I en undersøgelse foretaget af det danske firma Novozymes blev plectasin-peptidet opdaget i 2001 i Pseudoplectania nigrella – en bægersvamp (beskrevet i en publikation i 2005. [3])

Som et molekyle med en beskyttende virkning mod patogener er det et af defensinerne, og dets kemiske struktur ligner dem, der findes i edderkopper, skorpioner, insekter og muslinger. [3] (Genet er formentlig udviklet tidligt og videreført til disse meget forskellige dyregrupper). Molekylets virkningsmekanisme ville kunne have to virkninger i mennesker, som begge ville kunne virke som et forsvar mod bakterielle patogener. [4] På den ene side dræber molekylet de sygdomsfremkaldende bakterier ved at molekylet fastgør sig selv til cellevægskomponenten Lipid II og derved forhindrer, at denne cellevægskomponent Lipid II bygges ind i nye bakterievægge. Dette vil ske i forbindelse med at der ved infektionen skal opbygges ​​nye bakteriecellevægge, og denne bakterievæg-dannelse forstyrres derved markant, når bakterierne formerer sig. [4] Den anden funktion ved molekylet er, at der samtidig sker en aktivering af immunsystemet. Plectasin er særlig effektiv mod pneumokokker både in vitro og in vivo – ifølge en infektionsmodel fra mus. [5] Prækliniske undersøgelser har også vist, at multiresistente bakterier, såsom Staphylococcus aureus får betydelige vanskeligheder hvis de udsættes for Plectasin NZ2114 (som er en plectasin-variant, der blev udviklet af Novozymes). [6] [4] [7]

 

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5487675/
Antimicrob Agents Chemother. 2017 Jul; 61(7): e00604-17.
Published online 2017 Jun 27. Prepublished online 2017 May 15. doi: 10.1128/AAC.00604-17
PMCID: PMC5487675
PMID: 28507110
Controlled Release of Plectasin NZ2114 from a Hybrid Silicone-Hydrogel Material for Inhibition of Staphylococcus aureus Biofilm
Kasper Klein,a Rasmus Birkholm Grønnemose,a Martin Alm,b Karoline Sidelmann Brinch,c Hans Jørn Kolmos,a and Thomas Emil Andersencorresponding authora
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ABSTRACT
Staphylococcus aureus is a major human pathogen in catheter-related infections. Modifying catheter material with interpenetrating polymer networks is a novel material technology that allows for impregnation with drugs and subsequent controlled release. Here, we evaluated the potential for combining this system with plectasin derivate NZ2114 in an attempt to design an S. aureus biofilm-resistant catheter. The material demonstrated promising antibiofilm properties, including properties against methicillin-resistant S. aureus, thus suggesting a novel application of this antimicrobial peptide.

KEYWORDS: Staphylococcus aureus, antimicrobial peptides, plectasin, biofilm formation, catheter infections, biofilms, catheter, hydrogel

A decade ago, antimicrobial peptides (AMPs) were considered to be one of the most important new classes of drugs in the fight against antimicrobial resistance. Today, however, most AMPs have been removed from development pipelines, with only a few having reached actual clinical use (1).

Despite their obvious potential, the systemic use of AMPs is hampered by their intrinsic immunogenicity and low in vivo stability (2), which together with regulatory requirements of superior efficiency to current treatment have been major obstacles for obtaining approval (1). The use of AMPs in combination with medical devices to prevent device-associated infections is, however, an enticing alternative application that to a lesser extent is restricted by the above problems (3, 4, 5).

Device-associated and, specifically, catheter-related bloodstream infections (CRBSI) constitute a major problem in modern health care. Methicillin-sensitive Staphylococcus aureus (MSSA) and, in particular, methicillin-resistant S. aureus (MRSA) are among the most important pathogens in CRBSI. The plectasin derivate NZ2114 is a promising AMP that has been potentiated toward MSSA, MRSA, and vancomycin-resistant S. aureus with proven in vivo efficiency (6, 7) and, therefore, holds potential for use against device-associated infections, including CRBSI.

Here, we evaluated the properties of a novel hybrid catheter material loaded with plectasin NZ2114 in order to inhibit MRSA biofilms. The material consists of an interpenetrating polymer network (IPN) based on silicone elastomer (polydimethylsiloxane) as the host polymer and poly(2-hydroxyethyl methacrylate)-co-poly(ethylene glycol) methyl ether acrylate (PHEMA-co-PEGMEA) hydrogel as the guest polymer. This IPN material exhibits unique drug loading and release properties through adsorption of drugs into the hydrogel component in the bulk of the material and subsequent release upon exposure to aqueous solutions, such as bodily fluids (8, 9, 10, 11). Hypothetically, the catheter matrix protects the embedded drug, in this case plectasin NZ2114, from proteolytic degradation. Furthermore, the IPN matrix ensures a controlled release of the drug, potentially resulting in prolonged efficiency and fewer side effects compared to those of the burst release often associated with conventional drug coatings.

The IPN material was produced as previously described (8) with modifications. Briefly, silicone samples were punched out from 2-mm-thick sheets of silicone rubber (PE4062; Lebo Production, Skogås, Sweden) or cut from silicone tubing (outer/inner diameter, 1.65/0.76 mm; Helix Medical, VWR, Radnor, PA). The samples were placed in a high-pressure reactor with equal amounts of HEMA (2-hydroxyethyl methacrylate; Aldrich Chemistry, Germany) and EGMEA (ethylene glycol methyl ether acrylate; Aldrich, Germany) with supercritical carbon dioxide as an aiding solvent. After polymerization into PHEMA-co-PEGMEA inside the silicone material, the samples were placed in 96% ethanol for 7 days to remove residual monomer followed by drying at 50°C until final mass was reached. Two IPN sample sets with hydrogel contents of 23% and 28%, respectively, were produced and evaluated. The amount of PHEMA-co-PEGMEA hydrogel in the samples was determined by mass increase. IPN samples were drug loaded by immersion in Milli-Q water containing 10 mg/ml of plectasin NZ2114 (6) (Novozymes A/S, Bagsværd, Denmark) or 10 mg/ml of dicloxacillin (Bristol-Myers Squibb, New York City, NY) for 7 days.

To evaluate release from the catheter material, release sampling was performed daily over 14 days from catheter tubing, 23 mm in length, placed in phosphate-buffered saline (PBS) with a pH of 7.4. The plectasin NZ2114 concentration was quantified by ultraperformance liquid chromatography (UPLC) (Fig. 1). Mean totals of 46.1 µg ± 21.3 (mean ± standard deviation) and 42.9 µg ± 20.9 for 23% and 28% PHEMA-co-PEGMEA, respectively, were released from the catheter specimens during this time period. The release correlated well with first-order kinetics (R2 = 0.97 and 0.92 for 23% and 28% PHEMA-co-PEGMEA, respectively), with an approximate release of 40.7% and 31.9% of remaining loaded drug each day for the 23% and 28% samples, respectively, indicating a better long-term slow release profile for catheters with the higher IPN content.

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Object name is zac0071763390001.jpg(A) Mean accumulated release from catheter specimens measured in micrograms (n = 4). (B) Mean release per day measured in micrograms per milliliter (release volume = 1.5 ml; n = 4).

Antimicrobial activity of plectasin NZ2114 and dicloxacillin against MSSA and MRSA was determinated as MIC and minimum bactericidal concentration (MBC) by broth dilution according to ISO 20776-1 standards (Table 1).

Strain MIC (μg/ml)
MBC (μg/ml)
Dicloxacillin Plectasin NZ2114 Dicloxacillin Plectasin NZ2114
MSSA ATCC 29213 1 0.5 8 8
MRSA ATCC 33591 32 1 >1,024 16
The loaded catheter tubing was tested for bactericidal effect in a static setup. The catheter tubing was placed in test tubes containing 10% heparinized human plasma in PBS, inoculated with approximately 5.0 × 104 CFU/ml of MRSA strain ATCC 33591, and incubated overnight. The CFU count in the suspension was then estimated, and the catheter tubing was transferred to a new test tube containing inoculated plasma and the procedure repeated for 11 days (Fig. 2).

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Functional release assay (n = 3) using PEGMEA-co-PHEMA IPN material impregnated with dicloxacillin or plectasin for inhibition of MRSA ATCC 33591. The CFU values in vials containing test catheters were measured after daily challenge with 5 × 104 CFU per ml plasma medium and are shown in the graph as a percentage of this daily inoculum. In control vials containing pristine silicone and unloaded IPN samples, CFU reached >1,000% of the inoculum added on day 1 (data not shown).

As it appears in Fig. 2, some fluctuations occur in this type of experiment, a phenomenon we have observed in an earlier study as well (11). To evaluate whether this is due to plectasin NZ2114 destabilization during loading, storage in, and release from the IPN hydrogel, the activity of the compound after release in buffer was tested using MIC determination, and dilutions were matched to the high-pressure liquid chromatography (HPLC) release quantification data (Fig. 1). Comparing these data to the MIC values for the stock showed no loss of activity upon loading and release (data not shown). Together with the relatively low day-to-day fluctuation observed in the direct release measurements (Fig. 1), we speculate that the fluctuations in Fig. 2 occur due to biological variation in bacterial sensitivity, where subpopulations may enter biofilm or an otherwise more persistent growth mode differently from day to day.

Assuming that the catheters remain effective as long as the mean CFU counts are below the initial inoculum (i.e., the 100% point on the y axis in Fig. 2), the dicloxacillin-loaded tubing lasts for 2 days whereas the plectasin NZ2114-loaded specimens last until day 10 (Fig. 2).

In an in vivo setting, a considerable part of venous catheters is exposed to blood flow, placing high demands on this part of the material with respect to maintaining an effective release of drug. Bacteria spreading to catheters may, furthermore, originate from biofilms growing on skin/wound sites; i.e., they are already more resilient than traditional broth-cultured bacteria when reaching the catheter material. To account for these conditions, plectasin NZ2114-loaded IPN discs (hydrogel content of 24%) were challenged with MRSA ATCC 33591 in a flow chamber model as previously described (11, 12) using an initial bacterial seeding inoculum of optical density at 600 nm (OD600) of 0.100 in PBS for 30 min at 30 μl/min. This was followed by a growth phase in 10% heparinized human plasma in PBS at 30 μl/min, which leads to a continuous seeding over the catheter surface with resilient biofilm emboli (11). The plectasin NZ2114-loaded disks showed effective inhibition of bacterial surface colonization compared to unloaded IPN disks as visualized with the LIVE/DEAD BacLight bacterial viability kit (L7012; Molecular Probes, Eugene, OR) using fluorescence and confocal laser scanning microscopy (CLSM) (Fig. 3).

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Microscopy of biofilms formed by MRSA ATCC 33591 on IPN material either as unloaded control (A, B, C) or loaded with plectasin NZ2114 (D, E, F). (A, B, D, and E) 0.64 mm by 0.64 mm CLSM images obtained with an Olympus FV1000MPE. (C and F) Full fluorescence microscopy scans of the flow chamber test surface performed using an Olympus BX51 microscope with motorized stage and image processing using Olympus cellSens software (only the green/GFP channel is shown). Over the 24-h experiment, the surface was continuously seeded with biofilm emboli (11).

To quantitatively assess whether the catheter tubing exhibited adequate drug-release properties for biofilm inhibition despite its limited wall thickness, catheter samples loaded with plectasin NZ2114 were tested in a flow system seeded as the previous flow chamber assay with MRSA and subsequently grown in a flow of 10% heparinized plasma. CFU in the resulting biofilm inside the tubing were quantified by pipetting 0.1% Triton X-100 in PBS through the tubing followed by plating. This experiment demonstrated effective prevention of growth in the plectasin NZ2114-loaded catheters compared to unloaded and dicloxacillin-loaded catheters (Fig. 4), displaying a significant 3-log reduction compared to unloaded IPN (Table 2). Furthermore, plectasin NZ2114-loaded catheters exposed to prerelease in PBS for 6 days prior to testing still exhibited a significant 2-log reduction compared to the unloaded IPN, indicating a clinically relevant release after 1 week of catheter placement (Table 2).

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Vs Dicloxacillin Plectasin 1d Plectasin 6d + 1d Silicone
Plectasin 1d <0.01
Plectasin 6d + 1d NS <0.01
Silicone NS <0.01 0.05
Unloaded IPN NS <0.01 <0.01 NS
aOne-way analysis of variance (ANOVA) followed by Tukey's honest significant difference test. n = 4 for all 5 groups. Before testing, data were transformed by natural logarithm to obtain normal distributions. P values are shown. NS = not significant.
Induction of antibiotic resistance is a relevant concern for medical devices that passively release antimicrobials due to an unavoidable period of time near drug depletion when the release of drug becomes less than the MIC. To asses to what extent sub-MICs of plectasin NZ2114 may induce resistance in S. aureus, we applied a serial passage of increasing drug concentration modified from Hammer et al. (13). In brief, ∼105 bacteria were inoculated in 5 ml tryptic soy broth (TSB) overnight at 37°C on a shaker. The following day, 100 μl of the culture was transferred to new test tubes containing 4.9 ml TSB with either ciprofloxacin or plectasin NZ2114 at 25% of MIC and incubated overnight. Then, 100 μl was transferred to new TSB tubes in which drug concentrations were increased by 100%. This procedure continued until growth inhibition was observed compared to positive controls without antimicrobials (21 days in this experiment). At this point, 100 μl of the bacterial suspension was plated and MIC determined as described above. Results for MSSA ATCC 29213 and MRSA ATCC 33591 (Table 3) showed that plectasin NZ2114 induced drug resistance only to a minor extent compared to the control antibiotic ciprofloxacin.

Strain MIC before serial passing:
MIC after serial passing
Ciprofloxacin Plectasin NZ2114 Ciprofloxacin Plectasin NZ2114
MSSA ATCC 29213 0.25 (0.25) 0.5 (0.5) 32 (32) 4 (4–8)
MRSA ATCC 33591 0.25 (0.25) 1 (1.0) 32 (32–64) 8 (4–8)
aMIC values were determined according to ISO standard 20776-1. All numbers are in μg/ml and presented as median with range in parentheses (n = 3).
Lastly, to assess possible immune synergism of plectasin NZ2114, we determined the MBC for MSSA ATCC 29213 and MRSA ATCC 33591, respectively, in heat-inactivated and untreated 10% pooled human serum in PBS. Here, identical MBC values were measured in untreated and heat-inactivated serum (data not shown), indicating no synergistic effects with complement factors, in contrast to what has been reported for other antimicrobial peptides (14, 15).

Plectasin NZ2114 has previously shown promising results in the treatment of various S. aureus diseases in vivo (6, 7). Using this antimicrobial peptide as an active loading agent in a novel IPN-based device material showed promising results in a comprehensive in vitro test procedure, accounting for several relevant factors that influence the performance of the catheter in vivo. This suggests that the material-drug combination may be suitable for venous catheters for more effective prevention of colonization by staphylococci and other Gram-positive pathogens.

The study was funded by the Innovation Fund Denmark (grants 52-2014-1 and 041-2010-3) and Ph.D. grants from Odense University Hospital and the University of Southern Denmark.

Martin Alm is employed at the company Biomodics, the owner of the patent concerning IPN technology (10). Karoline Sidelmann Brinch is employed at Novozymes, former patent holder of plectasin NZ2114. The study was in no way funded by these or other companies.

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Weblinks
Plectasin: Neue Waffe gegen hochresistente Keime bei organische-chemie.ch

<1> InterPro-Eintrag

<2> LINK 2 Novozymes reveals knowledge on new antibiotic against resistant bacteria. In: Novozymes. 28. Mai 2010. 

 

<3> https://www.nature.com/articles/nature04051 Per H. Mygind1, Rikke L. Fischer et al.: Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus. Nature 437, vol 7061, 2005, S. 975–80 doi:10.1038/nature04051

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Per H. Mygind, Rikke L. Fischer, Kirk M. Schnorr, Mogens T. Hansen, Carsten P. Sönksen, Svend Ludvigsen, Dorotea Raventós, Steen Buskov, Bjarke Christensen, Leonardo De Maria, Olivier Taboureau, Debbie Yaver, Signe G. Elvig-Jørgensen, Marianne V. Sørensen, Bjørn E. Christensen, Søren Kjærulff, Niels Frimodt-Moller, Robert I. Lehrer, Michael Zasloff & Hans-Henrik Kristensen
Nature volume 437, pages 975–980 (2005

Animals and higher plants express endogenous peptide antibiotics called defensins. These small cysteine-rich peptides are active against bacteria, fungi and viruses. Plectasin is the first defensin to be isolated from a fungus, the saprophytic ascomycete Pseudoplectania nigrella. Plectasin has primary, secondary and tertiary structures that closely resemble those of defensins found in spiders, scorpions, dragonflies and mussels. Recombinant plectasin can be produced at a very high, and commercially viable, yield and purity. In vitro, the recombinant peptide is especially active against Streptococcus pneumoniae, including strains resistant to conventional antibiotics. Plectasin show extremely low toxicity in mice, and can cure them of experimental peritonitis and pneumonia caused by S. pneumoniae as efficaciously as vancomycin and penicillin. These findings identify fungi as a novel source of antimicrobial defensins, and show the therapeutic potential of plectasin. They also suggest that the defensins of insects, molluscs and fungi arose from a common ancestral gene.

<5>https://aac.asm.org/content/53/7/3003 D. Andes, W. Craig et al.: In Vivo Pharmacodynamic Characterization of a Novel Plectasin Antibiotic, NZ2114, in a Murine Infection Model. Antimicrobial Agents and Chemotherapy, Juli 2009, Vol.: 53, Nr.: 7, S. 3003–3009, doi:10.1128/AAC.01584-08. In Vivo Pharmacodynamic Characterization of a Novel Plectasin Antibiotic, NZ2114, in a Murine Infection Model
D. Andes, W. Craig, L. A. Nielsen, H. H. Kristensen
DOI: 10.1128/AAC.01584-08
NZ2114 is a novel plectasin derivative with potent activity against gram-positive bacteria, including multiply drug-resistant strains. The neutropenic murine thigh infection model can be used to characterize the time course of antimicrobial activity of NZ2114 and determine its pharmacokinetic/pharmacodynamic (PK/PD) index and efficacy. Serum drug levels following administration of three fourfold-escalating single-dose levels of NZ2114 has been measured by microbiologic assay. Single-dose time-kill studies following doses of 10, 40, and 160 mg/kg of body weight has demonstrated concentration-dependent killing over the dose range (0.5 to 3.7 log10 CFU/thigh) and prolonged postantibiotic effects (3 to 15 h) against both Staphylococcus aureus and Streptococcus pneumoniae. Mice had 106.3 to 106.8 CFU/thigh of strains of S. pneumoniae or S. aureus at the start of therapy when treated for 24 h with 0.625 to 160 mg/kg/day of NZ2114 fractionated for 4-, 6-, 12-, and 24-h dosing regimens. Nonlinear regression analysis has been used to determine which PK/PD index best correlated with microbiologic efficacy. Efficacies of NZ2114 were shown to be similar among the dosing intervals (P = 0.99 to 1.0), and regression with the 24-h area under the concentration-time curve (AUC)/MIC index was strong (R2, 0.90) for both S. aureus and S. pneumoniae. The maximum concentration of drug in serum/MIC index regression was also strong for S. pneumoniae (R2, 0.96). Studies to identify the PD target for NZ2114 utilized eight S. pneumoniae and six S. aureus isolates and an every-6-h regimen of drug (0.156 to 160 mg/kg/day). Treatment against S. pneumoniae required approximately twofold-less drug for efficacy in relationship to the MIC than did treatment against S. aureus. The free drug 24-h AUCs/MICs necessary to produce a stasis effect were 12.3 ± 6.7 and 28.5 ± 11.1 for S. pneumoniae and S. aureus, respectively. The 24-h AUC/MIC associated with a 1-log killing endpoint was only 1.6-fold greater than that needed for stasis. Resistance to other antimicrobial classes did not impact the magnitude of the PD target required for efficacy. The PD target in this model should be considered in the design of clinical trials with this novel antibiotic.

The epidemic of antimicrobial resistance is a growing public health threat. Unfortunately, few drug classes have been identified and brought to market in the last decade (15). One novel antibacterial compound is from the plectasin class (12). Plectasin antibiotics are defensin-like peptide antibiotics of fungal origin. These compounds exhibit broad-spectrum activity against gram-positive bacteria, including potency against multiply drug-resistant strains. The plectasins specifically bind a target molecule and interfere with bacterial biosynthesis, resulting in rapid cell death (T. Schneider et al.). Recent studies suggested the potential for both in vitro and in vivo efficacy with one derivative, NZ2114 (12).

Preclinical pharmacodynamic investigations have proven to be predictive of outcome in therapy of patients and thus important in the design of dosing regimens in the development of antimicrobial clinical trials (1, 2, 3, 5, 8, 10). The goals of the current experiments were to characterize the in vivo pharmacodynamic characteristics of this drug against Streptococcus pneumoniae and Staphylococcus aureus in order to identify the pharmacodynamic target for future clinical development.

Eight strains of Streptococcus pneumoniae with variable resistance to penicillin were used in this study. Six strains of Staphylococcus aureus (three methicillin-susceptible and three methicillin-resistant S. aureus strains) were also used for these experiments. Organisms were grown, subcultured, and quantified in Mueller-Hinton broth (Difco Laboratories, Detroit, MI) and Mueller-Hinton agar (Difco Laboratories, Detroit, MI) for all organisms except S. pneumoniae. Sheep blood agar plates (Remel, Milwaukee, WI) were utilized for S. pneumoniae. NZ2114 was supplied by Novozymes A/S.The MICs of NZ2114, penicillin, and methicillin for the various isolates were determined by standard Clinical and Laboratory Standards Institute microdilution methods (14). All MIC assays were performed in duplicate on three occasions. The reported MIC was the mean of the replicate assays.

The neutropenic mouse thigh infection model has been used extensively for determination of pharmacokinetic/pharmacodynamic (PK/PD) index determination and prediction of antibiotic efficacy in patients (3-5). Animals were maintained in accordance with the American Association for Accreditation of Laboratory Animal Care criteria (13). All animal studies were approved by the Animal Research Committee of the William S. Middleton Memorial VA Hospital. Six-week-old, specific-pathogen-free, female ICR/Swiss mice weighing 23 to 27 g were used for all studies (Harlan Sprague-Dawley, Indianapolis, IN). Mice were rendered neutropenic (neutrophils, <100/mm3) by injecting them with cyclophosphamide (Mead Johnson Pharmaceuticals, Evansville, IN) intraperitoneally 4 days (150 mg/kg of body weight) and 1 day (100 mg/kg) before thigh infection. Previous studies have shown that this regimen produces neutropenia in this model for 5 days (16). Broth cultures of freshly plated bacteria were grown to logarithmic phase overnight to an absorbance at 580 nm of 0.3 (Spectronic 88; Bausch and Lomb, Rochester, NY). After a 1:10 dilution into fresh Mueller-Hinton broth, bacterial counts of the inoculum ranged from 106.4 to 107.6 CFU/ml. Thigh infections with each of the isolates were produced by injection of 0.1 ml of inoculum into the thighs of isoflurane-anesthetized mice 2 h before therapy with NZ2114.

Single-dose serum pharmacokinetic studies were performed in thigh-infected mice. Animals were administered subcutaneous doses (0.2 ml/dose) of NZ2114 (10, 40, and 160 mg/kg). Two groups of three mice each were included for each dose studied. Blood was removed from three mice per time point at 0.25- to 1-h intervals over 6 h. Serum was collected and processed immediately for microbiologic assay using S. aureus 6538p as the assay organism. The lower limit of detection of NZ2114 in the microbiologic assay was 0.25 μg/ml, and the lower limit of quantitation was 1.0 μg/ml. The intraday variation was 4.3% at a concentration of 4 μg/ml. Pharmacokinetic constants, including elimination half-life, area under the concentration-time curve (AUC), and peak level, were calculated using a noncompartmental model. For doses used in treatments for which actual kinetic measurements were not made, estimates were based upon linear extrapolation or interpolation from the three studied dose levels. Protein binding in the serum of neutropenic infected mice was performed using ultrafiltration methods as previously described (7, 9). The degree of binding was measured using NZ2114 concentrations of 25 and 100 μg/ml.

Treatment protocols. (i) In vivo PAE.Two hours after infection with S. pneumoniae strain ATCC 10813 or S. aureus strain ATCC 25923, neutropenic mice were treated with single subcutaneous doses of NZ2114 (10, 40, or 160 mg/kg). Groups of two treated and untreated control mice each were sacrificed at sampling intervals ranging from 2 to 12 h. Control growth was determined at five sampling times over 24 h. The treated groups were sampled seven times over 24 h. The thighs were removed at each time point and processed immediately for CFU determination. The times for which the levels of NZ2114 (based on unbound drug concentrations) in the serum remained above the MIC for the organisms were calculated from the pharmacokinetic studies. The postantibiotic effect (PAE) was calculated by subtracting the time that it took for organisms to increase 1 log in level in the thighs of saline-treated animals from the time that it took organisms to grow the same amount in treated animals after serum levels fell below the MIC for the infecting organism (PAE = T − C, where C is the time for 1-log10 control growth and T is the time for 1-log10 treatment growth after levels have fallen below MIC) (6).

(ii) PK/PD index determination: Neutropenic mice were infected with either penicillin-susceptible S. pneumoniae ATCC 10813 or methicillin-susceptible S. aureus ATCC 25923. Treatment with NZ2114 was initiated 2 h after infection. Groups of two mice were treated for 24 h with 20 different dosing regimens using fourfold-increasing total doses divided into one, two, four, or six doses. Total doses of NZ2114 ranged 256-fold (0.625 to 160 mg/kg/24 h). Drug doses were administered subcutaneously in 0.2-ml volumes. The mice were sacrificed after 24 h of therapy, and the thighs were removed and processed for CFU determination. Untreated control mice were sacrificed just before treatment and after 24 h.

(iii) PK/PD index magnitude studies.Similar dosing studies using five or six fourfold-increasing NZ2114 doses administered every 6 h were utilized to treat thigh-infected neutropenic animals with eight strains of S. pneumoniae (two penicillin-susceptible, three penicillin-intermediate, and three penicillin-resistant strains) and six strains of S. aureus (three methicillin-susceptible and three methicillin-resistant strains). The total daily dose of NZ2114 used in these studies varied from 0.156 to 160 mg/kg/day.

Data analysis: The results of these studies were analyzed using the sigmoid dose-effect model. The model, as follows, is derived from the Hill equation: E = (Emax × DN)/(ED50N − DN), where E is the effect or, in this case, the log change in CFU per thigh between treated mice and untreated controls after the 24-h period of study, Emax is the maximum effect, D is the 24-h total dose, ED50 is the dose required to achieve 50% of Emax, and N is the slope of the dose-effect curve (4, 11). The indices Emax, ED50, and N were calculated using nonlinear least-squares regression. The correlation between efficacy and each of the three PK/PD indices (T > MIC, AUC/MIC, and peak/MIC) studied was determined by nonlinear regression (Sigma Stat; SPSS. Inc., San Rafael, CA). The coefficient of determination, or R2, was used to estimate the variance that could be due to regression with each of the PK/PD indices.

The scientists utilized the 24-h static dose as well as the doses necessary to achieve a 1-log10 reduction in colony counts compared to numbers at the start of therapy to compare the impacts of the dosing intervals on treatment efficacy. If these dose values remained similar among each of the dosing intervals, this would support the 24-h AUC/MIC as the predictive index. If the dose values increased as the dosing interval was lengthened, this would suggest that T > MIC is the predictive index. Lastly, if the dose values decreased as the dosing interval was increased, this would support peak/MIC as the pharmacodynamically important index. The static dose and 1-log kill values for each of the dosing intervals were compared statistically using analysis of variance.

To allow a comparison of the potencies of NZ2114 against a variety of organisms, the scientists utilized the 24-h static dose. The magnitude of the PK/PD index associated with each endpoint dose was calculated from the equation log10 D = log10 (E/Emax − E)/N + log10 ED50, where D is the drug dose, E is the control growth for dose (D), E is the control growth + 1 log for a D of 1-log kill, Emax is the maximal effect, N is the slope of the dose-response relationship, and ED50 is the dose needed to achieve 50% of the maximal effect. The significance of differences among the various dosing endpoints was determined by using analysis of variance on ranks.

In vitro susceptibility testing: The MICs of NZ2114 for the 14 study strains are shown in Table 1. NZ2114 MICs varied more than 30-fold (range, 0.06 to 2.0 μg/ml).

Pharmacokinetics: The time course of serum levels of NZ2114 in infected neutropenic mice following subcutaneous doses of 10, 40, and 160 mg/kg is shown in Fig. 1. Peak levels were observed by 30 min. The elimination half-life in the mice ranged from 0.38 to 1.0 h. The AUC and peak values for the escalating single doses ranged from 17 to 204 and 21 to 126, respectively. The protein binding of NZ2114 in mouse serum, as determined by ultrafiltration, was 80%. This is similar to the degree of binding in other animal species and in human serum (D. Sandvang, personal communication).

Serum pharmacokinetics of plectasin derivative NZ2114 following single subcutaneous doses. Each symbol represents the mean concentration from three mice. The error bars represent the standard deviations. Three dose levels (fourfold escalating) of NZ2114 were studied. The measured Cmax and calculated elimination half-life and AUC (zero to infinity) are shown in the table. Protein binding was determined by ultrafiltration using concentrations of 25 and 100 μg/ml. A microbiologic assay using S. aureus 6538p was used for determination of all NZ2114 concentrations.

In vivo PAE.At the start of therapy, mice had 106.6 to 106.7 CFU/thigh of S. pneumoniae and S. aureus, respectively. Growth of 1 log10 CFU/thigh in saline-treated animals occurred in 4.1 and 2.0 h in S. pneumoniae- and S. aureus-infected animals, respectively. Based upon the serum pharmacokinetic determinations, serum NZ2114 levels following the single doses of 10, 40, and 160 mg/kg remained above the MIC for S. pneumoniae strain ATCC 10813 (MIC, 0.06 μg/ml) for 2.3, 4.5, and 7.7 h based on free drug levels, respectively. Rapid, dose-dependent killing of organisms occurred following each of the dose levels for both strains. Maximal killing compared to organism burden at the start of therapy ranged from 2.7 to 3.7 log10 CFU/thigh over the dose range for S. pneumoniae. Over a similar dose range, maximal killing of S. aureus ranged from 0.5 to 1.7 log10 CFU/thigh. Organism regrowth with S. pneumoniae did not occur for more than 12 h. For the lowest dose in the S. aureus study, regrowth began 4 h after dosing. The times above the MIC for these doses against S. aureus strain ATCC 25923 (MIC, 1.0 μg/ml) were 1.2, 2.6, and 4.6 h based on free drug levels. The time-kill curves for both of the studies are shown in Fig. 2. Against S. pneumoniae, escalating doses produced free drug PAEs ranging from 12.2 to 15.4 h. The study with S. aureus produced free drug PAEs ranging from 2.8 to 14.3 h. No detectable drug carryover was observed in any of the treatment groups.

FIG. 2.
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FIG. 2.
Impact of plectasin derivative NZ2114 dose escalation on burden of either S. pneumoniae or S. aureus in the thighs of neutropenic mice over time. Each symbol represents the mean CFU/thigh from two mice (four thighs). The error bars represent the standard deviations. Solid symbols represent growth of organisms in control mice over time. Open symbols represent the burden of organisms in NZ2114-treated mice. The horizontal boxes represent the duration of time that free drug NZ2114 serum concentrations remained above the MIC of the infecting organism. The PAE is expressed in hours.

PK/PD index determination.At the start of therapy, mice had 6.8 ± 0.07 and 6.3 ± 0.32 log10 CFU/thigh of S. pneumoniae strain ATCC 10813 and S. aureus strain ATCC 29213, respectively. The organisms grew 2.1 ± 0.2 and 3.4 ± 0.03 log10 CFU/thigh after 24 h in untreated control mice, respectively. Escalating doses of NZ2114 resulted in the concentration-dependent killing of both strains. The highest doses studied reduced organism burden by 4.8 ± 0.26 and 1.7 ± 0.01 log10 CFU/thigh compared to numbers at the start of therapy for S. pneumoniae and S. aureus, respectively. The dose-response relationships for the four dosing intervals against S. pneumoniae and S. aureus are shown in Fig. 3. The curves were similar among each of the dosing intervals against S. aureus. In the study with S. pneumoniae, there was a slight shift in the dose-response curve to the left (indicating enhanced effect) as the dosing interval was lengthened. The dose levels required to produce stasis and a 1-log reduction were calculated for each dosing interval (data not shown). At each of these treatment endpoints, we did not observe a significant difference, as the dosing interval was lengthened from every 4 h to every 24 h (data not shown). These analyses suggest that treatment efficacy was dependent upon dose level and independent of the dosing intervals studied. The relationships between microbiologic effect and each of the pharmacodynamic indices, 24-h AUC/MIC, percent time above the MIC, and peak/MIC against S. pneumoniae strain ATCC 10813, are shown in Fig. 4. Therapeutic outcome correlated well with both the 24-h AUC/MIC and maximum concentration of drug in serum (Cmax)/MIC indices. For S. aureus the relationship was strongest for the 24-h AUC/MIC index (R2 = 0.90 for 24-h AUC/MIC; R2 = 0.85 for Cmax/MIC), and for S. pneumoniae the Cmax/MIC index correlated somewhat better (R2 = 0.89 for 24-h AUC/MIC; R2 = 0.97 for Cmax/MIC) (Fig. 5). Regression with the percent T > MIC index resulted in a less strong relationship (R2 = 0.74 for each organism). The reasonable fit of the data with each of the PK/PD indices is due to the interrelationships among each of the indices (5). Consideration of total or unbound drug levels did not appreciably impact the relationship between efficacy and percent T > MIC (data not shown).

FIG. 3.
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FIG. 3.
Impact of plectasin derivative NZ2114 dosing interval on efficacy against a strain of S. aureus and a strain of S. pneumoniae in the neutropenic murine thigh infection model. Five total (mg/kg/24-h) doses were fractionated into one, two, four, or six doses over the 24-h treatment period (q24 h, q12 h, q6 h, and q4 h, respectively). Total escalating doses varied fourfold. Efficacy is expressed as change in CFU/thigh compared to organism burden at the start of therapy. Each symbol represents the mean CFU/thigh from two mice (four thighs). The error bars represent the standard deviations. The horizontal dashed line represents the burden of organisms at the start of therapy.

Relationship between the NZ2114 pharmacodynamic index (24-h AUC/MIC, Cmax/MIC, and percent time above MIC) and efficacy over 24 h against S. pneumoniae 10813 in a neutropenic murine thigh infection model. Unbound (free drug) concentrations were used for index calculations. Efficacy is expressed as change in CFU/thigh compared to organism burden at the start of therapy. Each symbol represents the mean CFU/thigh from two mice (four thighs). The sigmoid line represents the best fit using the sigmoid Emax model. R2 is the coefficient of determination.

Relationship between the NZ2114 pharmacodynamic index (24-h AUC/MIC, Cmax/MIC, and percent time above MIC) and efficacy over 24 h against S. aureus 25923 in a neutropenic murine thigh infection model. Unbound (free drug) concentrations were used for index calculations. Efficacy is expressed as change in CFU/thigh compared to organism burden at the start of therapy. Each symbol represents the mean CFU/thigh from two mice (four thighs). The sigmoid line represents the best fit using the sigmoid Emax model. R2 is the coefficient of determination.

PK/PD magnitude determination.Calculation of the doses necessary to achieve a static effect and a 1-log10 kill against multiple organisms is shown in Table 1. The growth curves of the eight pneumococcal and six staphylococcal strains in the thighs of control animals were relatively similar. At the start of therapy, mice had 7.0 ± 0.44 (range, 5.6 to 7.6) log10 CFU of pneumococci or S. aureus/thigh. The organisms grew to 2.4 ± 0.54 log10 CFU/thigh (range, 1.5 to 3.4) in untreated control mice. The maximal reduction in S. pneumoniae with NZ2114-treated mice compared to untreated controls ranged from 2.8 ± 0.3 to 4.6 ± 0.2 log10 CFU/thigh (mean, 3.4 ± 0.60). Less killing was observed against the S. aureus strains (mean, 1.9 ± 0.42 log10 CFU/thigh).

The 24-h AUC/MIC index was used for determination of the pharmacodynamic magnitude of exposure associated with efficacy. Table 1 shows the 24-h dose and free drug 24-h AUC/MIC ratios necessary to achieve a net static effect and 1-log10 reduction in organism burden. The static doses varied from 1.1 mg/kg every 24 h to 218 mg/kg every 24 h against the strains of S. pneumoniae and S. aureus. The corresponding free drug 24-h AUC/MICs varied from 3.4 to 37.7. The mean 24-h AUC/MICs associated with a static effect were slightly lower for S. pneumoniae than for S. aureus (12.3 ± 6.7 versus 28.5 ± 11.1, respectively) (P = 0.006). The exposure associated with bactericidal activity or a 1-log10 reduction in organism burden compared to the start of therapy was less than twofold larger than that associated with a stasis endpoint. The presence of antimicrobial resistance in both bacterial species did not alter the 24-h AUC/MIC required to produce efficacy. The relationship between the 24-h free drug AUC/MIC and efficacy against the two organism groups is demonstrated graphically in Fig. 6. The exposure-response relationships were relatively strong, with R2 values of 0.85 and 0.82 for S. pneumoniae and S. aureus, respectively.

Relationship between NZ2114 free drug 24-h AUC/MIC and efficacy against eight S. pneumoniae (left) and six S. aureus (right) strains. Each symbol represents the mean CFU/thigh from two mice (four thighs). Efficacy on the y axis is expressed as the change in CFU/thigh compared to the burden of organisms at the start of therapy. The dashed horizontal line represents the burden of organisms in thighs at the start of therapy. The sigmoid line represents the best-fit curve using the sigmoid Emax model. R2 is the coefficient of determination.

DISCUSSION
The number of infections due to resistant gram-positive bacteria, including penicillin-resistant Streptococcus pneumoniae and methicillin-resistant Staphylococcus aureus, continues to increase (15). The development of effective antimicrobial agents to treat these infections is an area of intense research. Peptide antimicrobial agents represent a promising new class of compounds which collectively act at a number of different bacterial targets and have demonstrated potency against these emerging pathogens (12).

Previous in vivo studies in two pneumococcal murine infection models showed both enhanced animal survival and a rapid decline in organism burden following single doses of the native plectasin molecule (12). A logical next step in the evaluation of these molecules is to use animal infection molecules to understand the pharmacodynamic activity of this drug class. Antimicrobial pharmacodynamic studies characterize the time course of antibiotic effect. The time course of antimicrobial activity can be determined by two characteristics: (i) the effect of increasing drug concentrations on the extent of organism killing and (ii) the presence or absence of antimicrobial effects that persist after the levels in serum have fallen below the MIC (5). The use of animal infection models to understand the pharmacodynamic activity of antimicrobial agents is one approach that has proven helpful for (i) the design of effective dosing regimens in humans and (ii) the development of appropriate in vitro susceptibility breakpoints (3).

In these studies, escalating doses of NZ2114 produced a rapid reduction in viable counts. Each of the doses examined reduced the burden of organisms more than 2 log in the first 6 h after a single dose of NZ2114, and nearly 2 additional logs of killing was observed while free drug concentrations exceeded the MIC of the infecting S. pneumoniae strain. After drug concentrations fell below the MIC, we observed prolonged growth suppression. A similar time course pattern was observed in a study with a strain of S. aureus. However, the degree of organism killing was somewhat lower against the S. aureus strain. Prolonged PAEs were also seen against S. aureus, and the duration of the PAE was dose dependent. The efficacy of antibiotics characterized by this pattern of activity is best correlated with either the 24-h AUC/MIC or the Cmax/MIC index.

Results from analysis of the multiple-dosing regimen studies indicate that the 24-h AUC/MIC is the best index for predicting the efficacy of NZ2114. The dose-response relationship against S. aureus was not impacted by a change in dosing interval, and pharmacodynamic regression was clearly strongest with the 24-h AUC/MIC index. In the pneumococcal investigation, the dosing interval did appear to have some impact on the dose-response relationship. As the dosing strategy provided larger doses more infrequently, the dose-response curves shifted slightly to the left. This visual pharmacodynamic relationship would suggest that the Cmax/MIC is important (5). However, statistical comparison of dosing endpoints (static dose and 1-log kill) among the dosing intervals did not show significance. The regression analysis results based upon the coefficient of determination (R2) suggested that both the Cmax/MIC and 24-h AUC/MIC indices predict efficacy with NZ2114.

Next, studies were undertaken to identify the magnitude of the pharmacodynamic index needed for efficacy of NZ2114. We considered the 24-h AUC/MIC index for these analyses. Experiments included 14 organisms with a wide range of NZ2114 MICs (more than 30-fold). The strains were chosen to include those resistant to beta-lactams. In vitro studies with NZ2114 demonstrated antimicrobial activity against S. pneumoniae and S. aureus including strains resistant to other antimicrobial classes including beta-lactams, macrolides, quinolones, and vancomycin (14a). Similarly, in the current in vivo experiments penicillin resistance in S. pneumoniae and methicillin resistance in S. aureus had no impact upon the in vivo pharmacodynamic target of NZ2114.

The NZ2114 dose-response relationships for each species were very similar. However, the dose-response curves for S. pneumoniae were shifted somewhat to the left compared to those for S. aureus. This slight difference in activity is similar to that observed in the time course studies. The free (unbound) NZ2114 24-h AUC/MIC associated with a net static effect against S. pneumoniae was roughly twofold lower than that for S. aureus. The dose-response curves were relatively steep against both bacterial species, and the pharmacodynamic exposure associated with a killing endpoint (1-log kill) was less than twofold greater than that needed for a stasis effect.

The microbiologic outcomes in this infection model have correlated with clinical and microbiologic efficacy of several antimicrobial classes in humans (1, 2, 3, 4, 5). The results of these studies should be utilized to examine the pharmacokinetics of NZ2114 in humans in the context of MIC distribution of target organisms to help guide appropriate dosing regimen design and to determine preliminary susceptibility breakpoints.

This research above is from 22 April 2009.
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<Link 6> Plectasin NZ2114 – Novel Microbial Agent. In: Drug Development Technology.
Kristensen, HH et al: In Vivo Pharmacodynamic Characterization of a Novel Plectasin Antibiotic, NZ2114, in a Murine Infection Model. In: Antimicrob Agents Chemother.. 53, Nr. 7, 2009, S. 3003–3009. doi:10.1128/AAC.01584-08. PMC 2704636 (freier Volltext).

____DANSK MASKINOVERSÆTTELSE AF LITTERATURLISTEN:____
Litteratur
Karoline Sidelmann Brinch1, Paul M. Tulkens et al .: Intracellulær aktivitet af peptid-antibiotikum NZ2114: Undersøgelser med Staphylococcus aureus og humane THP-1-monocytter og sammenligning med daptomycin og vancomycin. Tidsskrift for antimikrobiel kemoterapi, august 2010, bind 65, nummer 8, s. 1720-1724

Weblinks

Plectasin: Nyt våben mod meget modstandsdygtige bakterier på organisk-chemie.ch

Novozymes afslører viden om nyt antibiotikum mod resistente bakterier . I: Novozymes. 28. maj 2010. Arkiveret fra originalen den 14. november 2012.

Per H. Mygind1, Rikke L. Fischer et al.: Plectasin er et peptid-antibiotikum med terapeutisk potentiale fra en saprofytisk svamp. Nature 437, vol 7061, 2005, s. 975-80 doi : 10.1038 / nature04051

T. Schneider et al .: Plectasin, et svampedefensin, der er målrettet bakteriecellevæggen, precursor Lipid II. Science , 28. maj 2010, bind .: 328, nr .: 5982, s. 1168 -1172, doi : 10.1126 / science.1185723

D. Andes, W. Craig et al .: In vivo farmakodynamisk karakterisering af et nyt antibiotikum Plectasin, NZ2114, i en Murine-infektionsmodel. Antimikrobielle midler og kemoterapi , juli 2009, bind .: 53, nr .: 7, s. 3003-3009, doi : 10.1128 / AAC.01584-08

<6> https://www.clinicaltrialsarena.com/projects/plectasin/ Plectasin NZ2114 – Ny mikrobiel agent . I: Drug Development Technology .
Plectasin NZ2114 – Novel Microbial Agent

Plectasin variant NZ2114
Novozymes A/S/ Sanofi-Aventis

Defensins

In a periode whis defensin was under development by Novozymes A/S, the Danish biotechnology company. Plectasin NZ2114 was a novel antimicrobial peptide. Pre-clinical studies suggested that it possess potent bactericidal activity against gram-positive pathogens.

Its excellent penetration into cerebrospinal fluid (CSF) suggests potential in the treatment of CNS infections caused by gram-positive pathogens such as pneumococcal meningitis.

In an era of rising rates of bacterial resistance to commonly used antibiotics, new agents are urgently needed to treat bacterial infections effectively and halt the spread of resistant strains. This need is arguably greatest in the hospital environment where rates of bacterial resistance are highest.

"Although still in early-stage development, defensins represent an exciting new approach to combating antibiotic resistance."
Today the threat of resistance is posed by gram-positive and gram-negative pathogens. Important examples include methicillin-resistant S. aureus (MRSA) and other multi-resistant gram-positive cocci, Clostridium difficile and extended-spectrum beta-lactamase (EBSL) producing gram-negative pathogens such as Acinetobacter baumannii, Pseudomonas aeruginosa and E. coli.

Although still in early-stage development, defensins represent an exciting new approach to combating antibiotic resistance. They are peptide antibiotics that are endogenously produced by certain animal and plant cells. This entirely new class of antimicrobial agents have potential broad-spectrum activity against bacteria, including strains resistant to conventional antibiotics, fungi and viruses.

Novozyme's plectasin NZ2114 was the first defensin-type antimicrobial peptide to be isolated from a fungus, the saprophytic ascomycete Pseudoplectania nigrella.

Plectasin NZ2114 – preclinical data
Novozyme's plectasin variant NZ2114 appears especially active against gram-positive bacteria. Pre-clinical studies show it is effective against Streptococcus pneumoniae, exerting bactericidal effects in experimental models of pneumococcal peritonitis and pneumonia.

It has also demonstrated potent bactericidal effects in experimental pneumococcal meningitis model where it was compared with ceftriaxone. In this model, plectasin NZ2114 achieved significantly higher penetration into the CSF of inflamed meninges compared with controls (non-inflamed meninges). In comparison with ceftriaxone, treatment with plectasin NZ2114 produced a significantly greater reduction in CSF bacterial concentration as well as complete bacterial eradication in some cases (sterile CSF).

These encouraging preliminary findings bode well for future clinical studies assuming this new antimicrobial proves safe and well tolerated.

Collaboration with Sanofi-Aventis: At the end of 2008, Novozymes A/S signed a global licensing agreement with Sanofi-Aventis for the further development and marketing of plectasin NZ2114 as a treatment for severe gram-positive bacterial infections. This represents the first drug candidate Novozymes A/S had out-licensed for clinical development.

"Novozyme's plectasin variant NZ2114 appears especially active against gram-positive bacteria."
Through this agreement, Sanofi-Aventis gains exclusive rights to development, registration and commercialisation of plectasin NZ2114. Its interest in this novel agent is said to stem from the fact that defensin antimicrobial peptides may have activity against bacteria that are commonly resistant to conventional antibiotics.

Both companies will be involved in commercial-scale manufacturing of plectasin NZ2114, which will use Novozymes' proprietary expression technology.

Antibiotics are often considered the poor relation in the field of drug research. Only three new antibiotic classes were approved between 1960 and 2000. Despite this seemingly gloomy scenario, the past decade has seen some important new additions to the range of antibiotics available to treat serious bacterial infections.

These have included not only new antibiotics from existing classes but also some from entirely new classes of antibiotics such as the glycylcyclines and licopeptides. The defensin antimicrobial peptides too represent a potentially important new class of antimicrobial agents, albeit still in the very early stages of development.

/a> Kristensen, HH et al : In Vivo farmakodynamisk karakterisering af et nyt antibiotikum Plectasin, NZ2114, i en Murine-infektionsmodel . I: Antimicrob Agents Chemother. , 53, nr. 7, 2009, s. 3003-3009. doi : 10.1128 / AAC.01584-08 . PMC 2704636 (gratis fuldtekst).

 

Antimicrob Agents Chemother. 2009 Jul; 53(7): 3003–3009.
Published online 2009 May 4. doi: 10.1128/AAC.01584-08
PMCID: PMC2704636
PMID: 19414576

In Vivo Pharmacodynamic Characterization of a Novel Plectasin Antibiotic, NZ2114, in a Murine Infection Model ▿
D. Andes,1,2,* W. Craig,1 L. A. Nielsen,3 and H. H. Kristensen3

NZ2114 is a novel plectasin derivative with potent activity against gram-positive bacteria, including multiply drug-resistant strains. The scientists used the neutropenic murine thigh infection model to characterize the time course of antimicrobial activity of NZ2114 and determine which pharmacokinetic/pharmacodynamic (PK/PD) index and magnitude best correlated with efficacy. Serum drug levels following administration of three fourfold-escalating single-dose levels of NZ2114 were measured by microbiologic assay. Single-dose time-kill studies following doses of 10, 40, and 160 mg/kg of body weight demonstrated concentration-dependent killing over the dose range (0.5 to 3.7 log10 CFU/thigh) and prolonged postantibiotic effects (3 to 15 h) against both Staphylococcus aureus and Streptococcus pneumoniae. Mice had 106.3 to 106.8 CFU/thigh of strains of S. pneumoniae or S. aureus at the start of therapy when treated for 24 h with 0.625 to 160 mg/kg/day of NZ2114 fractionated for 4-, 6-, 12-, and 24-h dosing regimens. Nonlinear regression analysis was used to determine which PK/PD index best correlated with microbiologic efficacy. Efficacies of NZ2114 were similar among the dosing intervals (P = 0.99 to 1.0), and regression with the 24-h area under the concentration-time curve (AUC)/MIC index was strong (R2, 0.90) for both S. aureus and S. pneumoniae. The maximum concentration of drug in serum/MIC index regression was also strong for S. pneumoniae (R2, 0.96). Studies to identify the PD target for NZ2114 utilized eight S. pneumoniae and six S. aureus isolates and an every-6-h regimen of drug (0.156 to 160 mg/kg/day). Treatment against S. pneumoniae required approximately twofold-less drug for efficacy in relationship to the MIC than did treatment against S. aureus. The free drug 24-h AUCs/MICs necessary to produce a stasis effect were 12.3 ± 6.7 and 28.5 ± 11.1 for S. pneumoniae and S. aureus, respectively. The 24-h AUC/MIC associated with a 1-log killing endpoint was only 1.6-fold greater than that needed for stasis. Resistance to other antimicrobial classes did not impact the magnitude of the PD target required for efficacy. The PD target in this model should be considered in the design of clinical trials with this novel antibiotic.

The epidemic of antimicrobial resistance is a growing public health threat. Unfortunately, few drug classes have been identified and brought to market in the last decade (15). One recently reported novel antibacterial compound is from the plectasin class (12). Plectasin antibiotics are defensin-like peptide antibiotics of fungal origin. These compounds exhibit broad-spectrum activity against gram-positive bacteria, including potency against multiply drug-resistant strains. The plectasins specifically bind a target molecule and interfere with bacterial biosynthesis, resulting in rapid cell death (T. Schneider et al., submitted for publication). Recent studies suggested the potential for both in vitro and in vivo efficacy with one derivative, NZ2114 (12).

Preclinical pharmacodynamic investigations have proven to be predictive of outcome in therapy of patients and thus important in the design of dosing regimens in the development of antimicrobial clinical trials (1, 2, 3, 5, 8, 10). The goals of the current experiments were to characterize the in vivo pharmacodynamic characteristics of this drug against Streptococcus pneumoniae and Staphylococcus aureus in order to identify the pharmacodynamic target for future clinical development.

Bacteria, media, and antibiotic: Eight strains of Streptococcus pneumoniae with variable resistance to penicillin were used. Six strains of Staphylococcus aureus (three methicillin-susceptible and three methicillin-resistant S. aureus strains) were also used for these experiments. Organisms were grown, subcultured, and quantified in Mueller-Hinton broth (Difco Laboratories, Detroit, MI) and Mueller-Hinton agar (Difco Laboratories, Detroit, MI) for all organisms except S. pneumoniae. Sheep blood agar plates (Remel, Milwaukee, WI) were utilized for S. pneumoniae. NZ2114 was supplied by Novozymes A/S.

In vitro susceptibility studies: The MICs of NZ2114, penicillin, and methicillin for the various isolates were determined by standard Clinical and Laboratory Standards Institute microdilution methods (14). All MIC assays were performed in duplicate on three occasions. The reported MIC was the mean of the replicate assays.

Murine infection model: The neutropenic mouse thigh infection model has been used extensively for determination of pharmacokinetic/pharmacodynamic (PK/PD) index determination and prediction of antibiotic efficacy in patients (3-5). Animals were maintained in accordance with the American Association for Accreditation of Laboratory Animal Care criteria (13). All animal studies were approved by the Animal Research Committee of the William S. Middleton Memorial VA Hospital. Six-week-old, specific-pathogen-free, female ICR/Swiss mice weighing 23 to 27 g were used for all studies (Harlan Sprague-Dawley, Indianapolis, IN). Mice were rendered neutropenic (neutrophils, <100/mm3) by injecting them with cyclophosphamide (Mead Johnson Pharmaceuticals, Evansville, IN) intraperitoneally 4 days (150 mg/kg of body weight) and 1 day (100 mg/kg) before thigh infection. Previous studies have shown that this regimen produces neutropenia in this model for 5 days (16). Broth cultures of freshly plated bacteria were grown to logarithmic phase overnight to an absorbance at 580 nm of 0.3 (Spectronic 88; Bausch and Lomb, Rochester, NY). After a 1:10 dilution into fresh Mueller-Hinton broth, bacterial counts of the inoculum ranged from 106.4 to 107.6 CFU/ml. Thigh infections with each of the isolates were produced by injection of 0.1 ml of inoculum into the thighs of isoflurane-anesthetized mice 2 h before therapy with NZ2114.

Drug pharmacokinetics: Single-dose serum pharmacokinetic studies were performed in thigh-infected mice. Animals were administered subcutaneous doses (0.2 ml/dose) of NZ2114 (10, 40, and 160 mg/kg). Two groups of three mice each were included for each dose studied. Blood was removed from three mice per time point at 0.25- to 1-h intervals over 6 h. Serum was collected and processed immediately for microbiologic assay using S. aureus 6538p as the assay organism. The lower limit of detection of NZ2114 in the microbiologic assay was 0.25 μg/ml, and the lower limit of quantitation was 1.0 μg/ml. The intraday variation was 4.3% at a concentration of 4 μg/ml. Pharmacokinetic constants, including elimination half-life, area under the concentration-time curve (AUC), and peak level, were calculated using a noncompartmental model. For doses used in treatments for which actual kinetic measurements were not made, estimates were based upon linear extrapolation or interpolation from the three studied dose levels. Protein binding in the serum of neutropenic infected mice was performed using ultrafiltration methods as previously described (7, 9). The degree of binding was measured using NZ2114 concentrations of 25 and 100 μg/ml.

Treatment protocols. (i) In vivo PAE: Two hours after infection with S. pneumoniae strain ATCC 10813 or S. aureus strain ATCC 25923, neutropenic mice were treated with single subcutaneous doses of NZ2114 (10, 40, or 160 mg/kg). Groups of two treated and untreated control mice each were sacrificed at sampling intervals ranging from 2 to 12 h. Control growth was determined at five sampling times over 24 h. The treated groups were sampled seven times over 24 h. The thighs were removed at each time point and processed immediately for CFU determination. The times for which the levels of NZ2114 (based on unbound drug concentrations) in the serum remained above the MIC for the organisms were calculated from the pharmacokinetic studies. The postantibiotic effect (PAE) was calculated by subtracting the time that it took for organisms to increase 1 log in level in the thighs of saline-treated animals from the time that it took organisms to grow the same amount in treated animals after serum levels fell below the MIC for the infecting organism (PAE = T − C, where C is the time for 1-log10 control growth and T is the time for 1-log10 treatment growth after levels have fallen below MIC) (6).

(ii) PK/PD index determination: Neutropenic mice were infected with either penicillin-susceptible S. pneumoniae ATCC 10813 or methicillin-susceptible S. aureus ATCC 25923. Treatment with NZ2114 was initiated 2 h after infection. Groups of two mice were treated for 24 h with 20 different dosing regimens using fourfold-increasing total doses divided into one, two, four, or six doses. Total doses of NZ2114 ranged 256-fold (0.625 to 160 mg/kg/24 h). Drug doses were administered subcutaneously in 0.2-ml volumes. The mice were sacrificed after 24 h of therapy, and the thighs were removed and processed for CFU determination. Untreated control mice were sacrificed just before treatment and after 24 h.

(iii) PK/PD index magnitude studies: Similar dosing studies using five or six fourfold-increasing NZ2114 doses administered every 6 h were utilized to treat thigh-infected neutropenic animals with eight strains of S. pneumoniae (two penicillin-susceptible, three penicillin-intermediate, and three penicillin-resistant strains) and six strains of S. aureus (three methicillin-susceptible and three methicillin-resistant strains). The total daily dose of NZ2114 used in these studies varied from 0.156 to 160 mg/kg/day.

Data analysis: The results of these studies were analyzed using the sigmoid dose-effect model. The model, as follows, is derived from the Hill equation: E = (Emax × DN)/(ED50N − DN), where E is the effect or, in this case, the log change in CFU per thigh between treated mice and untreated controls after the 24-h period of study, Emax is the maximum effect, D is the 24-h total dose, ED50 is the dose required to achieve 50% of Emax, and N is the slope of the dose-effect curve (4, 11). The indices Emax, ED50, and N were calculated using nonlinear least-squares regression. The correlation between efficacy and each of the three PK/PD indices (T > MIC, AUC/MIC, and peak/MIC) studied was determined by nonlinear regression (Sigma Stat; SPSS. Inc., San Rafael, CA). The coefficient of determination, or R2, was used to estimate the variance that could be due to regression with each of the PK/PD indices.

We utilized the 24-h static dose as well as the doses necessary to achieve a 1-log10 reduction in colony counts compared to numbers at the start of therapy to compare the impacts of the dosing intervals on treatment efficacy. If these dose values remained similar among each of the dosing intervals, this would support the 24-h AUC/MIC as the predictive index. If the dose values increased as the dosing interval was lengthened, this would suggest that T > MIC is the predictive index. Lastly, if the dose values decreased as the dosing interval was increased, this would support peak/MIC as the pharmacodynamically important index. The static dose and 1-log kill values for each of the dosing intervals were compared statistically using analysis of variance.

To allow a comparison of the potencies of NZ2114 against a variety of organisms, we utilized the 24-h static dose. The magnitude of the PK/PD index associated with each endpoint dose was calculated from the equation log10 D = log10 (E/Emax − E)/N + log10 ED50, where D is the drug dose, E is the control growth for dose (D), E is the control growth + 1 log for a D of 1-log kill, Emax is the maximal effect, N is the slope of the dose-response relationship, and ED50 is the dose needed to achieve 50% of the maximal effect. The significance of differences among the various dosing endpoints was determined by using analysis of variance on ranks.In vitro susceptibility testing.
The MICs of NZ2114 for the 14 study strains are shown in Table ​Table1.1. NZ2114 MICs varied more than 30-fold (range, 0.06 to 2.0 μg/ml).

The time course of serum levels of NZ2114 in infected neutropenic mice following subcutaneous doses of 10, 40, and 160 mg/kg is shown in Fig. ​Fig.1.1. Peak levels were observed by 30 min. The elimination half-life in the mice ranged from 0.38 to 1.0 h. The AUC and peak values for the escalating single doses ranged from 17 to 204 and 21 to 126, respectively. The protein binding of NZ2114 in mouse serum, as determined by ultrafiltration, was 80%. This is similar to the degree of binding in other animal species and in human serum (D. Sandvang, personal communication).

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FIG. 1.: Serum pharmacokinetics of plectasin derivative NZ2114 following single subcutaneous doses. Each symbol represents the mean concentration from three mice. The error bars represent the standard deviations. Three dose levels (fourfold escalating) of NZ2114 were studied. The measured Cmax and calculated elimination half-life and AUC (zero to infinity) are shown in the table. Protein binding was determined by ultrafiltration using concentrations of 25 and 100 μg/ml. A microbiologic assay using S. aureus 6538p was used for determination of all NZ2114 concentrations.

In vivo PAE: At the start of therapy, mice had 106.6 to 106.7 CFU/thigh of S. pneumoniae and S. aureus, respectively. Growth of 1 log10 CFU/thigh in saline-treated animals occurred in 4.1 and 2.0 h in S. pneumoniae- and S. aureus-infected animals, respectively. Based upon the serum pharmacokinetic determinations, serum NZ2114 levels following the single doses of 10, 40, and 160 mg/kg remained above the MIC for S. pneumoniae strain ATCC 10813 (MIC, 0.06 μg/ml) for 2.3, 4.5, and 7.7 h based on free drug levels, respectively. Rapid, dose-dependent killing of organisms occurred following each of the dose levels for both strains. Maximal killing compared to organism burden at the start of therapy ranged from 2.7 to 3.7 log10 CFU/thigh over the dose range for S. pneumoniae. Over a similar dose range, maximal killing of S. aureus ranged from 0.5 to 1.7 log10 CFU/thigh. Organism regrowth with S. pneumoniae did not occur for more than 12 h. For the lowest dose in the S. aureus study, regrowth began 4 h after dosing. The times above the MIC for these doses against S. aureus strain ATCC 25923 (MIC, 1.0 μg/ml) were 1.2, 2.6, and 4.6 h based on free drug levels. The time-kill curves for both of the studies are shown in Fig. ​Fig.2.2. Against S. pneumoniae, escalating doses produced free drug PAEs ranging from 12.2 to 15.4 h. The study with S. aureus produced free drug PAEs ranging from 2.8 to 14.3 h. No detectable drug carryover was observed in any of the treatment groups.

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FIG. 2.: Impact of plectasin derivative NZ2114 dose escalation on burden of either S. pneumoniae or S. aureus in the thighs of neutropenic mice over time. Each symbol represents the mean CFU/thigh from two mice (four thighs). The error bars represent the standard deviations. Solid symbols represent growth of organisms in control mice over time. Open symbols represent the burden of organisms in NZ2114-treated mice. The horizontal boxes represent the duration of time that free drug NZ2114 serum concentrations remained above the MIC of the infecting organism. The PAE is expressed in hours.

PK/PD index determination: At the start of therapy, mice had 6.8 ± 0.07 and 6.3 ± 0.32 log10 CFU/thigh of S. pneumoniae strain ATCC 10813 and S. aureus strain ATCC 29213, respectively. The organisms grew 2.1 ± 0.2 and 3.4 ± 0.03 log10 CFU/thigh after 24 h in untreated control mice, respectively. Escalating doses of NZ2114 resulted in the concentration-dependent killing of both strains. The highest doses studied reduced organism burden by 4.8 ± 0.26 and 1.7 ± 0.01 log10 CFU/thigh compared to numbers at the start of therapy for S. pneumoniae and S. aureus, respectively. The dose-response relationships for the four dosing intervals against S. pneumoniae and S. aureus are shown in Fig. ​Fig.3.3. The curves were similar among each of the dosing intervals against S. aureus. In the study with S. pneumoniae, there was a slight shift in the dose-response curve to the left (indicating enhanced effect) as the dosing interval was lengthened. The dose levels required to produce stasis and a 1-log reduction were calculated for each dosing interval (data not shown). At each of these treatment endpoints, we did not observe a significant difference, as the dosing interval was lengthened from every 4 h to every 24 h (data not shown). These analyses suggest that treatment efficacy was dependent upon dose level and independent of the dosing intervals studied. The relationships between microbiologic effect and each of the pharmacodynamic indices, 24-h AUC/MIC, percent time above the MIC, and peak/MIC against S. pneumoniae strain ATCC 10813, are shown in Fig. ​Fig.4.4. Therapeutic outcome correlated well with both the 24-h AUC/MIC and maximum concentration of drug in serum (Cmax)/MIC indices. For S. aureus the relationship was strongest for the 24-h AUC/MIC index (R2 = 0.90 for 24-h AUC/MIC; R2 = 0.85 for Cmax/MIC), and for S. pneumoniae the Cmax/MIC index correlated somewhat better (R2 = 0.89 for 24-h AUC/MIC; R2 = 0.97 for Cmax/MIC) (Fig. ​(Fig.5).5). Regression with the percent T > MIC index resulted in a less strong relationship (R2 = 0.74 for each organism). The reasonable fit of the data with each of the PK/PD indices is due to the interrelationships among each of the indices (5). Consideration of total or unbound drug levels did not appreciably impact the relationship between efficacy and percent T > MIC (data not shown).

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FIG. 3.: Impact of plectasin derivative NZ2114 dosing interval on efficacy against a strain of S. aureus and a strain of S. pneumoniae in the neutropenic murine thigh infection model. Five total (mg/kg/24-h) doses were fractionated into one, two, four, or six doses over the 24-h treatment period (q24 h, q12 h, q6 h, and q4 h, respectively). Total escalating doses varied fourfold. Efficacy is expressed as change in CFU/thigh compared to organism burden at the start of therapy. Each symbol represents the mean CFU/thigh from two mice (four thighs). The error bars represent the standard deviations. The horizontal dashed line represents the burden of organisms at the start of therapy.

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FIG. 4.: Relationship between the NZ2114 pharmacodynamic index (24-h AUC/MIC, Cmax/MIC, and percent time above MIC) and efficacy over 24 h against S. pneumoniae 10813 in a neutropenic murine thigh infection model. Unbound (free drug) concentrations were used for index calculations. Efficacy is expressed as change in CFU/thigh compared to organism burden at the start of therapy. Each symbol represents the mean CFU/thigh from two mice (four thighs). The sigmoid line represents the best fit using the sigmoid Emax model. R2 is the coefficient of determination.

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FIG. 5: Relationship between the NZ2114 pharmacodynamic index (24-h AUC/MIC, Cmax/MIC, and percent time above MIC) and efficacy over 24 h against S. aureus 25923 in a neutropenic murine thigh infection model. Unbound (free drug) concentrations were used for index calculations. Efficacy is expressed as change in CFU/thigh compared to organism burden at the start of therapy. Each symbol represents the mean CFU/thigh from two mice (four thighs). The sigmoid line represents the best fit using the sigmoid Emax model. R2 is the coefficient of determination.

PK/PD magnitude determination.
Calculation of the doses necessary to achieve a static effect and a 1-log10 kill against multiple organisms is shown in Table ​Table1.1. The growth curves of the eight pneumococcal and six staphylococcal strains in the thighs of control animals were relatively similar. At the start of therapy, mice had 7.0 ± 0.44 (range, 5.6 to 7.6) log10 CFU of pneumococci or S. aureus/thigh. The organisms grew to 2.4 ± 0.54 log10 CFU/thigh (range, 1.5 to 3.4) in untreated control mice. The maximal reduction in S. pneumoniae with NZ2114-treated mice compared to untreated controls ranged from 2.8 ± 0.3 to 4.6 ± 0.2 log10 CFU/thigh (mean, 3.4 ± 0.60). Less killing was observed against the S. aureus strains (mean, 1.9 ± 0.42 log10 CFU/thigh).

The 24-h AUC/MIC index was used for determination of the pharmacodynamic magnitude of exposure associated with efficacy. Table ​Table11 shows the 24-h dose and free drug 24-h AUC/MIC ratios necessary to achieve a net static effect and 1-log10 reduction in organism burden. The static doses varied from 1.1 mg/kg every 24 h to 218 mg/kg every 24 h against the strains of S. pneumoniae and S. aureus. The corresponding free drug 24-h AUC/MICs varied from 3.4 to 37.7. The mean 24-h AUC/MICs associated with a static effect were slightly lower for S. pneumoniae than for S. aureus (12.3 ± 6.7 versus 28.5 ± 11.1, respectively) (P = 0.006). The exposure associated with bactericidal activity or a 1-log10 reduction in organism burden compared to the start of therapy was less than twofold larger than that associated with a stasis endpoint. The presence of antimicrobial resistance in both bacterial species did not alter the 24-h AUC/MIC required to produce efficacy. The relationship between the 24-h free drug AUC/MIC and efficacy against the two organism groups is demonstrated graphically in Fig. ​Fig.6.6. The exposure-response relationships were relatively strong, with R2 values of 0.85 and 0.82 for S. pneumoniae and S. aureus, respectively.

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Figur 6: Relationship between NZ2114 free drug 24-h AUC/MIC and efficacy against eight S. pneumoniae (left) and six S. aureus (right) strains. Each symbol represents the mean CFU/thigh from two mice (four thighs). Efficacy on the y axis is expressed as the change in CFU/thigh compared to the burden of organisms at the start of therapy. The dashed horizontal line represents the burden of organisms in thighs at the start of therapy. The sigmoid line represents the best-fit curve using the sigmoid Emax model. R2 is the coefficient of determination.

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DISCUSSION
The number of infections due to resistant gram-positive bacteria, including penicillin-resistant Streptococcus pneumoniae and methicillin-resistant Staphylococcus aureus, continues to increase (15). The development of effective antimicrobial agents to treat these infections is an area of intense research. Peptide antimicrobial agents represent a promising new class of compounds which collectively act at a number of different bacterial targets and have demonstrated potency against these emerging pathogens (12).

Previous in vivo studies in two pneumococcal murine infection models showed both enhanced animal survival and a rapid decline in organism burden following single doses of the native plectasin molecule (12). A logical next step in the evaluation of these molecules is to use animal infection molecules to understand the pharmacodynamic activity of this drug class. Antimicrobial pharmacodynamic studies characterize the time course of antibiotic effect. The time course of antimicrobial activity can be determined by two characteristics: (i) the effect of increasing drug concentrations on the extent of organism killing and (ii) the presence or absence of antimicrobial effects that persist after the levels in serum have fallen below the MIC (5). The use of animal infection models to understand the pharmacodynamic activity of antimicrobial agents is one approach that has proven helpful for (i) the design of effective dosing regimens in humans and (ii) the development of appropriate in vitro susceptibility breakpoints (3).

In these studies, escalating doses of NZ2114 produced a rapid reduction in viable counts. Each of the doses examined reduced the burden of organisms more than 2 log in the first 6 h after a single dose of NZ2114, and nearly 2 additional logs of killing was observed while free drug concentrations exceeded the MIC of the infecting S. pneumoniae strain. After drug concentrations fell below the MIC, we observed prolonged growth suppression. A similar time course pattern was observed in a study with a strain of S. aureus. However, the degree of organism killing was somewhat lower against the S. aureus strain. Prolonged PAEs were also seen against S. aureus, and the duration of the PAE was dose dependent. The efficacy of antibiotics characterized by this pattern of activity is best correlated with either the 24-h AUC/MIC or the Cmax/MIC index.

Results from analysis of the multiple-dosing regimen studies indicate that the 24-h AUC/MIC is the best index for predicting the efficacy of NZ2114. The dose-response relationship against S. aureus was not impacted by a change in dosing interval, and pharmacodynamic regression was clearly strongest with the 24-h AUC/MIC index. In the pneumococcal investigation, the dosing interval did appear to have some impact on the dose-response relationship. As the dosing strategy provided larger doses more infrequently, the dose-response curves shifted slightly to the left. This visual pharmacodynamic relationship would suggest that the Cmax/MIC is important (5). However, statistical comparison of dosing endpoints (static dose and 1-log kill) among the dosing intervals did not show significance. The regression analysis results based upon the coefficient of determination (R2) suggested that both the Cmax/MIC and 24-h AUC/MIC indices predict efficacy with NZ2114.

Next, studies were undertaken to identify the magnitude of the pharmacodynamic index needed for efficacy of NZ2114. We considered the 24-h AUC/MIC index for these analyses. Experiments included 14 organisms with a wide range of NZ2114 MICs (more than 30-fold). The strains were chosen to include those resistant to beta-lactams. In vitro studies with NZ2114 demonstrated antimicrobial activity against S. pneumoniae and S. aureus including strains resistant to other antimicrobial classes including beta-lactams, macrolides, quinolones, and vancomycin (14a). Similarly, in the current in vivo experiments penicillin resistance in S. pneumoniae and methicillin resistance in S. aureus had no impact upon the in vivo pharmacodynamic target of NZ2114.

The NZ2114 dose-response relationships for each species were very similar. However, the dose-response curves for S. pneumoniae were shifted somewhat to the left compared to those for S. aureus. This slight difference in activity is similar to that observed in the time course studies. The free (unbound) NZ2114 24-h AUC/MIC associated with a net static effect against S. pneumoniae was roughly twofold lower than that for S. aureus. The dose-response curves were relatively steep against both bacterial species, and the pharmacodynamic exposure associated with a killing endpoint (1-log kill) was less than twofold greater than that needed for a stasis effect.

The microbiologic outcomes in this infection model have correlated with clinical and microbiologic efficacy of several antimicrobial classes in humans (1, 2, 3, 4, 5). The results of these studies should be utilized to examine the pharmacokinetics of NZ2114 in humans in the context of MIC distribution of target organisms to help guide appropriate dosing regimen design and to determine preliminary susceptibility breakpoints.

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In Vivo Pharmacodynamic Characterization of a Novel Plectasin Antibiotic, NZ2114, in a Murine Infection Model
D. Andes, W. Craig, L. A. Nielsen, H. H. Kristensen
Antimicrob Agents Chemother. 2009 Jul; 53(7): 3003–3009. Published online 2009 May 4. doi: 10.1128/AAC.01584-08
PMCID: PMC2704636

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Controlled Release of Plectasin NZ2114 from a Hybrid Silicone-Hydrogel Material for Inhibition of Staphylococcus aureus Biofilm
Kasper Klein,a Rasmus Birkholm Grønnemose,a Martin Alm,b Karoline Sidelmann Brinch,c Hans Jørn Kolmos,a and Thomas Emil Andersencorresponding authora
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ABSTRACT
Staphylococcus aureus is a major human pathogen in catheter-related infections. Modifying catheter material with interpenetrating polymer networks is a novel material technology that allows for impregnation with drugs and subsequent controlled release. Here, we evaluated the potential for combining this system with plectasin derivate NZ2114 in an attempt to design an S. aureus biofilm-resistant catheter. The material demonstrated promising antibiofilm properties, including properties against methicillin-resistant S. aureus, thus suggesting a novel application of this antimicrobial peptide.

KEYWORDS: Staphylococcus aureus, antimicrobial peptides, plectasin, biofilm formation, catheter infections, biofilms, catheter, hydrogel

A decade ago, antimicrobial peptides (AMPs) were considered to be one of the most important new classes of drugs in the fight against antimicrobial resistance. Today, however, most AMPs have been removed from development pipelines, with only a few having reached actual clinical use (1).

Despite their obvious potential, the systemic use of AMPs is hampered by their intrinsic immunogenicity and low in vivo stability (2), which together with regulatory requirements of superior efficiency to current treatment have been major obstacles for obtaining approval (1). The use of AMPs in combination with medical devices to prevent device-associated infections is, however, an enticing alternative application that to a lesser extent is restricted by the above problems (3, 4, 5).

Device-associated and, specifically, catheter-related bloodstream infections (CRBSI) constitute a major problem in modern health care. Methicillin-sensitive Staphylococcus aureus (MSSA) and, in particular, methicillin-resistant S. aureus (MRSA) are among the most important pathogens in CRBSI. The plectasin derivate NZ2114 is a promising AMP that has been potentiated toward MSSA, MRSA, and vancomycin-resistant S. aureus with proven in vivo efficiency (6, 7) and, therefore, holds potential for use against device-associated infections, including CRBSI.

Here, we evaluated the properties of a novel hybrid catheter material loaded with plectasin NZ2114 in order to inhibit MRSA biofilms. The material consists of an interpenetrating polymer network (IPN) based on silicone elastomer (polydimethylsiloxane) as the host polymer and poly(2-hydroxyethyl methacrylate)-co-poly(ethylene glycol) methyl ether acrylate (PHEMA-co-PEGMEA) hydrogel as the guest polymer. This IPN material exhibits unique drug loading and release properties through adsorption of drugs into the hydrogel component in the bulk of the material and subsequent release upon exposure to aqueous solutions, such as bodily fluids (8, 9, 10, 11). Hypothetically, the catheter matrix protects the embedded drug, in this case plectasin NZ2114, from proteolytic degradation. Furthermore, the IPN matrix ensures a controlled release of the drug, potentially resulting in prolonged efficiency and fewer side effects compared to those of the burst release often associated with conventional drug coatings.

The IPN material was produced as previously described (8) with modifications. Briefly, silicone samples were punched out from 2-mm-thick sheets of silicone rubber (PE4062; Lebo Production, Skogås, Sweden) or cut from silicone tubing (outer/inner diameter, 1.65/0.76 mm; Helix Medical, VWR, Radnor, PA). The samples were placed in a high-pressure reactor with equal amounts of HEMA (2-hydroxyethyl methacrylate; Aldrich Chemistry, Germany) and EGMEA (ethylene glycol methyl ether acrylate; Aldrich, Germany) with supercritical carbon dioxide as an aiding solvent. After polymerization into PHEMA-co-PEGMEA inside the silicone material, the samples were placed in 96% ethanol for 7 days to remove residual monomer followed by drying at 50°C until final mass was reached. Two IPN sample sets with hydrogel contents of 23% and 28%, respectively, were produced and evaluated. The amount of PHEMA-co-PEGMEA hydrogel in the samples was determined by mass increase. IPN samples were drug loaded by immersion in Milli-Q water containing 10 mg/ml of plectasin NZ2114 (6) (Novozymes A/S, Bagsværd, Denmark) or 10 mg/ml of dicloxacillin (Bristol-Myers Squibb, New York City, NY) for 7 days.

To evaluate release from the catheter material, release sampling was performed daily over 14 days from catheter tubing, 23 mm in length, placed in phosphate-buffered saline (PBS) with a pH of 7.4. The plectasin NZ2114 concentration was quantified by ultraperformance liquid chromatography (UPLC) (Fig. 1). Mean totals of 46.1 µg ± 21.3 (mean ± standard deviation) and 42.9 µg ± 20.9 for 23% and 28% PHEMA-co-PEGMEA, respectively, were released from the catheter specimens during this time period. The release correlated well with first-order kinetics (R2 = 0.97 and 0.92 for 23% and 28% PHEMA-co-PEGMEA, respectively), with an approximate release of 40.7% and 31.9% of remaining loaded drug each day for the 23% and 28% samples, respectively, indicating a better long-term slow release profile for catheters with the higher IPN content.

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Object name is zac0071763390001.jpg(A) Mean accumulated release from catheter specimens measured in micrograms (n = 4). (B) Mean release per day measured in micrograms per milliliter (release volume = 1.5 ml; n = 4).

Antimicrobial activity of plectasin NZ2114 and dicloxacillin against MSSA and MRSA was determinated as MIC and minimum bactericidal concentration (MBC) by broth dilution according to ISO 20776-1 standards (Table 1).

Strain MIC (μg/ml)
MBC (μg/ml)
Dicloxacillin Plectasin NZ2114 Dicloxacillin Plectasin NZ2114
MSSA ATCC 29213 1 0.5 8 8
MRSA ATCC 33591 32 1 >1,024 16
The loaded catheter tubing was tested for bactericidal effect in a static setup. The catheter tubing was placed in test tubes containing 10% heparinized human plasma in PBS, inoculated with approximately 5.0 × 104 CFU/ml of MRSA strain ATCC 33591, and incubated overnight. The CFU count in the suspension was then estimated, and the catheter tubing was transferred to a new test tube containing inoculated plasma and the procedure repeated for 11 days (Fig. 2).

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Functional release assay (n = 3) using PEGMEA-co-PHEMA IPN material impregnated with dicloxacillin or plectasin for inhibition of MRSA ATCC 33591. The CFU values in vials containing test catheters were measured after daily challenge with 5 × 104 CFU per ml plasma medium and are shown in the graph as a percentage of this daily inoculum. In control vials containing pristine silicone and unloaded IPN samples, CFU reached >1,000% of the inoculum added on day 1 (data not shown).

As it appears in Fig. 2, some fluctuations occur in this type of experiment, a phenomenon we have observed in an earlier study as well (11). To evaluate whether this is due to plectasin NZ2114 destabilization during loading, storage in, and release from the IPN hydrogel, the activity of the compound after release in buffer was tested using MIC determination, and dilutions were matched to the high-pressure liquid chromatography (HPLC) release quantification data (Fig. 1). Comparing these data to the MIC values for the stock showed no loss of activity upon loading and release (data not shown). Together with the relatively low day-to-day fluctuation observed in the direct release measurements (Fig. 1), we speculate that the fluctuations in Fig. 2 occur due to biological variation in bacterial sensitivity, where subpopulations may enter biofilm or an otherwise more persistent growth mode differently from day to day.

Assuming that the catheters remain effective as long as the mean CFU counts are below the initial inoculum (i.e., the 100% point on the y axis in Fig. 2), the dicloxacillin-loaded tubing lasts for 2 days whereas the plectasin NZ2114-loaded specimens last until day 10 (Fig. 2).

In an in vivo setting, a considerable part of venous catheters is exposed to blood flow, placing high demands on this part of the material with respect to maintaining an effective release of drug. Bacteria spreading to catheters may, furthermore, originate from biofilms growing on skin/wound sites; i.e., they are already more resilient than traditional broth-cultured bacteria when reaching the catheter material. To account for these conditions, plectasin NZ2114-loaded IPN discs (hydrogel content of 24%) were challenged with MRSA ATCC 33591 in a flow chamber model as previously described (11, 12) using an initial bacterial seeding inoculum of optical density at 600 nm (OD600) of 0.100 in PBS for 30 min at 30 μl/min. This was followed by a growth phase in 10% heparinized human plasma in PBS at 30 μl/min, which leads to a continuous seeding over the catheter surface with resilient biofilm emboli (11). The plectasin NZ2114-loaded disks showed effective inhibition of bacterial surface colonization compared to unloaded IPN disks as visualized with the LIVE/DEAD BacLight bacterial viability kit (L7012; Molecular Probes, Eugene, OR) using fluorescence and confocal laser scanning microscopy (CLSM) (Fig. 3).

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Microscopy of biofilms formed by MRSA ATCC 33591 on IPN material either as unloaded control (A, B, C) or loaded with plectasin NZ2114 (D, E, F). (A, B, D, and E) 0.64 mm by 0.64 mm CLSM images obtained with an Olympus FV1000MPE. (C and F) Full fluorescence microscopy scans of the flow chamber test surface performed using an Olympus BX51 microscope with motorized stage and image processing using Olympus cellSens software (only the green/GFP channel is shown). Over the 24-h experiment, the surface was continuously seeded with biofilm emboli (11).

To quantitatively assess whether the catheter tubing exhibited adequate drug-release properties for biofilm inhibition despite its limited wall thickness, catheter samples loaded with plectasin NZ2114 were tested in a flow system seeded as the previous flow chamber assay with MRSA and subsequently grown in a flow of 10% heparinized plasma. CFU in the resulting biofilm inside the tubing were quantified by pipetting 0.1% Triton X-100 in PBS through the tubing followed by plating. This experiment demonstrated effective prevention of growth in the plectasin NZ2114-loaded catheters compared to unloaded and dicloxacillin-loaded catheters (Fig. 4), displaying a significant 3-log reduction compared to unloaded IPN (Table 2). Furthermore, plectasin NZ2114-loaded catheters exposed to prerelease in PBS for 6 days prior to testing still exhibited a significant 2-log reduction compared to the unloaded IPN, indicating a clinically relevant release after 1 week of catheter placement (Table 2).

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Vs Dicloxacillin Plectasin 1d Plectasin 6d + 1d Silicone
Plectasin 1d <0.01
Plectasin 6d + 1d NS <0.01
Silicone NS <0.01 0.05
Unloaded IPN NS <0.01 <0.01 NS
aOne-way analysis of variance (ANOVA) followed by Tukey's honest significant difference test. n = 4 for all 5 groups. Before testing, data were transformed by natural logarithm to obtain normal distributions. P values are shown. NS = not significant.
Induction of antibiotic resistance is a relevant concern for medical devices that passively release antimicrobials due to an unavoidable period of time near drug depletion when the release of drug becomes less than the MIC. To asses to what extent sub-MICs of plectasin NZ2114 may induce resistance in S. aureus, we applied a serial passage of increasing drug concentration modified from Hammer et al. (13). In brief, ∼105 bacteria were inoculated in 5 ml tryptic soy broth (TSB) overnight at 37°C on a shaker. The following day, 100 μl of the culture was transferred to new test tubes containing 4.9 ml TSB with either ciprofloxacin or plectasin NZ2114 at 25% of MIC and incubated overnight. Then, 100 μl was transferred to new TSB tubes in which drug concentrations were increased by 100%. This procedure continued until growth inhibition was observed compared to positive controls without antimicrobials (21 days in this experiment). At this point, 100 μl of the bacterial suspension was plated and MIC determined as described above. Results for MSSA ATCC 29213 and MRSA ATCC 33591 (Table 3) showed that plectasin NZ2114 induced drug resistance only to a minor extent compared to the control antibiotic ciprofloxacin.

Strain MIC before serial passing:
MIC after serial passing
Ciprofloxacin Plectasin NZ2114 Ciprofloxacin Plectasin NZ2114
MSSA ATCC 29213 0.25 (0.25) 0.5 (0.5) 32 (32) 4 (4–8)
MRSA ATCC 33591 0.25 (0.25) 1 (1.0) 32 (32–64) 8 (4–8)
aMIC values were determined according to ISO standard 20776-1. All numbers are in μg/ml and presented as median with range in parentheses (n = 3).
Lastly, to assess possible immune synergism of plectasin NZ2114, we determined the MBC for MSSA ATCC 29213 and MRSA ATCC 33591, respectively, in heat-inactivated and untreated 10% pooled human serum in PBS. Here, identical MBC values were measured in untreated and heat-inactivated serum (data not shown), indicating no synergistic effects with complement factors, in contrast to what has been reported for other antimicrobial peptides (14, 15).

Plectasin NZ2114 has previously shown promising results in the treatment of various S. aureus diseases in vivo (6, 7). Using this antimicrobial peptide as an active loading agent in a novel IPN-based device material showed promising results in a comprehensive in vitro test procedure, accounting for several relevant factors that influence the performance of the catheter in vivo. This suggests that the material-drug combination may be suitable for venous catheters for more effective prevention of colonization by staphylococci and other Gram-positive pathogens.

The study was funded by the Innovation Fund Denmark (grants 52-2014-1 and 041-2010-3) and Ph.D. grants from Odense University Hospital and the University of Southern Denmark.

Martin Alm is employed at the company Biomodics, the owner of the patent concerning IPN technology (10). Karoline Sidelmann Brinch is employed at Novozymes, former patent holder of plectasin NZ2114. The study was in no way funded by these or other companies.

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