Assessment of the safety of the cationic arginine-rich peptides (CARPs) poly-arginine-18 (R18 and R18D) in ex vivo models of mast cell degranulation and red blood cell hemolysis

Abstract

Our laboratory focuses on the development of novel neuroprotective cationic peptides, such poly-arginine-18 (R18: 18-mer of l-arginine; net charge +18) and its d-enantiomer R18D in stroke and other brain injuries. In the clinical development of R18/R18D, their cationic property raises potential safety concerns on their non-specific effects to induce mast cell degranulation and hemolysis. To address this, we first utilised primary human cultured mast cells (HCMCs) to examine anaphylactoid effects. We also included as controls, the well-characterised neuroprotective TAT-NR2B9c peptide and the widely used heparin reversal peptide, protamine. Degranulation assay based on β-hexosaminidase release demonstrated that R18 and R18D did not induce significant mast cell degranulation in both untreated (naïve) and IgE-sensitised HCMCs in a dose-response study to a maximum peptide concentration of 16 μM. Similarly, TAT-NR2B9c and protamine did not induce significant mast cell degranulation. To examine hemolytic effects, red blood cells (RBCs), were incubated with the peptides at a concentration range of 1-16 μM in the absence or presence of 2% plasma. Measurement of hemoglobin absorbance revealed that only R18 induced a modest, but significant degree of hemolysis at the 16 μM concentration, and only in the absence of plasma. This study addressed the potential safety concern of the application of the cationic neuroprotective peptides, especially, R18D, on anaphylactoid responses and hemolysis. The findings indicate that R18, R18D, TAT-NR2B9c and protamine are unlikely to induce histamine mediated anaphylactoid reactions or RBC hemolysis when administered intravenously to patients.

Keywords: Cationic arginine-rich peptides; Hemolysis; Mast cells degranulation; Polyarginine-18 (R18); Protamine; TAT-NR2B9c.

Fig. 1

Effects of R18, R18D, TAT-NR2B9c and protamine on naïve mast cell degranulation. (A) R18 acetate and HCl salts, (B) R18D, and (D) TAT-NR2B9c and protamine. The data is presented as mean ± SEM from three independent experiments for each peptide (n = 3). Note: data in graphs A (R18; acetate/HCl), B (R18D) and C (TAT-NR2B9c and protamine) were collected in independent studies. *P < 0.001 when compared with untreated control. Cont = untreated control. Pos cont = positive control; PMA = phorbol 12-myristate 13-acetate (50 ng/mL) and calcium ionophore A23187 (10 μM).

Fig. 2

Effects of R18, R18D, TAT-NR2B9c and protamine on IgE-dependent mast cell degranulation. (A) R18 acetate and (B) HCl, (C) R18D, (D) TAT-NR2B9c and (E) protamine. The data is presented as mean ± SEM from three independent experiments for each peptide (n = 3). Note: data in graphs A (R18; acetate/HCl), B (R18D) and C (TAT-NR2B9c and protamine) were collected in independent studies. *P < 0.001 when compared with untreated control. Cont = untreated control. Pos cont = positive control; Anti-IgE at 1 μg/mL.

Fig. 3

Effects of R18, R18D, TAT-NR2B9c and protamine on red blood cell hemolysis (A) R18 acetate and (B) HCl salts, (C) R18D, (D) TAT-NR2B9c and (E) protamine. The data is presented as mean ± SEM; n = 4 for R18 and protamine and n = 8 for R18D and TAT-NR2B9c. Note: data for R18D and TAT-NR2B9c were collected from two independent studies. *P < 0.001 when compared with untreated control. Triton X-100 (TritonX) control represents 100% RBC lysis. Absorbance 490 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)