The small quinolone derived compound HT61 enhances the effect of tobramycin against Pseudomonas aeruginosa in vitro and in vivo.

HT61 is a small quinolone-derived compound previously demonstrated to exhibit bactericidal activity against gram-positive bacteria including methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA). When combined with the classical antibiotics and antiseptics neomycin, gentamicin, mupirocin and chlorhexidine, HT61 demonstrated synergistic bactericidal activity against both MSSA and MRSA infections in vitro. In this study, we investigated the individual antimicrobial activity of HT61 alongside its capability to potentiate the efficacy of tobramycin against both a tobramycin sensitive laboratory reference strain (PAO1) and tobramycin resistant clinical isolates (RP73, NN2) of the gram-negative bacteria Pseudomonas aeruginosa (P. aeruginosa). Using broth microdilution methods, the MICs of HT61 were assessed against all strains, as well as the effect of HT61 in combination with tobramycin using both the chequerboard method and bacterial time-kill assays. A murine model of pulmonary infection was also used to evaluate the combination therapy of tobramycin and HT61 in vivo. In these studies, we demonstrated significant synergism between HT61 and tobramycin against the tobramycin resistant P. aeruginosa strains RP73 and NN2, whilst an additive/intermediate effect was observed for P. aeruginosa strain PA01 which was further confirmed using bacterial time kill analysis. In addition, the enhancement of tobramycin by HT61 was also evident in in vitro assays of biofilm eradication. Finally, in vivo studies revealed analogous effects to those observed in vitro with HT61 significantly reducing bacterial load when administered in combination with tobramycin against each of the three P. aeruginosa strains at the highest tested dose (10 mg/kg).

Lipopolysaccharide (LPS) induced pulmonary neutrophil recruitment and platelet activation is mediated via the P2Y1 and P2Y14 receptors in mice.

Platelet activation occurs during host defence and in various inflammatory disorders. In animal models of infection and inflammation, experimental depletion of platelets leads to significantly reduced leukocyte recruitment and impaired clearance of pathogens from the lung. It is now appreciated that purinergic receptor activation is required for leukocyte activation, motility and adhesion, and platelet interactions with leukocytes can be modulated by purinergic stimulation of platelets. Here, we have investigated the role of platelet P2Y1, P2Y12, P2Y14, and P2X1 receptors on leukocyte recruitment and chemotaxis. Mice were administered either vehicle controls or selective P2Y1, P2Y12, P2Y14, or P2X1 antagonists intravenously before intranasal administration of lipopolysaccharide (LPS) to investigate the effect of these drugs on pulmonary leukocyte recruitment, peripheral platelet counts, bleeding times, and ex vivo platelet aggregation. Separately, platelets were incubated with P2Y1, P2Y12, P2X1 antagonists, or P2Y14 agonists to assess effects on platelet-induced neutrophil chemotaxis in vitro. Pulmonary neutrophil recruitment induced by intranasal LPS administration was inhibited in mice administered either with P2Y1 or P2Y14 antagonists, but not with P2Y12 or P2X1 antagonists. Furthermore, the administration of either a P2Y1 or a P2Y14 antagonist reversed the incidence of peripheral thrombocytopaenia associated with LPS exposure. Bleeding times were significantly increased in mice administered P2Y1, P2Y12, or P2X1 antagonists, whilst ex vivo platelet aggregation to ADP was significantly reduced. These haemostatic responses remained unaltered following antagonism of P2Y14. In vitro chemotaxis assays revealed direct antagonism of platelet P2Y1, but not P2Y12 or P2X1 receptors suppressed platelet-dependent neutrophil motility towards Macrophage derived chemokine (MDC, CCL22). Furthermore, the stimulation of platelets with selective P2Y14 agonists (UDP-glucose, MRS2690) resulted in significant platelet-dependent neutrophil chemotaxis. These results reveal a role for P2Y1 and P2Y14 activation of platelets following exposure to LPS, whilst haemostatic indices were unaffected by inhibition of platelet function with the P2Y14 antagonist in response to LPS.