Investigating dextrin-colistin conjugates as a novel treatment for sepsis

Objectives: The treatment of maxillofacial trauma patients with life-threatening sepsis associated with multi-drug resistant gram-negative infections is limited, due to the systemic toxicity associated with effective antibiotics such as colistin. We have previously generated novel dextrin-colistin polymer therapeutics which are activated by amylase degradation to release free colistin, thereby reducing clinical toxicity associated with colistin and improving targeting to sites of infection.  This study investigated the in vitro ability of these dextrin-colistin conjugates to bind and modulate bacterial lipopolysaccharides (LPS).

Methods: A chromogenic endotoxin assay was used to measure the clotting reaction of Limulus amoebocyte lysate (LAL) in the presence of LPS following pre-incubation with test compounds. Turbidity caused by precipitation of aggregates was measured spectrophotometrically at 620 nm over 2 h. HK-2 cells were seeded into 96-well plates (1 x 10⁵ cells/mL) in media supplemented with foetal calf serum, LPS and test compounds. Supernatants were collected after 24 h and assayed for TNFa content using a commercial ELISA kit (Thermo Scientific).

Results: LPS binding of both amylase-treated and untreated conjugate was reduced in E. coli (50%) and P. aeruginosa (20%) LPS in comparison to free colistin. Neutralisation of LPS by colistin required 20-fold more LPS to trigger LAL gelation compared to LPS in water, and was significantly greater than CMS. At the lower LPS concentrations, amylase-treated conjugates bound 20-50% more LPS than untreated conjugate. Amylase treatment demonstrated a time-dependent increase in endotoxin binding (6 h > 24 h > 0 h). In vitro studies showed that colistin, CMS and dextrin-colistin decreased LPS-induced production of TNFa in a concentration-dependent manner.

Conclusion: These studies demonstrate the potential of dextrin-colistin polymer therapeutics as intracorporeal neutralisers of bacterial LPS and support their development for further in vivo pharmacokinetic and activity studies.

Acknowledgements: This work was part-funded by MRC and SARTRE.