Activity of ALX-009, a novel combination of hypothiocyanite and , against clinical (CF) respiratory pathogens Payne, Joanna1; Ingram, Rebecca2; Elborn, J. Stuart1,2; Gilpin, Deirdre1; Juarez-Perez, Victor3; Tunney, Michael M.1 1Halo, Queen’s University Belfast, Belfast, UK. 2 Centre for Experimental Medicine, Queen’s University Belfast, Belfast, UK. 3Alaxia SAS, Lyon, France.

Introduction Given the problems with antibiotic resistance and the detection of new and emerging pathogens in the CF lung, there is a clear unmet need for novel antimicrobial agents for treatment of respiratory infection in CF patients. ALX-009, a combination of lactoferrin and hypothiocyanite, has been shown to have in vitro activity against a range of CF pathogens, notably and Burkholderiaisolates.

Aim: To determine if treatment of sputum from CF patients with ALX-009 results in a significant decrease in total sputum microbial load and microbial load of key CF pathogens, P. aeruginosa and Burkholderia cepacia complex (Bcc). Methods • Sputum samples, excess to clinical requirements, were collected from CF patients chronically colonized with P. aeruginosa (n=34) or Bcc (n=9). Sputum plugs were selected and homogenised by repeated passage through a 1ml syringe. • To determine the effect of a single treatment with ALX-009, ALX-009, tobramycin, ALX-009 and tobramycin or Phosphate buffered saline (PBS; control) were added to P. aeruginosa (n=24) and Bcc (n=9) positive sputum samples and total viable counts (TVC) measured at 0, 6 and 24 hours. To determine the effect of treating sputum with a second dose of ALX-009, CF sputum samples with a high P. aeruginosa load (≥105) (n=10) were used. ALX-009 was added at 0 and 12 hours with TVCs performed at 0, 6, 12, 18, 24 and 34 hours. A reduction, by any of the agents/combinations under test, of the original inoculum by ≥3 log10 cfu/g sputum was considered bactericidal. • TVCs for P. aeruginosa and Bcc count were performed using selective agar plates, with non-selective blood agar plates used to determine total sputum bacterial load. Results • Following a single dose, ALX-009 demonstrated bactericidal activity against P. aeruginosa in 18/24 sputum samples (Figure 1A). • In 10/24 samples the TVC of P. aeruginosa was reduced below the detectable limit at 24 hours (Figure 1B); initial P. aeruginosa bacterial burden was approximately 105 CFU/g sputum in these samples. In contrast, for the remaining 14/24 samples in which P. aeruginosa was detected at 24 hours (Figure 1C), initial P. aeruginosa bacterial burden was significantly higher at approximately 107 CFU/g sputum. • Treatment with a second dose of ALX-009 resulted in a further reduction in P. aeruginosa TVC at 34 hours in comparison to a single dose (Figure 1E). For 7/10 samples, P. aeruginosa TVC was below the detectable limit at 34 hours (Figure 1F); in the remaining 3 samples, P. aeruginosaTVC was reduced but was still detected. • Given the excellent activity of ALX-009 against P. aeruginosa growing in sputum, it was difficult to determine if there was a synergistic effect when ALX-009 was combined with tobramycin. However, synergistic activity was apparent for 4 samples at 24 hours. • ALX-009 demonstrated bactericidal activity for 4/9 Bcc samples at 24 hours; however, the TVC of Bcc was not reduced below the detectable limit for any sample at any timepoint (Figure 1D). B C D A 9 9 10 9 10 10 9 10 8 8 10 8 10 10 8 10 10 7 7 10 7 10 7 10 Control 10 6 6 10 6 10 6 10 5 ALX-009 5 10 5 10 5 10 10 Tobramycin 4 4 10 4 10 10 4 10 3 ALX-009 + 3 10 3 10 10 3 10 tobramycin

CFU/G sputum CFU/G 2

CFU/G sputum CFU/G 2 10 sputum CFU/G 2 10 sputum CFU/G 10 2 10 1 10 1 1 10 10 1 10 0 0 10 0 10 10 0 10 0 10 20 30 0 10 20 30 0 10 20 30 0 10 20 30 Time (hours) Time (hours) Time (hours) Time (hours)

E F 9 10 8 10 8 10 7 10 E 10 710 9 10 6 Control 10 610 8 10 5 ALX-009 10 510 7 10 4 (single dose) 410 6 10 ALX-009 10 3 5 10 310 (two doses) 2 4

CFU/G sputum CFU/G 10 10 CFU/G sputum CFU/G 10 2 1 3 10 10 110

CFU/G sputum CFU/G 2 0 10 10 10 0 0 10 20 30 40 1001 10 20 30 40 Time (hours) 10 0 Time (hours) 0 10 20 30 40 Time (hours) Figure 1. Antimicrobial activity of ALX-009 against (A) P. aeruginosa in sputum samples (n=24), P. aeruginosa in sputum at an initial bacterial burden of (B) ~105 CFU/g sputum (n=10) and (C) ~107 CFU/g sputum (n=14). (D) Antimicrobial activity of ALX-009 against Bcc in sputum samples (n=9). Activity of ALX- 009 (two doses) against (E) P. aeruginosa in sputum samples (n=10) and (F) representative graph for reduction of P. aeruginosa TVC below the limit of detection following two doses of ALX-009. Discussion This study demonstrates that treatment of sputum from CF patients with ALX-009 results in a significant decrease in the microbial load of key respiratory pathogens. Moreover, there was no significant change in the total load of bacteria present in the respiratory microbiota. A second dose of ALX-009, added within a similar time-frame to the administration of currently used inhaled antibiotics, inhibited the regrowth of P. aeruginosa in sputum samples with a high P. aeruginosaload.

Funded through a Department for Employment and Learning studentship and Alaxia