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. 2019 Feb 26:14:1563-1573.
doi: 10.2147/IJN.S187139. eCollection 2019.

Acute physiological changes caused by complement activators and amphotericin B-containing liposomes in mice

Erik Őrfi  1   2 , Tamás Mészáros  1   2 , Mark Hennies  3 , Tamás Fülöp  1   2 , László Dézsi  1   2 , Alexander Nardocci  1 , László Rosivall  1   2 , Péter Hamar  4   5 , Barry W Neun  6 , Marina A Dobrovolskaia  6 , János Szebeni  1   2   7 , Gábor Szénási  1   8
Affiliations

Acute physiological changes caused by complement activators and amphotericin B-containing liposomes in mice

Erik Őrfi et al. Int J Nanomedicine. .

Abstract

Purpose: Undesirable complement (C) activation by nanomedicines can entail an adverse immune reaction known as C activation-related pseudoallergy (CARPA) in sensitive patients. The syndrome includes cardiopulmonary, hemodynamic, and a variety of other physiological changes that have been well described in man, pigs, dogs, and rats. However, the information on CARPA is scarce and ambiguous in mice, a species widely used in preclinical studies. The present study aimed to fill this gap by exploring signs of CARPA in mice following i.v. administration of AmBisome and Abelcet, which are nano-formulations of Amphotericin B with high risk to cause CARPA.

Materials and methods: Anesthetized NMRI mice were intravenously injected with liposomal amphotericin B (Abelcet and AmBisome; 30-300 mg phospholipid/kg), drug-free high cholesterol multilamellar vesicles (HC-MLV), and positive controls, cobra venom factor (CVF) and zymosan, followed by the measurement of blood pressure (BP), heart rate, white blood cell, and platelet counts and plasma thromboxane B2 (TXB2) levels. C activation was assessed by C3a ELISA, a C3 consumption assay (PAN-C3) and a modified sheep red blood cell hemolytic assay.

Results: All test agents, except HC-MLV, caused transient hypertension, thrombocytopenia, and elevation of plasma TXB2, which were paralleled by significant rises of plasma C3a in CVF and zymosan-treated animals, wherein the initial hypertension turned into hypotension and shock. Abelcet and AmBisome caused minor, delayed rise of C3a that was not associated with hypertension. The C3a receptor inhibitor SB-290157 attenuated the hypertension caused by Abelcet and decreased the BP thereafter.

Conclusion: The parallelism between C3a anaphylatoxin production and severity of physiological changes caused by the different agents is consistent with CARPA underlying these changes. Although the reactive dose of liposomal phospholipids was substantially higher than that in other species (pigs, dogs), the mouse seems suitable for studying the mechanism of hypersensitivity reactions to liposomal formulations of amphotericin B, a frequent side effect of these drugs.

Keywords: TXB2; anaphylatoxins; cholesterol; cobra venom factor; hypersensitivity; infusion reactions; platelets; zymosan.

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Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Schematic outline of the study design. Abbreviations: BP, blood pressure; HR, heart rate.
Figure 2
Figure 2
Hemodynamic changes caused by complement activators and liposomes in male NMRI mice (n=5–6). Note: (A) Zymosan (30 mg/kg), (B) CVF 100 U/kg), (C) Abelcet (30 mg/kg), and (D) AmBisome (300 mg/kg) were administered as i.v. bolus at times indicated by the arrows. Abbreviations: CVF, cobra venom factor; NMRI, Naval Medical Research Institute.
Figure 3
Figure 3
Hematological changes (A–C), TXB2 levels (D), complement consumption (E), and plasma extravasation of Evans blue in various organs (F) of Zymosan-treated (30 mg/kg i.v.) male NMRI mice. Notes: The arrow indicates the time of treatment. The number of mice is n=3–5 for each time. Complement consumption was assessed using a modified sheep red blood cell hemolytic assay. The number of animals is eleven for the plasma extravasation test. Significant differences (*P<0.05; **P<0.01; ***P<0.001) are shown in comparison to the baseline or vehicle treated control group in the case of extravasation. Abbreviations: NMRI, Naval Medical Research Institute; TXB2, thromboxane B2 levels.
Figure 4
Figure 4
Hematological effects of C activators and liposomes. Notes: A–D show the maximal changes of platelets, WBC, RBC and hematocrit, respectively, following injection of the C activators and liposomes, specified on the x-axis. n=8–11 mice for each assay. Significant differences (**P<0.01; ***P<0.001) relative to the group treated with saline (NS). *?HC-MLV interfered with the detection of WBC, these data are not reliable. Abbreviations: CVF, cobra venom factor; HC-MLV, high cholesterol multilamellar vesicles; RBC, red blood cell; WBC, white blood cell.
Figure 5
Figure 5
Effects of C activators and liposomes on biomarkers of complement activation and TXB2 levels. (A) Hemolytic C consumed, measured in a modified sheep red blood cell hemolytic assay, (B) C3 consumption evaluated by the PAN-C3 assay, and (C) mouse C3a, measured by mouse specific ELISA. (D) TXB2 levels measured by ELISA. n=8–11 mice for each assay. (E) The effects of Abelecet and AmBisome on plasma C3a measured at 3 minutes as well as 10, 20, and 30 minutes after treatment (the results for 10–30 minutes are combined as C3a levels were similar at 10, 20, and 30 minutes). Notes: n=3–5 mice at each time. Significant differences (*P<0.05; ***P<0.001) relative to the group treated with saline (NS). Abbreviations: CVF, cobra venom factor; HC-MLV, high cholesterol multilamellar vesicles; RBC, red blood cell; TXB2, thromboxane B2 levels; WBC, white blood cell.
Figure 6
Figure 6
Effects of SB-290157 on the blood pressure and HR changes caused by Abelcet and comparison of the effects of Abelcet in NMRI and C57Bl/6N mice. Changes in mean arterial pressure (A) and HR (B) after pretreatment with SB-290157 at 10 mg/kg i.v. or its vehicle (5% DMSO in saline) and then administration of Abelcet at 30 mg/kg i.v. Comparison of changes in mean arterial pressure (C) and HR (D) in NMRI and C57Bl/6N mice after administration of Abelcet at 30 mg/kg i.v. Note: *P<0.05; **P<0.01; significant differences between the two groups. Abbreviations: HR, heart rate; MAP, mean arterial pressure; NMRI, Naval Medical Research Institute.
Figure 7
Figure 7
Double hit hypothesis of C activator- and liposome-induced circulatory changes in mice. Notes: Cells (blue) that can secrete vasoactive mediators (orange) can be stimulated either via anaphylatoxin-receptor (ATR, “AT hit”), or directly ((“C-independent hit”) leading to CARPA and C-independent pseudoallergy (CIPA)). Zymosan and CVF act via ATR, while AmBisome and Abelcet via a direct hit. The entailing physiological changes are listed on a black background. Italicized entries are hypothetical. Abbreviations: AT, anaphylatoxin; ATR, anaphylatoxin receptor; CARPA, C activation-related pseudoallergy; CIPA, C-independent pseudoallergy; CVF, cobra venom factor.
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References

    1. Szebeni J. Complement activation-related pseudoallergy: a new class of drug-induced acute immune toxicity. Toxicology. 2005;216(2–3):106–121. - PubMed
    1. Szebeni J. Complement activation-related pseudoallergy: a stress reaction in blood triggered by nanomedicines and biologicals. Mol Immunol. 2014;61(2):163–173. - PubMed
    1. Dézsi L, Rosivall L, Hamar P, Szebeni J, Szénási G. Rodent models of complement activation-related pseudoallergy: inducers, symptoms, inhibitors and reaction mechanisms. Eur J Nanomed. 2015;7(1):15.
    1. Proctor LM, Moore TA, Monk PN, Sanderson SD, Taylor SM, Woodruff TM. Complement factors C3a and C5a have distinct hemodynamic effects in the rat. Int Immunopharmacol. 2009;9(6):800–806. - PubMed
    1. Engström G, Hedblad B, Berglund G, Janzon L, Lindgärde F. Plasma levels of complement C3 is associated with development of hypertension: a longitudinal cohort study. J Hum Hypertens. 2007;21(4):276–282. - PubMed

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