Transcript ICAAC 2010 peritonitis

F1-2074
A Variant of Arenicin-3, NZ17074 Shows Efficacy
Against E. coli in the Mouse Peritonitis/Sepsis Model
K. S. BRINCH1, B. T. RAVN1, C. V. LUNDBERG2, S. NEVE1 and H.-H. KRISTENSEN1
1Novozymes
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.
Contact information:
[email protected]
Phone: +45 4446 4787
A/S, Bagsvaerd, Denmark, 2Statens Serum Institut, Copenhagen, Denmark
Results
Results - continued
Dose-response (ED50) study
Time-kill 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.
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.
Treatment with NZ17074 resulted in significantly (p<0.001) lower CFU/ml
compared to the vehicle-treated group at 6 and 12 mg/kg.
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; multiresistant (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 doseresponse curve (Hill’s regression) with variable slope. Furthermore, the
maximal effect (Emax) and the dose resulting in 1 log10 kill were calculated.
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.
Time-kill peritonitis/sepsis study
Conclusions.
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.
• Arenicin-3 variant, NZ17074, shows potent in vivo activity against a multiresistant 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.
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.
• 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.