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).
During the past few years, numerous clinical studies have been performed with liposomal formulations of anticancer drugs and other therapeutic agents. These studies attest to the general safety of intravenous liposomes, as 4 liposomal drugs entrapping doxorubicin (Doxil), daunorubicin (DaunoXome), and amphotericin B (Abelcet and Ambisome) are already licensed in several countries and many others are in advanced clinical trials.1 Nevertheless, some of the studies2 3 4 5 6 7 8 9 have also revealed a hypersensitivity reaction to liposomes that develops immediately after the start of infusion and includes symptoms of cardiopulmonary distress, such as dyspnea, tachypnea, tachycardia, hypotension and hypertension, chest pain, and back pain. Unlike IgE-mediated (type I) allergy, the reaction to liposomes arises at first exposure to the drug without prior sensitization, and the symptoms usually lessen or disappear on later treatments. Because of these unusual features, the reaction has recently been called “pseudoallergic.”9 The frequency of such reactions among 705 patients treated with Doxil was 6.8%,8 which is comparable to the incidence rate reported in other liposome trials.3 4 6 7 9 With the underlying cause not being understood, at present it is impossible to anticipate or specifically treat these reactions, which are severe, life-threatening in some 0.9% of patients, precluding further treatment with the liposomal formulation.8
It is known that certain liposomes can activate the complement system10 and that complement activation can lead to cardiovascular and pulmonary adverse responses very similar to those described above.11 12 13 Nevertheless, complement activation has not been implicated previously in the above-described clinical reactions.2 3 4 5 6 7 8 9 In an effort to test the hypothesis that complement activation plays a causal role in the cardiopulmonary reaction to intravenous liposomes, we extended here an earlier report from our laboratory on liposome-induced anaphylactoid reaction in miniature pigs.14 It was suggested in that study that the reaction was due to complement activation; however, direct, conclusive evidence regarding the causal role of complement was not available.
Another goal of the present study was to examine the acute physiological effects of the oxygen-carrying blood substitute liposome-encapsulated hemoglobin (LEH)15 16 in pigs. One of the potential applications of LEH is substitution of shed blood in trauma patients, who are prone to develop adult respiratory distress syndrome partly as a consequence of injury-related complement activation.12 13 Liposome-induced complement activation with additional cardiopulmonary distress therefore represents a critical safety issue that could be usefully addressed in a model sensitive to complement-mediated vasoactivity.
Closed-Chest Instrumented Pig
Experiments were performed in accordance with guidelines of the Committee on Animal Care of the Uniformed Services University of the Health Sciences. Female Yorkshire swine (32 to 48 kg) were sedated with intramuscular ketamine, anesthetized with halothane (1%), and instrumented as described previously in detail.17 In brief, a catheter was advanced via the right internal jugular vein into the pulmonary artery to measure pulmonary artery pressure (PAP), central venous pressure (CVP), and cardiac output (CO); another was advanced through the right femoral artery into the proximal aorta to measure systemic arterial pressure (SAP) and for blood sampling; and a third catheter was placed into the left ventricle through the left femoral artery to monitor left ventricular end-diastolic pressure (LVEDP). Systemic vascular resistance (SVR) and pulmonary vascular resistance (PVR) were calculated from SAP, PAP, CO, CVP, and LVEDP by standard formulas.17 Blood pressure and leads II and V5 of the ECG were recorded continually.
Preparation of Liposomes and LEH
The preparation and characteristics of large multilamellar liposomes consisting of dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, and cholesterol (50:5:45 mole ratios) with 0.5% α-tocopherol and 40 mg/mL heat-sterilized, diaspirin-crosslinked human hemoglobin (in LEH) were described previously.18 Liposomes were suspended in normal saline or PBS at 1 to 100 mg/mL lipid (≈1 to 100 mmol/L phospholipid).
Liposomes were injected into the jugular vein in 1-mL boluses containing 5 mg lipid (3.4 mg phospholipid and 1.6 mg cholesterol). These injections were repeated several times at 20- to 60-minute intervals. Before each injection, at 1-minute intervals up to 10 to 15 minutes, and finally at 30 minutes, hemodynamic parameters were recorded and arterial samples (2 to 3 mL) were withdrawn into EDTA-containing vials for white blood cell, platelet, total serum hemolytic complement/mL, and thromboxane B2 (TXB2) measurements, as described previously.19 Blood was also collected for hemoglobin, arterial O2, mixed venous O2, pH, and plasma HCO3 measurements, which remained in the normal range throughout the studies.
In further experiments, 2-mL aliquots from freshly prepared pig serum were incubated with liposomes (10 mg/mL lipid) at 37°C for 10 minutes with shaking. After addition of 4 volumes of PBS, vesicles were pelleted and the supernatant was immediately injected into pigs as described above for liposomes.
Murine anti-porcine C5a (GS1, Chemicon) was prepared from tissue culture or ascites, purified by protein G affinity chromatography, and dialyzed against PBS (purity, >95%). It was shown previously to inhibit C5a-induced porcine neutrophil aggregation with an IC50 of 3 μg/mL and to significantly inhibit polymorphonuclear leukocyte (PMN) chemotaxis at a dose of 17 μg/mL.20 Administration in pigs at 1.6 mg/kg maintains plasma GS1 levels >40 μg/mL for at least 3 hours.20a
Recombinant soluble complement receptor type 1 (sCR1)21 was obtained from T Cell Sciences, Inc. Its plasma clearance in pigs was reported to have α- and β-phase t1/2 values of 8.3 and 363 minutes, respectively, with 31% of drug clearing slowly (US patent 5,456,909). Previous studies showed 0.8 to 20 μg/mL sCR1 to effectively suppress LEH-induced complement activation in human serum in vitro.18
C5a Production and Immunoglobulin Binding by Liposomes In Vitro
Liposomes were incubated with pig serum with or without 10 mmol/L EGTA/2.5 mmol/L Mg2+ for 10 minutes at 37°C with shaking, and after centrifugal separation of vesicles, C5a was measured in serum by a chemotaxis assay.22 To measure liposome-bound IgG and IgM, vesicles were fixed in 1% paraformaldehyde (30 minutes, 4°C), washed with PBS, and stained with class-specific anti-swine antibodies (Kirkegaard). Fluorescence labeling was done with FITC-conjugated F(ab′)2 (Jackson) directed against anti-swine antibodies. Fluorescence-activated cell sorting (FACS) analysis was performed in a FACSort flow cytometer with live gating set on the forward scatter parameter.
Data are presented as single values or mean±SEM. Differences were analyzed by Student’s t tests or by ANOVA followed by Newman-Keuls correction. Fitting of nonlinear equations was done as described previously23 by use of maximum-likelihood algorithms of Gauss System 3.02 and Gaussx 3.5 (Aptech Systems). Confidence limits and standard errors of coefficients were obtained by computations of multiple regression coefficient (R2), residual-sum squares, and Durban-Watson statistics for serial errors.23 Randomness of residuals and error variance were examined by Wilk-Shapiro statistics (Statistix 4.1, NH Analytical Software) and the heteroscedasticity routines of Gaussx 3.5, respectively.
Effects of Liposomes and LEH on Pulmonary and Systemic Circulation
Figure 1⇓ demonstrates typical hemodynamic changes caused by injections of liposome boluses containing 5 mg lipid, defined as “standard” injection. These changes were transient and included a 50% to 250% increase in PAP (A), a 0% to 80% decline in CO (B), a 2- to 6-fold increase in PVR (C), a 5% to 10% increase in heart rate (D), a 20% to 40% fall or rise or biphasic changes in SAP (E), and a 0% to 400% rise of systemic vascular resistance (F). The reactions started within 1 to 2 minutes after injection, reached peaks within 5 to 6 minutes, and returned to respective baselines within 10 to 15 minutes.
Figure 2⇓ illustrates that 8 repetitive injections of standard liposome boluses into a pig at 30- to 60-minute intervals produced virtually identical rises in PAP, implying a lack of tachyphylaxis and remarkable quantitative reproducibility of hypertensive response. The latter properties were verified for most animals examined by injection of the standard boluses 2 to 3 times at the beginning and toward the end of the 6- to 8-hour experiments.
Table 1⇓ summarizes all hemodynamic data obtained in 18 pigs injected several times with the standard liposome boluses or an equivalent amount of LEH. The changes, expressed as percentage of baseline for peak responses, were significantly different from baseline (P<0.01, paired t test), except for SAP. Furthermore, the hemodynamic effects of empty liposomes were not significantly different from those caused by LEH, indicating that the changes were accounted for primarily by the phospholipid bilayer of liposomes. Nevertheless, there was a tendency for larger increases and greater variabilities in PAP and PVR in the case of LEH, suggesting some influence of surface-exposed hemoglobin.18
In addition to the above changes, we also observed that the most intense reactions were associated with transient ST-segment depression and T-wave changes on the ECG (data not shown), implying cardiac ischemia. Furthermore, the most marked elevations of PAP and declines of CO were associated with an initial decline of SAP (Figure 1E⇑), with increased CVP and decreased LVEDP (data not shown); these observations point to increased PVR as the primary effect of liposomes. The observation that the increase in heart rate (Figure 1D⇑) occurred independently of the changes in CO and/or SAP (Figure 1B⇑ and E) raises the possible involvement of mechanisms other than baroreflex response, for example, transient blockage of coronary circulation and/or direct humoral effects (complement split products, catecholamines) on the heart.
Figure 3A⇓ shows that the pulmonary hypertensive effect of liposomes displayed linear dependence on lipid dose in the 0- to 20-mg range, with an estimated ED50 of 4.5±1.4 mg lipid. This provided the rationale for using 5-mg lipid boluses as standard test dose. With boluses containing ≥20 mg lipid, the dose-response curve reached its plateau, indicating saturation of response. We also observed a dose-dependent change in the kinetics of PAP response, with readily reversible (within 10 minutes), symmetrical peaks after the injection of ≤10-mg liposome boluses and slowly reversing, asymmetrical waves after administration of 50 and 100 mg lipid (Figure 3B⇓).
Involvement of Serum in Mediating the Hemodynamic Effects of Liposomes
Intravenous injection of the pigs’ own serum after in vitro incubation with liposomes caused significant increases in PAP (65±16%, n=5 pigs), whereas untreated serum or serum that had been heat-inactivated at 55°C for 30 minutes before incubation with liposomes caused no or significantly less pulmonary hypertension (10±2% and 21±9%, respectively, n=4 pigs). These observations suggest that the pulmonary hypertensive effect of liposomes was linked primarily to an interaction of the vesicles with serum rather than to physical obstruction of pulmonary microcirculation or direct effects on tissue or blood cells. The heat-sensitivity of serum elements that are involved in this interaction points to a key role of complement proteins.
Effects of Liposomes on Platelets, White Blood Cells, and Serum Complement Levels
Figure 4⇓ shows early (2 to 5 minutes), minor (5% to 18%) decreases in platelet counts in 4 of 8 tested animals (A), with a parallel, minor decline of white blood cell count in 1 pig (B). Similar measurements after 2 to 3 subsequent liposome injections produced essentially identical results, with transient, <20% drops in cell counts. Measurements of hemolytic complement levels in pig plasma before and at 5 and 30 minutes after injection of standard boluses indicated no significant complement consumption (total hemolytic complement/mL values were 147±28, n=6 pigs; 162±26, n=5; and 137±26, n=3, respectively).
Liposome-Induced Changes in Plasma TXA2
Figure 5A⇓ shows that injection of the standard bolus in a pig caused massive (30-fold) increase in plasma TXB2 levels with a time course that exactly mirrored the rise of PAP. A second injection 30 minutes later, as well as several injections over the course of hours (Figure 5B⇓), produced essentially identical, parallel rises of PAP and TXB2.
Figure 6⇓ plots PAP peak responses versus plasma TXB2, using all matched preinjection and postinjection readings from 7 pigs. The best fit is a sigmoidal dose-response curve that shows no correlation between PAP and TXB2 below ≈1 ng/mL TXB2 but a strong, linear correlation above these values until the pulmonary response reaches saturation around 80 mm Hg. These data suggest that TXA2-induced vasoconstriction is likely to be a major mechanism of pulmonary hypertension.
Effects of Complement Inhibitors and Indomethacin on Liposome-Induced Pulmonary Hypertension
Direct evidence for causal roles of both complement activation and TXA2 release in liposome-induced pulmonary hypertension came from experiments using the specific complement inhibitors GS1 and sCR1 and the cyclooxygenase inhibitor indomethacin. These blockers inhibited the liposome-induced rises in PAP relative to preinhibitor (baseline) response (Figure 7A⇓), most efficiently indomethacin. The suppression of hypertensive response was not due to nonspecific toxicity or tachyphylaxis, because the inhibitory effects of these agents could be overcome by increasing the liposome doses (illustrated for sCR1). Figure 7B⇓ demonstrates the time points and extent of maximal inhibition that we observed in 4 pigs treated with each of the above inhibitors. We found that 5 mg/kg indomethacin completely blunted the pulmonary reaction to 5-mg liposome boluses in all pigs tested, whereas 1.6 mg/kg GS1 exerted 25% to 60% inhibition, and 0.2 and 2 mg/kg sCR1 (in 2 to 2 pigs) caused 30% to 100% inhibition. These differences between preinhibitor and postinhibitor rises in PAP were significant (P<0.01) by Student’s paired t test.
Pulmonary Vascular Effects of Zymosan
Injection of the (alternative pathway) complement activator zymosan in pigs in a fashion and at a dose level (5 mg) that simulated the administration of liposomes caused a 53±13% increase in PAP (n=7 injections in 4 pigs) with a time course that was indistinguishable from that observed with the standard liposome injections.
In Vitro Studies on the Mechanism of Liposome-Induced Complement Activation
Table 2⇓ shows that (1) incubation of pig serum with liposomes in vitro increased the leukocyte chemotactic activity of serum, (2) this increase was inhibited in the presence of GS1, and (3) the chemotaxis-promoting effect of liposomes was inhibited by EGTA/Mg2+. The first 2 observations provide evidence that liposomes can trigger complement activation in pig blood with resultant production of C5a, whereas the inhibitory effect of EGTA/Mg2+ on this process shows that this activation was Ca2+-dependent, a characteristic of classic pathway activation.
One possibility for complement activation via the classic pathway is the binding of natural anti-lipid antibodies to liposomes. To test this possibility, we measured the amount of immunoglobulins on the surface of liposomes after incubation with pig serum in vitro. The FACS analysis shown in Figure 8⇓ indicated binding of both IgG (A) and IgM (B) antibodies to liposomes, implying that preconditions for classic pathway activation exist in pigs.
A Novel Porcine Model of Pseudoallergic Cardiopulmonary Distress
The advance of liposomal drugs into clinical trials brought attention to an unusual hypersensitivity reaction that included hemodynamic changes with cardiopulmonary distress.3 4 5 6 7 8 9 Although in most cases the symptoms are manageable with corticosteroids and antihistamines, the reaction can be life-threatening in an occasional patient with a history of allergy and cardiopulmonary disease.3 4 5 8 The phenomenon has been called pseudoallergy9 ; however, its mechanism has not been clarified and, to the best of our knowledge, has never been studied in an animal model.
The present experiments extended a previous study from our laboratory reporting that intravenous injection of liposomes in miniature pigs triggered a dramatic anaphylactoid reaction.14 The liposomes were the same as applied here but were injected slowly (3 to 5 minutes) at a 500-fold higher dose. The reaction was associated with hemodynamic and TXB2 changes similar to those described here, except that they were prolonged for 30 to 60 minutes and were associated with significant (36% to 38%) leukopenia and thrombocytopenia. Furthermore, unlike in the present study, repeat injections of liposomes caused death in 3 of 4 animals.14 Thus, the reduction of liposome dose and a change in administration protocol resulted in improved reproducibility and control of hemodynamic changes, providing a sensitive animal model for liposome-induced cardiopulmonary distress.
Evidence for a Causal Role of Complement Activation
Based on the symptoms, laboratory changes, and evidence of serum-induced, anti-cholesterol antibody– and complement-dependent immune damage to liposomes in vitro, we proposed previously that the liposome-induced anaphylactoid reaction in pigs could be due to complement activation.14 The present work obtained the following new, more direct support for this concept: (1) the pulmonary response to liposomes was mediated by a heat-sensitive serum component; (2) the hemodynamic effects of liposomes were mimicked by the complement activator zymosan; (3) incubation of pig serum with liposomes in vitro led to the formation of C5a; (4) the liposome-induced rises of plasma TXB2, one of the secondary mediators produced in response to anaphylatoxin binding to responsive cells,11 closely paralleled the rises of PAP; and (5) the specific complement inhibitors GS1 and sCR1 caused significant inhibition of liposome-induced pulmonary hypertension. This latter observation was of particular importance because it provided direct evidence for a causal role of complement activation in the hemodynamic changes.
Figure 9⇓ provides a hypothetical reaction scheme for liposomal complement activation and subsequent cellular and molecular interactions that may underlie the hemodynamic response. With regard to the mechanism of complement activation, our data are consistent with natural antibody–mediated classic pathway activation, as described previously for liposome and LEH-induced complement activation in human serum.18 24 However, the involvement of other mechanisms, such as the alternative pathway amplification loop or direct binding of C1q and/or C3 to the phospholipid bilayer,10 cannot be excluded.
The efficient coupling of relatively weak complement signal to massive hemodynamic changes was most likely achieved through the actions of vasoactive mediators from PMNs, platelets, macrophages, basophils, and mast cells released in response to the binding of anaphylatoxins and C5b-9 to these cells.11 25 26 Among the secondary mediators, we focused here on TXA2, a potent vasoconstrictor eicosanoid that was shown previously to rise in the blood of pigs14 and rats19 after the injection of liposomes. Our data showed remarkable quantitative and temporal correlation between elevations of plasma TXB2 and PAP, which, together with the inhibitory effect of indomethacin on the reaction, provides evidence that a prostaglandin, most likely TXA2, was a major mediator in the amplification process.
In addition to TXA2-mediated vasoconstriction, complement activation can also lead to increased PAP through another mechanism: upregulation of adhesion and other surface molecules on endothelial cells, PMNs, and platelets, leading to PMN trapping in the microcirculation and microthrombus formation from PMN-platelet aggregates.27 28 29 In our study, however, the lack of major leukopenic or thrombocytopenic effects of low-dose liposome boluses, combined with the fact that indomethacin completely suppressed the liposome-induced rise in PAP (although it does not inhibit microcirculatory trapping of PMNs30 ) argues against a major involvement of this mechanism. In fact, indomethacin did not inhibit the 36% to 38% leukopenic and thrombocytopenic effects of liposomes in our previous pig study, although it reduced the rise of PVR from 317% to 20%.14 Nevertheless, it is possible that microcirculatory stasis may be a contributing factor to the prolonged hypertensive responses to larger liposome doses, as suggested by the 20% persistent elevation of PVR despite treatment with indomethacin in our previous study,14 and the prolongation of hypertensive response to 50- to 100-mg lipid boluses (Figure 3B⇑) in the present study.
Implications of the Porcine Liposome-Induced Cardiopulmonary Distress Model
The present experiments highlight a little-known yet clinically important interaction between the immune and pulmonary circulatory systems, whereby a minimal exposure of foreign particles to blood leads to substantial circulatory derangements. The reaction is a major manifestation of pseudoallergy,9 a poorly understood immediate hypersensitivity syndrome. Our evidence that complement activation is causally involved in the phenomenon provides a rationale to tentatively define it as “complement activation–related pseudoallergy,” or “CARPA,” and to use complement and cyclooxygenase inhibitors for the prevention or alleviation of symptoms.
Our finding that LEH causes cardiopulmonary distress in pigs suggests that the formulation tested may aggravate the clinical state of trauma patients.12 13 Therefore, it seems critical to reduce or eliminate the complement-activating potency of this or similar blood substitutes. The porcine model presented affords a uniquely sensitive bioassay for this purpose, as well as for the screening of liposomal drugs for potential cardiopulmonary side effects. The model could also be used for the biocompatibility testing of colloidal dispersions, particulate biomaterials, oil-based drug vehicles (such as Cremophor EL31 ), and many other pharmaceutical products that may cause unexplained hypersensitivity reactions.
This study was supported by a grant from the US Naval Medical Research and Development Command and the Naval Research Laboratory (program 0603706N, project M2336.001.9717) and by grants from the NIH to Dr Stahl (HL-52886, HL-56086) and Dr Bünger (RO-7638). The authors thank Dr John Hess for providing cross-linked hemoglobin, Drs Lajos Baranyi and Sandor Savay for helpful comments, and Ruoyen Cheng and Eva Fleischmann for technical assistance. Dr Stahl is an Established Investigator of the American Heart Association.
- Received July 23, 1998.
- Revision received December 30, 1998.
- Accepted January 4, 1999.
- Copyright © 1999 by American Heart Association
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Szebeni J, Muggia FM, Alving CR. Complement activation by cremophor EL as a possible contributor to hypersensitivity to paclitaxel: an in vitro study. J Natl Cancer Inst. 1998;90:300–306.Intravenous injection of milligram amounts of liposomes in pigs caused significant hemodynamic changes, most importantly a 79±9% rise in pulmonary arterial pressure and 30±7% decline in cardiac output; consequences of a primary, 236±54% increase in pulmonary vascular resistance. The reaction was transient and could be repeatedly induced in the same animal over several hours. The strong correlation between the rises of pulmonary arterial pressure and plasma thromboxane B2, together with the inhibitory effects of complement blockers and indomethacin, provides strong evidence that the cardiopulmonary reaction to liposomes is due to complement activation with subsequent secretion of thromboxane A2.