Endotoxin Binding By the Cationic Amphiphilic Peptide WLBU2 in Relation to Polymyxin B

Introduction: Sepsis is a blood infection that in the US alone affects about 750,000 people each year, killing 28-50% of them.^1-3 Passing blood through a sorbent device (hemoperfusion) to specifically remove targets such as endotoxin and bacterial cells holds promise for rapid treatment of acute sepsis. Clinical use of hemoperfusion in this context is based on immobilized polymyxin B (PMB),^4,5 but remains limited owing to ineffective endotoxin removal, and serious complications (e.g., nephrotoxicity, neurotoxicity, monocyte stimulation, substantial protein loss) associated with PMB and PMB-based devices^4,6-9. The synthetic, cationic amphiphilic peptide (CAP) WLBU2 has greater antimicrobial activity than PMB, works against a much broader spectrum of Gram-positive and Gram-negative bacteria, and shows higher selectivity for pathogenic entities over host cells.^10,11 In this research WLBU2 was compared to PMB with respect to its ability to bind endotoxin in the presence and absence of blood proteins, while in solution or immobilized at surfaces.  Endotoxin binding of WLBU2 while covalently tethered to a surface was also investigated.

Materials and Methods: Optical waveguide lightmode spectroscopy (OWLS) was used to detect adsorption on hydrophilic and silanized (hydrophobic) SiO₂ coated OW2400c waveguides under steady flow conditions. Vapor-phase silanization was carried out with trichlorovinylsilane (TCVS). Peptide (WLBU2 or PMB) and endotoxin (lipopolysaccharide, LPS, isolated from Pseudomonas aeruginosa), in PBS or diluted human plasma, were introduced to the OWLS flow cell in sequence or in mixed suspensions. Each adsorption step was followed by an elution step in peptide-free, LPS-free PBS buffer. LPS vesicle adsorption and spreading behavior under conditions representative of the OWLS experiments were also recorded, using interfacial tensiometry.

Results and Discussion: The antimicrobial effect of PMB and many CAPs is attributed to membrane disruption in susceptible bacteria. Assuming a multi-step mechanism for binding and disruption, our results strongly indicate that in contrast to PMB, WLBU2 binding is not accompanied by rapid disruption of LPS bilayer vesicles. In particular, competitive adsorption results showed adsorption of WLBU2 to hydrophobic surfaces from peptide-LPS mixtures is reduced by an order of magnitude relative to WLBU2 in the absence of LPS. Peptide-LPS and LPS-peptide sequential adsorption results were consistent with this outcome. Figure 1 shows that after LPS layering at a hydrophobic surface, WLBU2 binding was entirely irreversible with respect to buffer dilution.

Figure 1:  Sequential adsorption of LPS and WLBU2 (solid line) or PMB (dashed line). After introduction of peptide, the resistance to elution by PBS is much more pronounced in the case of WLBU2, indicating peptide “entrapment” within the LPS layer.  This is consistent with binding affinity of WLBU2 for LPS.

Conclusions:  Over the timescales of these experiments, we have seen that WLBU2 tightly binds LPS in a fashion which leaves the LPS largely intact. In contrast, PMB binding to LPS is associated with vesicle disruption and evolution of disruption products. Use of WLBU2 to capture pathogenic entities in a hemoperfusion device would avoid adverse clinical effects due to reintroduction of such entities to circulating blood.  These early investigations of use of tethered WLBU2 to capture LPS are promising.  Further investigation will be conducted using tethered constructs in the presence of blood proteins, as well as against whole bacterial cells.

References

1.            Czura CJ. “Merinoff Symposium 2010: Sepsis”—Speaking with One Voice. Molecular Medicine 2011;17(1-2):2.

2.            Dombrovskiy VY, Martin AA, Sunderram J, Paz HL. Rapid increase in hospitalization and mortality rates for severe sepsis in the United States: A trend analysis from 1993 to 2003*. Critical care medicine 2007;35(5):1244-1250.

3.            Wood KA, Angus DC. Pharmacoeconomic implications of new therapies in sepsis. Pharmacoeconomics 2004;22(14):895-906.

4.            Davies B, Cohen J. Endotoxin removal devices for the treatment of sepsis and septic shock. The Lancet Infectious Diseases 2011;11(1):65-71.

5.            Shoji H. Extracorporeal endotoxin removal for the treatment of sepsis: endotoxin adsorption cartridge (Toraymyxin). Therapeutic Apheresis and Dialysis 2003;7(1):108-114.

6.            Matsuno N, Ikeda T, Ikeda K, Hama K, Iwamoto H, Uchiyama M, Kozaki K, Narumi Y, Kikuchi K, Degawa H. Changes of cytokines in direct endotoxin adsorption treatment on postoperative multiple organ failure. Therapeutic Apheresis 2001;5(1):36-39.

7.            Vincent J-L, Laterre P-F, Cohen J, Burchardi H, Bruining H, Lerma FA, Wittebole X, De Backer D, Brett S, Marzo D. A pilot-controlled study of a polymyxin B-immobilized hemoperfusion cartridge in patients with severe sepsis secondary to intra-abdominal infection. Shock 2005;23(5):400-405.

8.            Ikeda T. Hemoadsorption in critical care. Therapeutic Apheresis 2002;6(3):189-192.

9.            Ueno T, Sugino M, Nemoto H, Shoji H, Kakita A, Watanabe M. Effect over time of endotoxin adsorption therapy in sepsis. Therapeutic Apheresis and Dialysis 2005;9(2):128-136.

10.          Gough M, Hancock RE, Kelly NM. Antiendotoxin activity of cationic peptide antimicrobial agents. Infection and immunity 1996;64(12):4922-4927.

11.          Scott MG, Yan H, Hancock REW. Biological properties of structurally related α-helical cationic antimicrobial peptides. Infection and immunity 1999;67(4):2005-2009.