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. 2020 Jan 28;25(3):558.
doi: 10.3390/molecules25030558.

PEGylated Liposomal Methyl Prednisolone Succinate does not Induce Infusion Reactions in Patients: A Correlation Between in Vitro Immunological and in Vivo Clinical Studies

Yaelle Bavli  1 , Bing-Mae Chen  2 , Steve R Roffler  2 , Marina A Dobrovolskaia  3 , Eldad Elnekave  4 , Shifra Ash  5 , Yechezkel Barenholz  1 , Keren Turjeman  1
Affiliations

PEGylated Liposomal Methyl Prednisolone Succinate does not Induce Infusion Reactions in Patients: A Correlation Between in Vitro Immunological and in Vivo Clinical Studies

Yaelle Bavli et al. Molecules. .

Abstract

PEGylated nanomedicines are known to induce infusion reactions (IRs) that in some cases can be life-threatening. Herein, we report a case study in which a patient with rare mediastinal and intracardiac IgG4-related sclerosing disease received 8 treatments of intravenously administered PEGylated liposomal methylprednisolone-succinate (NSSL-MPS). Due to the ethical requirements to reduce IRs, the patient received a cocktail of premedication including low dose of steroids, acetaminophen and H2 blockers before each infusion. The treatment was well-tolerated in that IRs, complement activation, anti-PEG antibodies and accelerated blood clearance of the PEGylated drug were not detected. Prior to the clinical study, an in vitro panel of assays utilizing blood of healthy donors was used to determine the potential of a PEGylated drug to activate complement system, elicit pro-inflammatory cytokines, damage erythrocytes and affect various components of the blood coagulation system. The overall findings of the in vitro panel were negative and correlated with the results observed in the clinical phase.

Keywords: IgG4 related disease; PEGylated nanodrugs; anti-PEG antibodies; complement activation; hypersensitive reactions; liposomal steroids.

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Conflict of interest statement

Yechezkel Barenholz is an inventor of two patents on NSSL-MPS owned by Yissum TTO of the Hebrew University. Yechezkel Barenholz, Yaacov Naparstek, Yuval Avnir and Rina Ulmansky: “The use of Liposomal Glucocorticoids for Treating Inflammatory States.” US Patent 7,744,920, 2010, June 29, 2010 and Yechezkel Barenholz, Alberto A. Gabizon and Yuval Avnir: “Liposomal Compositions of Glucocorticoid and Glucocorticoid Derivatives”. US Patent 8,932,627, January 13, 2015. These 2 granted patents were not yet licensed.The other authors declare no competing interests.

Figures

Figure 1
Figure 1
Evaluation of in vitro activation of the complement system and hemolytic properties of PEGylated liposomal methylprednisolone-succinate NSSL-MPS. (A) Different concentrations of NSSL-MPS were incubated in human plasma for 30 min and the increase in iC3b was measured and compared to iC3b activation by Cobra Venom Factor (CVF) (B). Different concentrations of NSSL-MPS were incubated in whole blood and the free hemoglobin released by damaged erythrocytes was quantified. BLOQ, below limit of quantitation; Cre, Cremophor-EL; CVF, Cobra Venom Factor; NC, Negative Control; PC, Positive Control (Triton-X-100). BLOQ is < 0.13 µg/mL.
Figure 2
Figure 2
Individual cytokines/chemokines/interferon release tests in the presence of the NSSL-MPS formulation. NSSL-MPS at different concentrations (0.304, 1.52, 7.62 or 76.2 µg/mL MP) were incubated for 24 h in whole human blood from at least 3 different healthy donors. After incubation, the supernatant was tested for each relevant signaling molecule with a corresponding ELISA assay. NC, Negative Control; PC, Positive Control, 20 ng/mL of E.coli K12 LPS.
Figure 3
Figure 3
NSSL-MPS effect on platelet aggregation and collagen-induced platelet aggregation. The capacity of NSSL-MPS to induce platelet aggregation (A) and modify collagen-induced platelet aggregation (B) were assessed. Briefly, NSSL-MPS at different concentrations (0.304, 1.52, 7.62 or 76.2 µg/mL MPS) were incubated in platelet-rich plasma for 15 min at 37 °C. Platelet aggregation was monitored in real time using light transmission aggregometry. AUC, Area Under the Curve; PC, Positive Control (positive control is collagen).
Figure 4
Figure 4
Leucocyte pro-coagulant activity in the presence of NSSL-MPS. Different concentrations of NSSL-MPS were tested for their ability to induce leukocyte pro-coagulant activity. Briefly, peripheral blood mononuclear cells (PBMC) isolated from healthy blood donor volunteers and HL-60 cells were incubated (24 h for PBMC and 5 h for HL-60 cells) with NSSL-MPS at 0.304, 1.52, 7.62 or 76.2 µg/mL MPS. Isolated cells from the different conditions were then used to initiate plasma coagulation. PC, Positive Control; PCA, Pro-Coagulant Activity.
Figure 5
Figure 5
Markers of in vivo complement activation in plasma. Immediately before the beginning of NSSL-MPS infusion (0) and 10, 20, 30 and 60 min after the beginning of the infusion, a 2 mL sample of blood was withdrawn in K2EDTA blood collection tubes. The blood was kept on ice and centrifuged at 960 xg for 10 min at 4 °C as soon as possible after collection. Immediately after centrifugation, the plasma was collected and frozen on dry ice. The following markers of complement activation were assayed using commercial kits (detailed in the Materials section): SC5b-9 (A), Bb (B), TXB2 (C) and iC3b (D). The dash line indicates the upper limit of normal values. TXB, Thromboxane.
Figure 6
Figure 6
Screening of healthy donors for pre-existing antibodies against polyethylene glycol (anti-PEG IgG and IgM). Blood was collected from healthy donors (n = 28, 14 men and 14 women) and the sera were assessed for the presence of natural anti-PEG antibodies using a direct ELISA against immobilized PEG. The positive threshold was defined as 3 times the value of background for each antibody. Each positive result was confirmed by competition assay against PEGylated liposomes. Dashed line indicates the positive threshold for IgG and dotted line the positive threshold for IgM detection.
Figure 7
Figure 7
Levels of MPS and MP in the patient’s serum for treatments 1–3. (A) MP to MPS ratio (mole %) during time following 1-hour slow infusion of NSSL-MPS for treatment 1, 2 and 3. (B) Serum levels (as percent of injected dose) of methylprednisolone hemisuccinate following 1-hour slow infusion of NSSL-MPS for treatment 1, 2 and 3 (treatment with 50, 100 and 150 mg liposomal MPS respectively).
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