taxol-induced C activation was manifested in varying rises of serum C3a-desarg, iC3b and SC5b-9. The causal role of CrEL micelles in C activation was demonstrated by the fact that filtration of aqueous solutions of Taxol or pure CrEL via 30-kDa cutoff filters eliminated, while the filter retentate restored C activation. C activation by Taxol was also inhibited by 10 mg/ml human immunoglobulin (IVIG). If proven clinically, HSRs to Taxol may represent a hitherto vaguely classified adverse drug reaction recently called C activation-related pseudoallergy (CARPA) [Circulation 99 (1999) 2302].

Complement activation-related pseudoallergy caused by liposomes, micellar carriers of intravenous drugs, and radiocontrast agents.

There is growing awareness that numerous drug-induced immediate hypersensitivity reactions (HSRs) do not fit in Gell and Coombs' Type I category of drug allergies, characterized by the pivotal pathogenic role of allergen-specific IgE. Such non-IgE-mediated "pseudoallergic" reactions are primarily caused by (1) certain liposomal formulations of intravenous drugs and imaging agents, (2) infusion liquids containing micelle-forming amphiphilic lipids or synthetic block-copolymer emulsifiers, and (3) iodinated radiocontrast media with limited solubility in water. Common features of the latter "pseudoallergens" include the capacity to activate the complement (C) system; also, the symptoms they cause are often typical manifestations of excessive anaphylatoxin generation in blood. Hence, these reactions have been called "C activation-related pseudoallergy" (CARPA). The present review surveys the experimental and clinical evidence for the involvement of C activation in HSRs caused by pseudoallergens in the above, three categories. To fit CARPA within the classical scheme of HSRs, a subdivision of Type I allergy is proposed on the basis of the mechanism of mast cell (and basophil) activation. The new scheme distinguishes direct and receptor-mediated HSRs, with the latter category subdivided to true IgE-mediated allergy; anaphylatoxin-mediated CARPA; and IgE plus anaphylatoxin double-triggered reactions. Further issues addressed in the review include animal models, risk factors, laboratory predictive tests, and pharmacological prevention of CARPA.

Liposome-induced pulmonary hypertension: properties and mechanism of a complement-mediated pseudoallergic reaction.

Intravenous injection of liposomes can cause significant pulmonary hypertension in pigs, a vasoconstrictive response that provides a sensitive model for the cardiopulmonary distress in humans caused by some liposomal drugs. The reaction was recently shown to be a manifestation of "complement activation-related pseudoallergy" (CARPA; Szebeni J, Fontana JL, Wassef NM, Mongan PD, Morse DS, Dobbins DE, Stahl GL, Bünger R, and Alving CR. Circulation 99: 2302-2309, 1999). In the present study we demonstrate that the composition, size, and administration method of liposomes have significant influence on pulmonary vasoactivity, which varied between instantaneously lethal (following bolus injection of 5 mg lipid) to nondetectable (despite infusion of a 2,000-fold higher dose). Experimental conditions augmenting the pulmonary hypertensive response included the presence of dimyristoyl phosphatidylglycerol, 71 mol% cholesterol, distearoyl phosphatidylcholine, and hemoglobin in liposomes, increased vesicle size and polydispersity, and bolus injection vs. slow infusion. The vasoactivity of large multilamellar liposomes was reproduced with human C3a, C5a, and xenoreactive immunoglobulins, and it correlated with the complement activating and natural antibody binding potential of vesicles. Unilamellar, monodisperse liposomes with 0.19 +/- 0.10 microm mean diameter had no significant vasoactivity. These data indicate that liposome-induced pulmonary hypertension in pigs is multifactorial, it is due to natural antibody-triggered classic pathway complement activation and it can be prevented by appropriate tailoring of the structure and administration method of vesicles.

Hemodynamic changes induced by liposomes and liposome-encapsulated hemoglobin in pigs: a model for pseudoallergic cardiopulmonary reactions to liposomes. Role of complement and inhibition by soluble CR1 and anti-C5a antibody.

BACKGROUND: Intravenous administration of some liposomal drugs can trigger immediate hypersensitivity reactions that include symptoms of cardiopulmonary distress. The mechanism underlying the cardiovascular changes has not been clarified. METHODS AND RESULTS: Anesthetized pigs (n=18) were injected intravenously with 5-mg boluses of large multilamellar liposomes, and the ensuing hemodynamic, hematologic, and laboratory changes were recorded. The significant (P<0.01) alterations included 79+/-9% (mean+/-SEM) rise in pulmonary arterial pressure, 30+/-7% decline in cardiac output, 11+/-2% increase in heart rate, 236+/-54% increase in pulmonary vascular resistance, 71+/-27% increase in systemic vascular resistance, and up to a 100-fold increase in plasma thromboxane B2. These changes peaked between 1 and 5 minutes after injection, subsided within 10 to 20 minutes, were lipid dose-dependent (ED50=4. 5+/-1.4 mg), and were quantitatively reproducible in the same animal several times over 7 hours. The liposome-induced rises of pulmonary arterial pressure showed close quantitative and temporal correlation with elevations of plasma thromboxane B2 and were inhibited by an anti-C5a monoclonal antibody (GS1), by sCR1, or by indomethacin. Liposomes caused C5a production in pig serum in vitro through classic pathway activation and bound IgG and IgM natural antibodies. Zymosan- and hemoglobin-containing liposomes and empty liposomes caused essentially identical pulmonary changes. CONCLUSIONS: The intense, nontachyphylactic, highly reproducible, complement-mediated pulmonary hypertensive effect of minute amounts of intravenous liposomes in pigs represents a unique, unexplored phenomenon in circulation physiology. The model provides highly sensitive detection and study of cardiopulmonary side effects of liposomal drugs and many other pharmaceutical products due to "complement activation-related pseudoallergy" (CARPA).

Complement-mediated acute effects of liposome-encapsulated hemoglobin.

Recent studies on liposome-encapsulated hemoglobin (LEH) have indicated that this potential blood substitute can activate the complement (C) system of rats, pigs and man. The reaction can involve both the classical and the alternative pathways, and is mediated, in part, by the binding of natural anti-lipid antibodies to the lipid membrane of liposomes. The significance of these discoveries lies in the fact that C activation appears to be the primary cause of the acute physiological, hematological and laboratory changes that have been observed previously in rats and pigs following the administration of LEH or liposomes, which changes include pulmonary vasoconstriction with decreased cardiac output. In light of the proposed use of LEH as an emergency blood substitute, the latter impairment of cardiopulmonary function may warrant particular circumspection as it could aggravate the clinical state of trauma patients who are prone to develop respiratory distress partly as a consequence of C activation by the injury. Our studies on rats and pigs suggest that the above acute side effects of LEH, including the cardiopulmonary distress, can be efficiently inhibited with soluble complement receptor type I, a specific inhibitor of C activation.

The interaction of liposomes with the complement system.

Activation of complement (C) by haptenized liposomes has been utilized for a long time to study the interaction of biological membranes with C proteins, or as a sensitive immunoassay for the measurement of specific antigens, antibodies, or serum hemolytic C levels. However, it has been increasingly recognized that regardless of antigenicity, C activation is an intrinsic property of all charged phospholipid/cholesterol bilayers. Liposome-induced C activation and its biological consequences show significant interspecies and interindividual variation, and critically depend on the properties of vesicles as well. Activation can proceed through both the classical and the alternative pathways, with or without the involvement of antibodies. The practical significance of the phenomenon is twofold: 1) opsonization of the vesicles promotes their clearance from the circulation and 2) the liberation of C3a and C5a can cause numerous, potentially adverse, biological reactions. In fact, many cardiovascular and hematological changes observed following administration of liposomes in vivo can be explained by C activation; a fact that has not yet gained wide recognition in the field of drug-carrier liposomes.

Complement activation by Cremophor EL as a possible contributor to hypersensitivity to paclitaxel: an in vitro study.

BACKGROUND: Cancer patients treated with the anticancer drug, paclitaxel (Taxol) often experience mild to severe hypersensitivity reactions. It is not known how these reactions are induced and whether the inducer is paclitaxel or its vehicle (i.e., Cremophor EL in 50% ethanol). Molecules present in Cremophor EL are similar in structure to certain nonionic block copolymers that activate complement proteins (i.e., proteins involved in various immune processes). To explore the role of complement in the observed hypersensitivity reactions, we studied the effects of paclitaxel and Cremophor EL plus ethanol on human complement in vitro. METHODS: Serum specimens from healthy individuals and cancer patients were incubated with paclitaxel or with relevant control compounds (Cremophor EL with ethanol, ethanol only, docetaxel, and cyclosporine), and markers of complement activation (SC5b-9 and Bb) were measured by enzyme-linked immunosorbent assay. Similar incubations were performed in the presence of inhibitors of complement activation (i.e., EGTA/Mg2+ and soluble complement receptor type 1 [sCR1]). RESULTS: Paclitaxel in Cremophor EL plus ethanol caused increased formation of SC5b-9 in serum specimens from 10 of 10 healthy control subjects and from five of 10 cancer patients. Experiments with one or more individual sera indicated the above effect was due to Cremophor EL plus ethanol, that increased formation of Bb also occurred, that the drug-induced rise in SC5b-9 was inhibited by sCR1, and that EGTA/Mg2+ partially inhibited SC5b-9 formation and stimulated Bb formation. IMPLICATION: The role of complement activation in hypersensitivity reactions associated with administration of paclitaxel in Cremophor EL plus ethanol should be studied in vivo.

Comparison of the lymphoid toxicities of mitobronitol and busulphan in mice: reduced B cell toxicity and improved thymic recovery as possible contributors to the reduced risk for complications following BMT with mitobronitol preconditioning.

It has previously been reported that the use of mitobronitol (dibromomannitol, DBM) instead of busulphan (BU) for myelosuppression is associated with significantly decreased risk for several complications of allogeneic bone marrow transplantation in accelerated chronic granulocytic leukemia. In exploring the pharmacologic basis for this observation, we have compared the acute and subacute cytotoxicities of DBM and BU on the spleen and thymus of mice. While there was comparable early (day 3) weight loss in both organs following these treatments, splenic B cells exhibited significantly less damage, and thymic regeneration (over weeks) was significantly faster following DBM treatment than with BU. These observations raise the possibility that improved post-BMT immune recovery could contribute to the clinical benefits observed with DBM-preconditioning.

Complement activation and thromboxane secretion by liposome-encapsulated hemoglobin in rats in vivo: inhibition by soluble complement receptor type 1.

Intravenous administration of liposome-encapsulated hemoglobin (LEH) in rats led to an early (within 15 min) decline of hemolytic complement (C) activity in the plasma along with a significant, parallel rise in thromboxane B2 (TXB2) levels. The TXB2 response was inhibited by co-administration of soluble C receptor type 1 (sCR1) with LEH, as well as by C depletion with cobra venom factor. These observations provide evidence for a causal relationship between LEH-induced C activation and TXB2 release, and suggest that sCR1 could be useful in attenuating the acute respiratory, hematological and hemodynamic side effects of LEH described earlier in the rat.

Complement activation in vitro by the red cell substitute, liposome-encapsulated hemoglobin: mechanism of activation and inhibition by soluble complement receptor type 1.

BACKGROUND: Liposome-encapsulated hemoglobin (LEH) has been developed as an emergency blood substitute, yet its effect on human complement has never been explored. Considering that complement activation is a major pathogenic factor in the respiratory distress syndrome that often develops in trauma and shock, LEH-induced complement activation may be a critical safety issue. STUDY DESIGN AND METHODS: Various LEH and corresponding empty liposomes were incubated with normal human sera, and various markers of complement activation (serum levels of C4d, Bb, SC5b-9, and CH50; C5a-induced granulocyte aggregation; membrane deposition of C3b) were measured. Incubations were also performed in the presence of (ethylene-bis[oxyethylenenitrilo]tetraacetic acid) (EGTA) and Mg++ (EGTA/Mg++) and soluble complement receptor type 1. RESULTS: LEH and liposomes activated human complement, as indicated by significant changes in one or more markers. The effect was primarily due to the presence of the phospholipid vehicle; small, unilamellar, highly homodispersed vesicles induced the greatest degree of complement activation. Complement activation was partially inhibited by EGTA/Mg++. The latter finding, together with the parallel increases in serum C4d and Bb, suggests activation of both the classical and alternative pathways. Soluble complement receptor type 1 (0.05-20 micrograms/mL) efficiently inhibited all vesicle-induced complement activation. CONCLUSION: Because of complement activation, the use of LEH for transfusion may require careful evaluation of safety. Soluble complement receptor type 1 may be useful as a prophylactic agent for complement activation-related complications of liposome infusions.

Complement activation in human serum by liposome-encapsulated hemoglobin: the role of natural anti-phospholipid antibodies.

In exploring the occurrence and mechanism of liposome-encapsulated hemoglobin (LEH)-induced complement (C) activation, we found that normal human serum contained low titers of IgG and IgM class natural antibodies with reactivity against LEH, and that the amount of vesicle-bound IgM significantly correlated with LEH-induced C consumption. IgM binding to LEH was inhibited by phosphocholine and ATP, but not by choline chloride. These data suggest that naturally occurring antibodies play a key role in LEH-induced C activation, and that a major portion of these antibodies are directed against the phosphate moiety on the phospholipid headgroups of liposome bilayers.

CAG repeat mutation in the mouse IL-2 gene enables concurrent assessment of T- and B-cell-specific gene expression by northern analysis.

The unique presence of CAG triplet repeats in the mouse IL-2 gene, lending the IL-2 cDNA a second specificity to MHC class II invariant chain (Ii) mRNA, coincides with another unusual genetic feature of the mouse, the lack of Ii gene upregulation in stimulated T cells. While the former anomaly allows simultaneous measurement of IL-2 and Ii mRNAs by Northern analysis, the latter condition ensures that Ii mRNA remains a T-cell-independent, specific marker of Ii positive, primarily B cell function. Thus, Northern analysis using CAG repeat containing IL-2 cDNA enables concurrent assessment of T and B cell activities in the spleen or in other mixtures of mouse lymphocytes.