Background

1. F1-2074 A Variant of Arenicin-3, NZ17074 Shows Efficacy Against E. coli in the Mouse Peritonitis/Sepsis ModelK. S. BRINCH1, B. T. RAVN1, C. V. LUNDBERG2, S. NEVE1 and H.-H. KRISTENSEN1 1Novozymes A/S, Bagsvaerd, Denmark, 2Statens Serum Institut, Copenhagen, Denmark Contact information: KBri@novozymes.com Phone: +45 4446 4787 Results Dose-response (ED50) study The ED50 values for the E. coli strain were calculated to be 3.1 mg/kg in peritoneal fluid (Fig. 1a) and 3.2 mg/kg in blood (Fig. 1b). The dose resulting in a 1 log killing effect was determined to 1.1 mg/kg (peritoneum) and 0.25 mg/kg (blood). Emax was calculated to 4.8 (peritoneum) and 3.1 (blood) log10 CFU/ml. Treatment with NZ17074 resulted in significantly (p<0.001) lower CFU/ml compared to the vehicle-treated group at 6 and 12 mg/kg. Figure 1. Dose-response curve of NZ17074 against a multi-resistant strain of E. coli in peritoneal fluid (a) and in blood (b) five hours after treatment in a neutropenic peritonitis/sepsis model. Background Arenicin-3 is a ß-hairpin antimicrobial peptide isolated from the lugworm (Arenicola marina). NZ17074, a variant of arenicin-3 (amino acid sequence: GFCWNVCVYRNGVRVCHRRCN), has shown in vitro activity against a range of Gram-negative bacteria including resistant strains of E. coli (please refer to posters F1-2070 and F1-2071 for further information). The purpose of the studies was to investigate the in vivo efficacy of the arenicin-3 variant, NZ17074, against a strain of E. coli through time-kill and dose-response studies in the murine peritonitis/sepsis model. Methods Dose-response (ED50) peritonitis/sepsis study Neutropenia was induced by dosing the mice intraperitoneally (i.p.) with cyclophosphamide on Day -4 (200 mg/kg) and on Day -1 (100 mg/kg). Day 0: Time=-1 hrs: The mice were inoculated i.p. with a bacterial suspension of 2 x 10E6 CFU/ml of E. coli (clinical isolate, 2003, from a human wound; multi-resistant (ampicillin, ceftazidime, aztreonam, gentamicin, ciprofloxacin). MICs: NZ17074: 0.5 mg/L, meropenem: 0.25 mg/L. Time =0 hrs: The mice (n=3) were treated with single doses of NZ17074 i.v. ranging from 0.18 – 12 mg/kg. Time=5 hrs: The mice were anaesthetized and blood was collected. Subsequently, the mice were sacrificed and a peritoneal wash performed. Quantitative bacterial counts were determined in both blood and peritoneal fluid and compared to untreated controls before start of treatment (T=0 hrs) and five hours later. The 50% effective dose (ED50) is defined as the dose required to obtain 50% of the maximum log CFU difference compared to start of treatment. The ED50 was calculated in GraphPad Prism using a sigmoidal dose-response curve (Hill’s regression) with variable slope. Furthermore, the maximal effect (Emax) and the dose resulting in 1 log10 kill were calculated. Time-kill peritonitis/sepsis study Neutropenia was induced with cyclophosphamide as described above. On Day 0 the mice (n=3) were infected i.p. with 2 x 10E6 CFU/ml of E. coli. One hour later the mice were treated with a single i.v. dose of 7.5 mg/kg NZ17074. After 2 and 5 hours mice were euthanized and CFU counts were determined in peritoneal fluid and blood. The results were compared to untreated control mice by one-way ANOVA. Mice dosed with meropenem at 40 mg/kg served as positive controls. • Results - continued • Time-kill study • After treatment with a single dose of 7.5 mg/kg NZ17074 a significant (p<0.001) reduction of E. coli was observed both in peritoneal fluid (3.8 log10 CFU/ml, Fig. 2a) and in blood (3.7 log10 CFU/ml, Fig. 2b) at T=5 hrs, when compared to vehicle controls. Also at T=2 hrs there was a significant reduction (p<0.01) in CFU/ml both in peritoneum (1.6 log10) and in blood (2.5 log10). Meropenem (40 mg/kg) also reduced the CFU/ml significantly (p<0.01) both in blood (1.6 log10) and in peritoneal fluid (1.5 log10) at T=5 hrs. At T=2 hrs the effect was only significant in the peritoneal fluid (1.5 log10, p<0.01), whereas the reduction in blood was only 0.8 log10 CFU/ml. • Figure 2. Time-kill study: Effect (CFU/ml) of single dose treatment with 7.5 mg/kg NZ17074 against E. coli in peritoneal fluid (a) and in blood (b) at 2 and 5 hrs after treatment in a neutropenic peritonitis/sepsis model. Treatment with meropenem at 40 mg/kg served as positive control. **: p<0.01; ***: p<0.001. • Conclusions. • Arenicin-3 variant, NZ17074, shows potent in vivo activity against a multi-resistant strain of E. coli in a mouse peritonitis/sepsis model. The ED50 in peritoneum and blood was 3.1 and 3.2 mg/kg, respectively. • NZ17074 at 7.5 mg/kg shows significant and rapid activity against E. coli in a time-kill study performed in the peritonitis/sepsis model. The effect was comparable to, or even superior to, the effect of meropenem at 40 mg/kg.