Received 5 April 2011/Returned for modification 26 July 2011/Accepted 13 August 2011

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 2011, p. 5325 5330 Vol. 55, No. 11 0066-4804/11/$12.00 doi:10.1128/aac.00453-11 Copyright 2011, American Society for Microbiology. All Rights Reserved. Efficacy of NZ2114, a Novel Plectasin-Derived Cationic Antimicrobial Peptide Antibiotic, in Experimental Endocarditis Due to Methicillin-Resistant Staphylococcus aureus Yan Q. Xiong, 1,2 * Wessam Abdel Hady, 1 Antoine Deslandes, 3 Astrid Rey, 4 Laurent Fraisse, 4 Hans-Henrik Kristensen, 5 Michael R. Yeaman, 1,2 and Arnold S. Bayer 1,2 Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California 1 ; Geffen School of Medicine at UCLA, Los Angeles, California 2 ; Sanofi-Aventis Recherche & Development, Alfortville, France 3 ; Sanofi-Aventis Recherche & Development, Toulouse Cedex 1, France 4 ; and Novozymes A/S, Krogshoejvej 36, 2880 Bagsvaerd, Denmark 5 Received 5 April 2011/Returned for modification 26 July 2011/Accepted 13 August 2011 Cationic antimicrobial peptides (CAPs) play important roles in host immune defenses. Plectasin is a defensin-like CAP isolated from the saprophytic fungus Pseudoplectania nigrella. NZ2114 is a novel variant of plectasin with potent activity against Gram-positive bacteria. In this study, we investigated (i) the in vivo pharmacokinetic and pharmacodynamic (PK/PD) characteristics of NZ2114 and (ii) the in vivo efficacy of NZ2114 in comparison with those of two conventional antibiotics, vancomycin or daptomycin, in an experimental rabbit infective endocarditis (IE) model due to a methicillin-resistant Staphylococcus aureus (MRSA) strain (ATCC 33591). All NZ2114 regimens (5, 10, and 20 mg/kg of body weight, intravenously [i.v.], twice daily for 3 days) significantly decreased MRSA densities in cardiac vegetations, kidneys, and spleen versus those in untreated controls, except in one scenario (5 mg/kg, splenic MRSA counts). The efficacy of NZ2114 was clearly dose dependent in all target tissues. At 20 mg/kg, NZ2114 showed a significantly greater efficacy than vancomycin (P < 0.001) and an efficacy similar to that of daptomycin. Of importance, only NZ2114 (in 10- and 20-mg/kg regimens) prevented posttherapy relapse in cardiac vegetations, kidneys, and spleen, while bacterial counts in these target tissues continued to increase in vancomycin- and daptomycin-treated animals. These in vivo efficacies were equivalent and significantly correlated with three PK indices investigated: fc max /MIC (the maximum concentration of the free, unbound fraction of a drug in serum divided by the MIC), fauc/mic (where AUC is the area under the concentration-time curve), and f%t >MIC (%T >MIC is the cumulative percentage of a 24-h period that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions), as analyzed by a sigmoid maximum-effect (E max ) model (R 2 > 0.69). The superior efficacy of NZ2114 in this MRSA IE model suggests the potential for further development of this compound for treating serious MRSA infections. Endovascular infections are very prevalent, life-threatening infectious syndromes (13). Staphylococcus aureus is the most common cause of such infections worldwide, with an unacceptably high morbidity and mortality, especially when caused by antimicrobial-resistant strains (e.g., methicillin-resistant S. aureus [MRSA] and vancomycin-intermediate S. aureus [VISA]) (3, 8, 15, 17, 18, 26). Therefore, there is an urgent need to develop new antimicrobial agents for the prevention and treatment of these syndromes. Plectasin is a defensin-like, cationic antimicrobial peptide (CAP) isolated from the saprophytic ascomycete Pseudoplectania nigrella. This CAP contains 40 amino acids and has potent activity against Gram-positive bacteria in vitro and in vivo (19, 21). In addition, plectasin shares primary-structure features with defensin-like peptides from spiders, scorpions, dragonflies, and mussels and folds into a cystine-stabilized alpha-beta conformation (CS- ). Most CAPs have been thought to target the bacterial cell membrane. However, plectasin has a novel mode of antimicrobial action, specifically binding to the key bacterial cell wall precursor, lipid II (21), and thereby interfering with bacterial cell wall biosynthesis. NZ2114 is a new variant of plectasin with improved in vitro activity against staphylococci and streptococci, including those resistant to clinically available antibiotics (1, 4, 5, 19 21). Recent studies suggested that NZ2114 has potent activities both in vitro and in a number of animal models, including rabbit meningitis, murine peritonitis, and thigh infections and against various strains of Staphylococcus aureus and of Streptococcus pneumoniae (1, 5, 20). The purpose of the current study was to (i) compare the in vivo antimicrobial efficacy of NZ2114 with those of two standard anti-mrsa antibiotics, vancomycin and daptomycin, in an experimental rabbit MRSA infective endocarditis (IE) model and (ii) characterize the in vivo pharmacokinetic and pharmacodynamic (PK/PD) profiles of NZ2114 in this model. (This work was presented in part at the 50th Interscience Conference on Antimicrobial Agents and Chemotherapy, Boston, MA, 12 to 15 September 2010 [25a].) * Corresponding author. Mailing address: Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, 1124 West Carson Street, Bldg. RB-2, Torrance, CA 90502. Phone: (310) 222-3545. Fax: (310) 782-2016. E-mail: yxiong@ucla.edu. Published ahead of print on 22 August 2011. MATERIALS AND METHODS Bacteria, media, and antibiotics. A vancomycin-susceptible, nonheteroresistant, hospital-associated MRSA strain (ATCC 33591) was employed in this study (16, 22). Mueller-Hinton broth (MHB) and Mueller-Hinton agar (MHA) (both 5325

5326 XIONG ET AL. ANTIMICROB. AGENTS CHEMOTHER. from Difco Laboratories, Detroit, MI) were used to culture the bacterial strain. NZ2114 was supplied by Sanofi-Aventis (Toulouse, France). Vancomycin and daptomycin were purchased from APP Pharmaceuticals (Schaumburg, IL) and Cubist Pharmaceuticals (Lexington, MA), respectively. In vitro susceptibility studies. The MICs of NZ2114, vancomycin, and daptomycin against the MRSA ATCC 33591 strain (5 10 5 CFU/ml) were determined by standard Clinical and Laboratory Standards Institute microdilution methods (11). All MIC assays were performed in duplicate on three occasions. In vitro time-kill curves of NZ2114. Time-kill curves of NZ2114 (range, 0.5 to 5 MIC) were performed in glass flasks containing a final inoculum of 5 10 5 CFU/ml of the study MRSA strain at 37 C with shaking for 24 h. At 0, 2, 4, 6, and 24 h of incubation, 0.1-ml aliquots were taken from each group, serially diluted in sterile phosphate-buffered saline, plated onto MHA plates, and incubated at 37 C for 24 h for a viable count enumeration. Each time-kill experiment was carried out at least in duplicate on separate days. Experimental IE model. A well-characterized rabbit model of catheter-induced infective endocarditis (IE) was used to study both the pharmacokinetic and pharmacodynamic (PK/PD) profiles of NZ2114 and to compare its anti- MRSA efficacy with those of vancomycin and daptomycin. Animals were maintained in accordance with the American Association for Accreditation of Laboratory Animal Care criteria. All animal studies were approved by the Animal Research Committee (IACUC) of the Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center. Briefly, anesthetized rabbits (New Zealand White; Harlan Laboratory, Inc., CA) underwent transcarotid-transaortic valve catheterization. IE was produced by the intravenous (i.v.) injection of MRSA ATCC 33591 ( 1 10 6 CFU, the 95% infective dose [ID 95 ] inoculum for this strain in the IE model as determined from pilot studies) at 24 h after catheterization. Antibiotic treatments. At 24 h after infection, animals were randomized to receive (i) no therapy (controls), (ii) NZ2114 (5, 10, or 20 mg/kg, i.v., twice a day [b.i.d.]; the dose range was established based on pilot PK/PD studies and MRSA MICs), (iii) vancomycin (15 mg/kg, i.v., b.i.d.; the dose regimen was based on prior efficacy studies of an experimental model of MRSA IE [12]), or (iv) daptomycin (12 mg/kg, i.v., once daily; the dose regimen mimics human-like PK at 6 mg/kg, i.v., once daily [7]). Each treatment regimen was administered for 3 days. Untreated controls were sacrificed at 24 h postinfection to establish the target tissue MRSA densities at the outset of antibiotic therapies. One-half of the antibiotic-treated animals were sacrificed at 24 h after the last antibiotic dose (to minimize tissue antibiotic carryover effects) for evaluating the treatment efficacies. The remaining antibiotic-treated animals were maintained drug free for three additional days for analysis of microbiologic relapse as previously described (25). At the time of sacrifice, target tissues were aseptically removed and quantitatively cultured as previously described (24). Culture results are expressed as mean log 10 numbers of CFU per gram of tissue ( standard deviations [SD]). The lower limit of microbiologic detection of the organism density in the abovedescribed target tissues is 1 log 10 CFU per gram of tissue. This value was assigned to all culture-negative (sterile) target tissues for the purposes of calculating the mean log 10 numbers of CFU per gram of tissue ( SD) for eventual statistical comparisons. PK/PD studies. The NZ2114 pharmacokinetics were determined after single i.v. bolus injections of 5, 10, or 20 mg/kg at 24 h postinfection in animals with established MRSA IE (three rabbits per group). Blood samples were collected at 5 min, as well as 0.5, 1, 2, 4, 6, 8, 12, and 24 h after NZ2114 administration. After centrifugation of the heparinized blood samples, the plasma was immediately separated and frozen at 20 C until analysis. NZ2114 plasma concentrations were determined by using a qualified liquid chromatography/tandem mass spectrometry (LC/MS-MS) assay (Applied Biosystems API 4000 mass spectrometer) with a lower limit of detection of 50 ng/ml (10). The PK parameters were calculated from the individual free-drug plasma concentrations using the WinNonlin Professional 5.2 software package, based on noncompartmental model 201. Because the PK of vancomycin and that of daptomycin at the dosages used in this study have been previously determined in the IE model (2, 7, 12, 24), we did not repeat them in this study. Briefly, using vancomycin at 15 mg/kg, i.v., mean serum levels are routinely greater than 50 g/ml at 1 to 2 h postdose ( 100 times of the MIC of MRSA ATCC 33591) and 2 to 4 g/ml at 6 h postdose, with no detectable trough levels at 12 h postdose (12). Daptomycin at a dose of 12 mg/kg in rabbits mimics the PK/PD profile observed in humans administered a dose of 6 mg/kg, the currently recommended dose for S. aureus bacteremia and rightsided IE (7). PK/PD index data analysis. The relationship between each PK/PD index, fc max /MIC (the maximum concentration of the free, unbound fraction of a drug FIG. 1. In vitro time-kill curve of NZ2114. Concentrations equal 0, 0.5, 1, and 5 MIC against MRSA ATCC 33591. }, control; f, 0.5 MIC of NZ2114; Œ, 1 MIC of NZ2114; and, 5 MIC of NZ2114. in serum divided by the MIC), fauc/mic (where AUC is the area under the concentration-time curve), and f%t MIC (the cumulative percentage of a 24-h period that the free-drug concentration exceeds the MIC under steady-state pharmacokinetic conditions), and the efficacy were best fitted using a sigmoid maximum-effect (E max ) model. The PK/PD correlations were calculated from the individual efficacy data using the WinNonlin Professional 5.2 software with pharmacodynamic model 107. Statistical analysis. The in vivo efficacy data in terms of reductions in target organ bacterial counts were analyzed by a one-way analysis of variance (ANOVA), followed by Dunnett s adjustment for multiplicity, whereas noninferiority analyses were based on the comparison of an upper 90% confidence limit to a predefined data limit. For the analysis of the proportion of sterile tissue cultures, Fisher s exact test was used. P values of 0.05 were considered statistically significant. All statistical analyses were conducted using SAS software version 9.2. RESULTS In vitro susceptibility studies. The MICs of NZ2114, vancomycin, and daptomycin versus the MRSA ATCC 33591 study strain were 0.5, 2.0, and 0.125 g/ml, respectively. In vitro time-kill curves of NZ2114. In the absence of NZ2114, the bacterial counts reached their maximum at 24 h after inoculation (Fig. 1). In the presence of 0.5 MIC of NZ2114, the bacterial counts grew more slowly than with the controls. NZ2114 at 1 and 5 MIC reduced bacterial densities by up to 4 log 10 CFU/ml at 6 h incubation. However, NZ2114 at 1 MIC failed to inhibit bacterial regrowth at the 24-h time point (Fig. 1). The efficacies of NZ2114, vancomycin, and daptomycin in the IE model. MRSA densities in the different therapy groups are shown in Fig. 2. A dose-dependent efficacy of NZ2114 was clearly observed in all target tissues at the end of treatment in a comparison with CFU counts for untreated controls. NZ2114 at dosing regimens of 5, 10, and 20 mg/kg significantly reduced the mean MRSA vegetation counts by 2, 3, and 6 log 10 CFU/g, respectively, compared to counts in untreated controls. At 10 and 20 mg/kg, the effect was also significant for kidney and spleen MRSA counts. In addition, NZ2114 at 20 mg/kg, had a significantly greater efficacy in all target tissues than vancomycin (P 0.05). NZ2114 at 20 mg/kg demonstrated an efficacy equivalent to that of daptomycin at 12 mg/kg (Fig. 2). Relapse analysis indicated that for vancomycin and daptomycin, the bacterial load in all three target tissues was higher

VOL. 55, 2011 MRSA ENDOCARDITIS MODEL AND ANTIMICROBIAL PEPTIDES 5327 FIG. 2. Efficacy of NZ2114, vancomycin (VAN), and daptomycin (DAP) in an experimental IE model. MRSA densities in target tissues after a 3-day NZ2114, VAN, or DAP treatment in the IE model. The values are shown as the mean log 10 numbers of CFU of MRSA/g target tissue SD. Asterisks indicate statistical significance ( *, P 0.05; **, P 0.001; and ***, P 0.00001 versus untreated control groups). P 0.05, NZ2114 at 20 mg/kg compared to VAN. than the end-of-treatment counts. In contrast, only NZ2114 regimens (10 and 20 mg/kg) prevented microbiologic relapse in this experimental IE model (Fig. 3). Moreover, NZ2114 at 20 mg/kg yielded the highest percentages of sterile target tissue cultures in both the treatment and relapse groups (Table 1). PK. The free-drug plasma concentration-time curve profiles and the PK parameters of NZ2114 administered i.v. at 5, 10, and 20 mg/kg in experimental MRSA IE are shown in Fig. 4. Peak NZ2114 plasma levels were observed by 5 min after i.v. administration (first sampling time). The free-nz2114 half-life (t 1/2 ), following single doses of 5, 10, or 20 mg/kg, was approximately 2.3 h in the IE model. The AUC for the free, unbound fraction of the drug from 0 to 12 h (fauc 0 12 ) and peak values for the escalating single doses ranged from 3.09 to 10.3 mg h/liter and 1.78 to 8.44 mg/liter, respectively. Over the range of the doses tested, plasma exposures increased with each dose, with the ratio of the mean fauc 0-12 for the 10-mg/kg dose to that for the 5-mg/kg dose being 1.4 and the ratio of the mean fauc 0-12 for the 20-mg/kg dose to that for the 5-mg/kg dose being 3.3. The protein binding of NZ2114 in rabbit serum was 91.6 to 94.5%, which is consistent with that of the parent plectasin compound in human and murine sera (5). NZ2144 PK/PD index determination. The relationships between values for each free-drug PK/PD index, namely, f%t MIC, fc max /MIC, and fauc 0 12 /MIC, and the efficacy of NZ2114 against MRSA are shown in Fig. 5. Therapeutic outcomes correlated significantly with the f%t MIC, fc max /MIC, and fauc 0 12 /MIC indices (R 2 0.69). DISCUSSION The growing problem of resistance to conventional antibiotics and the need for new antibiotics has stimulated great interest in the development of antimicrobial peptides, especially congeners of native host defense peptides, as novel human therapeutic agents. This impetus has been driven in part by the demonstration that the emergence of resistance in vitro to antimicrobial peptides is less common and rapid than that of conventional antibiotics (14, 27). However, significant issues in the development of antimicrobial peptides, including potential host toxicities, durability in vivo, and formulation strategies, have hampered their development as therapeutic agents to date. Previous studies have shown that plectasin, a defensin-like CAP, and its derivative NZ2114 have potent in vivo activities and low systemic toxicities in several animal models, including models of meningitis, murine peritonitis-sepsis, pneumonia, and thigh infections (1, 19). However, such studies have not evaluated NZ2114 in a multiorgan model of endovascular infection due to a drug-resistant, Gram-positive pathogen. Thus, our present study evaluated the efficacy of NZ2114 versus conventional therapy in experimental model of MRSA IE. Several interesting findings emerged from the present study. First, in general, all drugs, especially NZ2114 and daptomycin, were significantly effective during therapy in reducing MRSA densities in all three target tissues compared to those in untreated controls. Second, the efficacy of NZ2114 against MRSA IE was clearly dose dependent in terms of reducing target tissue MRSA burdens and in terms of organ sterilizations. Third, NZ2114 (20 mg/kg) was significantly more effective than van-

5328 XIONG ET AL. ANTIMICROB. AGENTS CHEMOTHER. Downloaded from http://aac.asm.org/ FIG. 3. Treatment and relapse comparison of NZ2114, vancomycin, and daptomycin in the IE model. The values shown are log 10 numbers of CFU/g target tissue (MRSA density) SD. P was 0.05 for NZ2114 (at 10 and 20 mg/kg) and daptomycin versus vancomycin relapse groups. comycin in reducing MRSA counts in target tissues after 3 days of treatment and comparable to daptomycin. Finally, NZ2114 and daptomycin (but not vancomycin) had equivalent efficacies in preventing posttherapy target tissue microbiologic relapses. TABLE 1. Percentages of sterile tissue cultures after 3 days of treatment and at posttherapy relapse in an experimental model of MRSA IE Regimen (no. of animals at treatment/no. at relapse) Dose (mg/kg) % of cultures that were sterile during treatment/at relapse a Vegetation Kidney Spleen NZ2114 (8/7) 5 0/0 0/0 0/0 NZ2114 (8/8) 10 0/0 0/63 0/38 NZ2114 (8/8) 20 50*/50* 100*/100** 100*/100* Vancomycin (6/6) 15 0/0 0/0 0/0 Daptomycin (6/6) 12 33/33 83/50 67/50 a, P 0.05 versus 5 mg/kg and 10 mg/kg of NZ2114 and vancomycin treatment and relapse groups., P 0.05 versus 5 mg/kg of NZ2114 and vancomycin relapse groups. Notably, after discontinuation of therapy, only NZ2114 at 10 and 20 mg/kg completely prevented posttherapy bacteriologic relapse in target tissues during a 3-day drug-free period. Of interest, following NZ2114 therapy, reductions in vegetation MRSA counts of approximately 2 log 10 CFU/g were observed, despite NZ2114 serum concentrations falling below the MIC for the infecting MRSA strain. The mechanism(s) of NZ2114- mediated relapse prevention may be multifactorial. For example, Andes et al. have documented a prolonged postantibiotic effect (3 to 15 h) for NZ2114 against S. aureus (1). Such prolonged postantibiotic effects may well provide an advantage for infrequent dosing strategies for this compound, despite its relatively short t 1/2. In addition, Brinch et al. recently showed that NZ2114 and daptomycin (but not vancomycin) exhibited similar extracellular and intracellular anti-mrsa activities against vancomycin-susceptible strains (6). Given the capacity of staphylococci to penetrate and persist within endovascular cells as a means of establishing a reservoir for persistent infection (9, 23), the excellent intracellular bactericidal activities of on April 25, 2018 by guest

VOL. 55, 2011 MRSA ENDOCARDITIS MODEL AND ANTIMICROBIAL PEPTIDES 5329 FIG. 4. Free-NZ2114 plasma pharmacokinetics after a single i.v. administration. Regimens consisted of a 5-, 10-, or 20-mg/kg bolus in animals with established (24-h) experimental MRSA IE. Each symbol represents the mean of concentrations from three animals. Error bars represent SD. t 1/2, apparent half-life of the free-drug plasma concentration; C max, calculated as the initial concentration, estimated by back extrapolation for bolus i.v. models; AUC 0 12, area under the free-drug concentration-time curve from 0 to 12 h. Downloaded from http://aac.asm.org/ NZ2114 and daptomycin might yield both favorable therapeutic and relapse prevention outcomes in patients with IE. In conclusion, the present findings demonstrate that NZ2114 has a dose-dependent efficacy in vivo against MRSA in this experimental IE model. In addition, the treatment efficacy of NZ2114 was significantly greater than that of vancomycin and equivalent to that of daptomycin in this IE model. Together, these results suggest that NZ2114 may be a promising template on April 25, 2018 by guest FIG. 5. Relationship between the NZ2114 PK/PD indices (fauc/mic, fc max /MIC, and f%t MIC ) and NZ2114 s efficacy against MRSA in an experimental IE model. The unbound-drug (free-drug) concentrations were used for index calculations. Efficacy is expressed as the change in the number of CFU/g of vegetation from the organism burden at the initiation of therapy. Each symbol represents the result for an individual animal. The sigmoid line represents the best fit using the sigmoid E max model. R 2 is the coefficient of determination, with values of 0.5 typically considered significant correlations.

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