Efficacy of NZ 2114 , a Novel Plectasin - Derived Cationic Antimicrobial Peptide Antibiotic , 1 in Experimental Endocarditis due to Methicillin - Resistant Staphylococcus aureus 2 3 4

25 26 Cationic antimicrobial peptides (CAPs) play important roles in host immune defenses. 27 Plectasin is a defensin-like CAP isolated from the saprophytic fungus, Pseudoplectania nigrella. 28 NZ2114 is a novel variant of plectasin with potent activity against Gram-positive bacteria. In this 29 study, we investigated: i) the in vivo pharmacokinetic and pharmacodynamic (PK/PD) 30 characteristics of NZ2114; and ii) the in vivo efficacy of NZ2114 in comparison with two 31 conventional antibiotics, vancomycin or daptomycin, in a rabbit experimental endocarditis model 32 (IE) due to a methicillin-resistant Staphylococcus aureus (MRSA) strain (ATCC33591). All 33 NZ2114 regimens (5, 10 and 20 mg/kg, iv, twice daily for three days), significantly decreased 34 MRSA densities in cardiac vegetations, kidneys and spleen vs. untreated controls, except in one 35 scenario (5 mg/kg vs. splenic MRSA counts). The efficacy of NZ2114 was clearly dose36 dependent in all target tissues. At 20 mg/kg, NZ2114 showed a significantly greater efficacy vs. 37 vancomycin (P < 0.001), and similar efficacy to daptomycin. Of importance, only NZ2114 (at 10 38 and 20 mg/kg regimen) prevented post-therapy relapse in cardiac vegetations, kidneys and 39 spleen, while bacterial counts in these target tissues continued to increase in vancomycinand 40 daptomycin-treated animals. These in vivo efficacies were equivalent and significantly correlated 41 with three PK indices investigated: fCmax/MIC, fAUC/MIC and f%T>MIC, as analyzed by a 42 sigmoid Emax model (R 2 > 0.69). The superior efficacy of NZ2114 in this MRSA IE model 43 suggest the potential for further development of this compound for treating serious MRSA 44 infections. 45 46 47 48 on Jne 5, 2017 by gest httpaac.asm .rg/ D ow nladed fom INTRODUCTION 49 50 Endovascular infections are very prevalent, life-threatening infectious syndromes (13). 51 Staphylococcus aureus is the most common cause of such infections worldwide, with an 52 unacceptably high morbidity and mortality, especially when caused by antimicrobial-resistant 53 strains (e.g., methicillin-resistant [MRSA] and vancomycin-intermediate resistant S. aureus 54 [VISA]) (3, 8, 15, 17, 18, 26). Therefore, there is an urgent need to develop new antimicrobial 55 agents for the prevention and treatment of these syndromes. 56 57 Plectasin is a defensin-like, cationic antimicrobial peptide (CAP) isolated from the 58 saprophytic ascomycete, Pseudoplectania nigrella. This CAP contains 40 amino acids and has 59 potent activity against Gram-positive bacteria in vitro and in vivo (19, 21). In addition, plectasin 60 shares primary structure features with defensin-like peptides from spiders, scorpions, dragonflies 61 and mussels, and folds into a cystine-stabilized alpha-beta conformation (CS-αβ). Most CAPs 62 have been thought to target the bacterial cell membrane. However, plectasin has a novel mode of 63 antimicrobial action, specifically binding to the key bacterial cell wall precursor, lipid II (21), 64 and thereby interfering with bacterial cell wall biosynthesis. NZ2114 is a new variant of 65 plectasin with improved in vitro activity against staphylococci and streptococci, including those 66 resistant to clinically available antibiotics (1, 4, 5, 19-21). Recent studies suggested that NZ2114 67 has potent activities both in vitro and in a number of animal models, including rabbit meningitis, 68 murine peritonitis and thigh infections, and against various strains of Staphylococcus aureus and 69 of Streptococcus pneumoniae (1, 5, 20). The purpose of the current study was to: i) compare the 70 in vivo antimicrobial efficacy of NZ2114 with two standard anti-MRSA antibiotics, vancomycin 71 on Jne 5, 2017 by gest httpaac.asm .rg/ D ow nladed fom and daptomycin, in a rabbit experimental MRSA endocarditis (IE) model; and ii) characterize the 72 in vivo pharmacokinetic and pharmacodynamic (PK/PD) profiles of NZ2114 in this model. 73 74 This work was presented in part at the 50th Interscience Conference on Antimicrobial 75 Agents and Chemotherapy. Abstract #F1-2076, Boston, September 12-15, 2010. 76 77 on Jne 5, 2017 by gest httpaac.asm .rg/ D ow nladed fom MATERIALS AND METHODS 78 79 Bacteria, media, and antibiotic. A vancomycin-susceptible, non-heteroresistant, hospital80 associated MRSA strain (ATCC33591) was employed in this study (16, 22). Mueller-Hinton 81 broth (MHB, Difco Laboratories, Detroit, MI) and Mueller-Hinton agar (MHA, Difco 82 Laboratories, Detroit, MI) were used to culture the bacterial strain. NZ2114 was supplied by 83 Sanofi-Aventis (Toulouse, France). Vancomycin and daptomycin were purchased from APP 84 Pharmaceuticals (Schaumburg, IL 60173), and Cubist Pharmaceuticals (Lexington, MA 02421), 85 respectively. 86 87 In vitro susceptibility studies. The minimal inhibitory concentrations (MICs) of NZ2114, 88 vancomycin and daptomycin against the MRSA ATCC33591 (5x10 5 CFU/ml) were determined 89 by the standard Clinical and Laboratory Standards Institute microdilution methods (11). All MIC 90 assays were performed in duplicate on three occasions. 91 92 In vitro time-kill curves of NZ2114. Time-kill curves of NZ2114 (range 0.5 5x MIC) were 93 performed in glass flasks containing a final inoculum of 5 x 10 5 CFU/ml of the study MRSA 94 strain at 37 o C with shaking for 24 hr. At 0, 2, 4, 6 and 24 hr of incubation, 0.1 ml aliquots were 95 taken from each group, serially diluted in sterile phosphate-buffered saline, plated onto MHA 96 plates and incubated at 37 o C for 24 h for viable count enumeration. Each time-kill experiment 97 was carried out in at least duplicate on separated days. 98 99 on Jne 5, 2017 by gest httpaac.asm .rg/ D ow nladed fom Experimental IE model. A well-characterized rabbit model of catheter-induced infective 100 endocarditis (IE) was used to study both the pharmacokinetic and pharmacodynamic (PK/PD) 101 profiles of NZ2114, and to compare its anti-MRSA efficacy with vancomycin and daptomycin. 102 Animals were maintained in accordance with the American Association for Accreditation of 103 Laboratory Animal Care criteria. All animal studies were approved by the Animal Research 104 Committee (IACUC) of the Los Angeles Biomedical Research Institute at Harbor-UCLA 105 Medical Center. Briefly, anesthetized rabbits (New Zealand white; Harland Laboratory Inc, 106 California) underwent transcarotid-transaortic valve catheterization. IE was produced by the iv 107 injection of MRSA ATCC33591 (~1 x 10 6 CFU; ID95 inoculum for this strain in the IE model as 108 determined from pilot studies) at 24 h after catheterization. 109 110 Antibiotic treatments. At 24 hrs after infection, animals were randomized to receive: 111 i) no therapy (controls); ii) NZ2114 (5, 10 or 20 mg/kg, iv, bid; dose-range established based on 112 pilot PK/PD studies and MRSA MICs); iii) vancomycin (15 mg/kg, iv, bid); dose-regimen based 113 on prior efficacy studies in experimental MRSA IE (12)); or iv) daptomycin (12 mg/kg, iv, once 114 daily; dose-regimen mimics human-like PK at 6 mg/kg/d iv, once daily (7)). Each treatment 115 regimen was administered for 3 days. Untreated controls were sacrificed at 24 hr post-infection 116 to establish the target tissue MRSA densities at the outset of antibiotic therapies. One-half of the 117 antibiotic-treated animals were sacrificed at 24 hr after the last antibiotic dose (to minimize 118 tissue antibiotic carryover effects) for evaluating the treatment efficacies. The remaining 119 antibiotic-treated animals were maintained drug-free for three additional days for analysis of 120 microbiologic relapse as previously described (25). At sacrifices, target tissues were aseptically 121 removed and quantitatively cultured as previously described (24). Culture results were expressed 122 on Jne 5, 2017 by gest httpaac.asm .rg/ D ow nladed fom as mean log10 CFU per gram of tissue (± SD). The lower limit of microbiologic detection of the 123 organism density in the above target tissues is ≤ 1 log10 CFU per gram of tissue. This value was 124 assigned to all culture-negative (‘sterile’) target tissues for the purposes of calculating the mean 125 log10 CFU per gram of tissue (± SD) for eventual statistical comparisons. 126 127 PK/PD studies. The NZ2114 pharmacokinetics were determined after single iv bolus 128 injections of 5, 10 or 20 mg/kg at 24 hr post-infection in animals with established MRSA IE 129 (three rabbits per group). Blood samples were collected at 5 minutes, as well as 0.5, 1, 2, 4, 6, 8, 130 12 and 24 h postNZ2114 administration. After centrifugation of the heparinized blood samples, 131 the plasma was immediately separated and frozen at -20 o C until analysis. NZ2114 plasma 132 concentrations were determined by using a qualified liquid chromatography-mass spectrometry 133 (LC/MS-MS) assay (Applied Biosystems API4000) with a lower limit of detection of 50 ng/ml 134 (10). The PK parameters were calculated from the individual free plasma concentrations using 135 the WinNonlin Professional 5.2 software package, based on the non-compartmental model #201. 136 137 Because the PK of vancomycin and daptomycin at the dosages used in this study have 138 been previously determined in the IE model (2, 7, 12, 24), we did not repeat them in this study. 139 Briefly, using vancomycin at 15 mg/kg, iv., mean serum levels are routinely greater than 50 140 μg/ml at 1-2 h post-dose (> 100 times of the MIC of MRSA ATCC33591), and 2-4 μg/ml at 6 h 141 post-dose, with no detectable trough levels at 12 h post-dose (12). Daptomycin at a dose of 12 142 mg/kg in rabbits mimics the PK-PD profile observed in humans administered a dose of 6 mg/kg, 143 the currently recommended dose for S. aureus bacteremia and right-sided IE (7). 144 145 on Jne 5, 2017 by gest httpaac.asm .rg/ D ow nladed fom PK/PD index data analysis. The relationship between each PK/PD index, f%T>MIC, free 146 Cmax/MIC (fCmax/MIC), free AUC/MIC (fAUC/MIC), and the efficacy were best fitted using a 147 sigmoid Emax model. The PK/PD correlations were calculated from the individual efficacy data 148 using the WinNonlin Professional 5.2 software, with pharmacodynamic model #107. 149 150 Statistical analysis. The in vivo efficacy data in terms of reductions in target organ bacterial 151 counts were analyzed by a one-way ANOVA followed by Dunnett’s adjustment for multiplicity, 152 whereas non-inferiority analyses were based on the comparison of upper 90% confidence limit to 153 a predefined limit data. For the analysis of the proportion of sterile tissue cultures, the Fisher’s 154 exact test was used. P values of < 0.05 were considered statistically significant. All statistical 155 analyses were conducted using Software SAS® v9.2. 156 157 158 on Jne 5, 2017 by gest httpaac.asm .rg/ D ow nladed fom RESULTS 159 160 In vitro susceptibility studies. The MICs of NZ2114, vancomycin and daptomycin versus the 161 MRSA ATCC33591 study strain were 0.5, 2.0 and 0.125 μg/ml, respectively. 162 163 In vitro time-kill curves of NZ2114. In the absence of NZ2114, the bacterial counts reached 164 their maximal at 24 hr after inoculation (Figure 1). In the presence of 0.5x MIC of NZ2114, the 165 bacterial counts grew more slowly than with the controls. NZ2114 at 1x and 5x MIC reduced 166 bacterial densities by up to 4 log10 CFU/ml at 6 hr incubation. However, NZ2114 at 1x MIC 167 failed to inhibit bacterial re-growth at the 24 hr time point (Figure 1). 168 169 The efficacy of NZ2114, vancomycin and daptomycin in the IE model. MRSA densities in 170 the different therapy groups are shown in Figure 2. A dose-dependent efficacy of NZ2114 was 171 clearly observed in all target tissues at end-of-treatment as compared with untreated controls. 172 NZ2114, at 5, 10 and 20 mg/kg dosing regimens, significantly reduced mean MRSA vegetation 173 counts by ~2, 3 and 6 log10 CFU/g, respectively, as compared to untreated controls. At 10 and 20 174 mg/kg, the effect was also significant for kidneys and spleen MRSA counts. In addition, NZ2114 at 175 20 mg/kg, had a significantly greater efficacy in all target tissues as compared to vancomycin 176 (P < 0.05). NZ2114 at 20mg/kg demonstrated an equivalent efficacy to daptomycin at 12mg/kg 177 (Figure 2). 178 179 Relapse analysis indicated that for vancomycin and daptomycin, the bacterial load in all 180 three target tissues was higher than the end-of-treatment counts. In contrast, only NZ2114 regimens 181 on Jne 5, 2017 by gest httpaac.asm .rg/ D ow nladed fom (10 and 20 mg/kg) prevented microbiologic relapse in this experimental IE model (Figure 3). 182 Moreover, NZ2114 at 20 mg/kg yielded the highest percent of sterile target tissue cultures in 183 both treatment and relapse groups (Table 1). 184 185 PK. The free plasma time-concentration profiles and PK parameters of NZ2114 administered iv 186 at 5, 10 and 20 mg/kg in experimental MRSA IE are shown in Figure 4. Peak NZ2114 plasma 187 levels were observed by 5 min post iv administration (first sampling time). The free NZ2114 188 half-life (T1⁄2) following single doses of 5, 10 or 20 mg/kg was approximately 2.3 h in the IE 189 model. The free AUC0-12h and peak values for the escalating single doses ranged from 3.09 to 190 10.3 mg.h/l and 1.78 to 8.44 mg/l, respectively. Over the range of the doses tested, plasma 191 exposures increased with dose, with a mean fAUC0-12h for 10/5 mg/kg and 20/5 mg/kg ratios of 192 1.4 and 3.3, respectively. The protein binding of NZ2114 in rabbit serum was 91.6-94.5%, which 193 is consistent with the parent plectasin compound in human and murine serum (5). 194 195 NZ2144 PK/PD index determination. The relationships between each free PK/PD index, 196 f%T>MIC, fCmax/MIC and fAUC0-12h/MIC vs. the efficacy of NZ2114 against MRSA are shown 197 in Figure 5. Therapeutic outcomes correlated significantly with the f%T>MIC, fCmax/MIC and 198 fAUC0-12h/MIC indices (R 2 =0.69). 199 200 on Jne 5, 2017 by gest httpaac.asm .rg/ D ow nladed fom DISCUSSION 201 The growing problem of resistance to conventional antibiotics, and the need for new 202 antibiotics has stimulated great interest in development of antimicrobial peptides, especially 203 congeners of native host defense peptides, as novel human therapeutics. This impetus has been 204 driven in-part by the demonstration that the emergence of resistance in vitro to antimicrobial 205 peptides is less common and rapid as compared to conventional antibiotics (14, 27). However, 206 significant issues in development of antimicrobial peptides, including potential host toxicities 207 and durability in vivo, and formulation strategies, have hampered their development as 208 therapeutics to date. 209 210 Previous studies have shown that plectasin, a defensin-like CAP, and its derivative 211 NZ2114, have potent in vivo activity and low systemic toxicities in several animal models, 212 including meningitis, murine peritonitis-sepsis, pneumonia, and thigh infections (1, 19). 213 However, such studies have not evaluated NZ2114 in a multi-organ model of endovascular 214 infection due to a drug-resistant Gram-positive pathogen. Thus, our present study evaluated the 215 efficacy of NZ2114 vs. conventional therapy in experimental MRSA IE. 216 217 Several interesting findings emerged from the present study. Firstly, in general, all 218 regimens, especially NZ2114 and daptomycin were significantly effective in reducing MRSA 219 densities in all three target tissues as compared to untreated controls during therapy. Secondly, 220 the efficacy of NZ2114 against MRSA IE was clearly dose-dependent in terms of reducing target 221 tissue MRSA burdens and in organ sterilizations. Thirdly, NZ2114 (20 mg/kg) was significantly 222 more effective than vancomycin in reducing MRSA counts in target tissues after three days of 223 on Jne 5, 2017 by gest httpaac.asm .rg/ D ow nladed fom treatment, and comparable to daptomycin. Finally, NZ2114 and daptomycin (but not 224 vancomycin) had equivalent efficacy in preventing post-therapy target tissue microbiologic 225 relapses. Notably, after discontinuation of therapy, only NZ2114 at 10 and 20 mg/kg regimens 226 completely prevented post-therapy bacteriologic relapse in target tissues during a three days 227 drug-free period. Of interest, following NZ2114 therapy, approximately 2 logs10 CFU/g 228 reductions in vegetation MRSA counts were observed, despite NZ2114 serum concentrations 229 falling below the MIC of the infecting MRSA strain. The mechanism(s) of NZ2114-mediated 230 relapse prevention may be multifactorial. For example, Andes et al have documented a 231 prolonged post-antibiotic effect (3-15 hr) for NZ2114 against S. aureus (1). Such prolonged post232 antibiotic effects may well provide an advantage for infrequent dosing strategies for this 233 compound, despite its relatively short T1/2. In addition, Brinch et al recently showed that NZ2114 234 and daptomycin (but not vancomycin) exhibited similar extracellular and intracellular anti235 MRSA activities against vancomycin-susceptible strains (6). Given the capacity of staphylococci 236 to penetrate and persist within endovascular cells as a means of establishing a reservoir for 237 persistent infection (9, 23), the excellent intracellular bactericidal activities of NZ2114 and 238 daptomycin might yield both favorable therapeutic and relapse-prevention outcomes in IE. 239 240 In conclusion, the present findings demonstrate that NZ2114 has dose-dependent efficacy 241 in vivo against MRSA in this experimental IE model. In addition, the treatment efficacy of 242 NZ2114 was significantly greater than vancomycin, and equivalent to daptomycin, in this IE 243 model. Together, these results suggest that NZ2114 could be a promising template for further 244 development as a novel anti-MRSA therapeutics. 245 246 on Jne 5, 2017 by gest httpaac.asm .rg/ D ow nladed fom ACKNOWLEDGMENTS 247 This work was supported by grants from Sanofi-Aventis Recherche & Development, France, and 248 Novozymes, Copenhagen, Denmark (to YQX and ASB, respectively). 249 onJne5,2017bygesthttpaac.asm.rg/Downladedfom REFERENCES2501. Andes, D., W. Craig, L. A. Nielsen, and H. H. Kristensen. 2009. In vivo251pharmacodynamic characterization of a novel plectasin antibiotic, NZ2114, in a murine252infection model. Antimicrob Agents Chemother 53:3003-9.2532. Bayer, A. S., and K. Lam. 1985. Efficacy of vancomycin plus rifampin in experimental254aortic-valve endocarditis due to methicillin-resistant Staphylococcus aureus: in vitro-in255vivo correlations. J Infect Dis 151:157-65.2563. Bengualid, V., V. B. Hatcher, B. Diamond, E. A. Blumberg, and F. D. 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Antimicrob Agents336Chemother 49:380-7.33726. Young, L. S., F. Perdreau-Remington, and L. G. Winston. 2004. Clinical,338epidemiologic, and molecular evaluation of a clonal outbreak of methicillin-resistant339Staphylococcus aureus infection. Clin Infect Dis 38:1075-83.34027. Zasloff, M. 2002. Antimicrobial peptides of multicellular organisms. Nature 415:389-95.341onJne5,2017bygesthttpaac.asm.rg/Downladedfom Table 1. Percent of sterile tissue cultures after three days of treatment, and at post-therapy342relapse in experimental MRSA IE.343344 Regimen% sterile cultures (treatment/relapse) (# animals; treatment/relapse) Vegetations KidneysSpleen NZ2114 (5 mg/kg, n=8/7)0/00/00/0 NZ2114 (10 mg/kg, n=8/8)0/00/630/38 NZ2114 (20 mg/kg, n=8/8)50*/50* 100*/100** 100*/100* Vancomycin (15 mg/kg, n=6/6)0/00/00/0 Daptomycin (12 mg/kg, n=6/6) 33/3383/5067/50345* P < 0.05 vs. NZ2114 5 mg/kg and 10 mg/kg, and vancomycin treatment and relapse346groups.347** P < 0.05 vs. NZ2114 5 mg/kg and vancomycin relapse groups.348onJne5,2017bygesthttpaac.asm.rg/Downladedfom FIGURES349350Figure 1. In vitro time-kill curve of NZ2114. Concentrations = 0, 0.5x, 1x, and 5x MIC351against MRSA ATCC33591.352Control ; NZ2114 0.5xMIC ; NZ2114 1xMIC ; NZ2114 5xMIC .353354 355Time (h)onJne5,2017bygesthttpaac.asm.rg/Downladedfom Figure 2. Efficacy of NZ2114, vancomycin (VAN) and daptomycin (DAP) in an356experimental IE model. MRSA densities in target tissues after three-day NZ2114, VAN or357DAP treatment in the IE model. The values are shown as mean log10 CFU/g target tissue MRSA358 ± SD. Asterisks indicate statistical significance (*P < 0.05; **P < 0.001; and ***P < 0.00001 vs.359 untreated control groups). P < 0.05 = NZ2114 at 20 mg/kg as compared to VAN.360361 362onJne5,2017bygesthttpaac.asm.rg/Downladedfom Figure 3. Treatment and relapse comparison of NZ2114, vancomycin and daptomycin in363 the IE model. The values shown are log10 CFU/g target tissue MRSA density ± SD. P < 0.05 for364 NZ2114 (at 10 and 20 mg/kg) and daptomycin vs. vancomycin relapse groups.365366 367onJne5,2017bygesthttpaac.asm.rg/Downladedfom Figure 4. Free plasma pharmacokinetics of NZ2114 after a single iv administration.368Regimens: 5, 10 or 20 mg/kg bolus in animals with 24h-established experimental MRSA IE.369Each symbol represents the mean concentration from three animals. Error bars represent SDs.370 371372T1⁄2: apparent half-life of free plasma concentration; Cmax: calculated as C0, initial concentration,373estimated by back extrapolation for bolus iv models; AUC0-12h area under the free concentration-374time curve from 0 to 12h.375376onJne5,2017bygesthttpaac.asm.rg/Downladedfom Figure 5. Relationship between the NZ2114 PK/PD indices (fAUC/MIC, fCmax/MIC, and377f%T>MIC, percent time above MIC) and efficacy against MRSA in an experimental IE378model. Unbound (free drug) concentrations were used for index calculations. Efficacy is379expressed as change in CFU/g. vegetation compared to organism burden at initiation of therapy.380Each symbol represents an individual animal. The sigmoid line represents the best fit using the381sigmoid Emax model. R2is the coefficient of determination., with values > 0.5 typically382considered significant correlations.383384 385onJne5,2017bygesthttpaac.asm.rg/Downladedfom