AAC Accepts, published online ahead of print on 3 September 2013 Antimicrob. Agents Chemother. doi:10.1128/aac.00285-13 Copyright 2013, American Society for Microbiology. All Rights Reserved. 1 1 2 Efficacy of Ceftaroline Fosamil against Escherichia coli and Klebsiella pneumoniae Strains in a rabbit meningitis model. 3 4 A. Stucki, F. Acosta, M. Cottagnoud and Ph. Cottagnoud* 5 6 * Corresponding author 7 8 9 10 11 12 13 14 Prof. Dr. Ph. Cottagnoud Clinic of Internal Medicine Clinic Sonnenhof Buchserstrasse 30 3006 Bern Email: philippecottagnoud@sonnenhof.ch Tel.: +41-31-358 12 82 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
2 30 31 32 33 34 35 36 37 38 Abstract In this study the efficacy of ceftaroline fosamil was compared with cefepime in the experimental rabbit meningitis model against two Gram-negative strains (Escherichia coli and Klebsiella pneumoniae). Penetration of ceftaroline into inflamed and uninflamed meninges was also investigated in this study. Both regimens were bactericidal but ceftaroline fosamil was significantly superior to cefepime against K. pneumonia and E. coli in this experimental rabbit meningitis model( p<0,0007 against K. Pneumoniae and p<0.0016 against E.coli). The penetration of ceftaroline into inflamed meninges was approximately 15% and approximately 3% in uninflamed meninges. 39 40 41 42 43 44 45 46 47 48
3 49 50 51 52 53 54 55 56 57 Introduction Among the less common causative agents of bacterial meningitis, Klebsiella pneumoniae, a Gram-negative microorganism, plays an increasing role in adult community-acquired or hospital-acquired meningitis in the last years (1-3). Major risk factors for K. pneumoniae meningitis are diabetes mellitus, debilitating diseases, extrameningeal K. pneumoniae infections including bacteremia, and patients who have undergone neurosurgical procedures (4). In Taiwan, K. pneumoniae is one of the most frequent microorganisms causing meningitis (5). Escherichia coli is the most common Gram-negative pathogen causing meningitis in the neonatal period (6). 58 59 60 61 62 63 64 65 66 67 68 69 70 Ceftaroline fosamil, a prodrug, is rapidly converted in plasma to the microbiologically active ceftaroline, a novel cephalosporin with broad-spectrum in vitro activity against multidrugresistant Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae, and common Gram-negative organisms (7-9). Ceftaroline fosamil, administered parentally, is approved by the United States Food and Drug Administration for treatment of acute bacterial skin and skin structure infections and community-acquired bacterial pneumonia. Little is known about the efficacy of ceftaroline fosamil in bacterial meningitis due to Gram-negative microorganisms. The aim of this study was to test the efficacy of ceftaroline, following ceftaroline fosamil administration compared with cefepime against K. pneumoniae and E. coli strains in an experimental rabbit meningitis model and to determine the penetration of ceftaroline into inflamed and uninflamed meninges in the rabbit. 71 72 Material and Methods 73 74 75 76 77 78 Strains The strains, E. coli (QK-9) and K. pneumoniae (1173687), were kindly provided by the Institute for Infectious Diseases, University of Bern (Bern, Switzerland). Both strains were isolated in blood cultures from patients with sepsis without meningitisof the University of Bern hospital, grown in Muller-Hinton broth, and used only after several passages in the
4 79 80 81 82 rabbits. Minimum inhibitory concentrations (MICs) were determined by agar dilution according to NCCLS recommendations published literature (Lorian, Antibiotics in Laboratory Medicine, 5 th edition, Philadeaphia, Lippincott, Williams & Wilkins). Inocula for use in rabbit infection studies were prepared from stocks stored at -80 C. 83 84 85 86 87 88 89 Study drugs The prodrug form of ceftaroline, ceftaroline fosamil (602 µg/mg), was used for in vivo studies, and microbiologically active ceftaroline (lot # FMD-CEF-019, 813 µg/mg) was used for in vitro studies. Ceftaroline and Ceftaroline fosamil was obtained from Forest Laboratories (New York, NY), and cefepime was commercially purchased. Both were used according to manufacturer s recommendations. 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 Experimental meningitis model The experimental rabbit meningitis model described by Dacey and Sande (10) was employed in this study. The experimental protocols were approved by the Kantonales Veterinäramt des Kantons Bern. Pathogen-free New Zealand rabbits were provided by the Zentraltierställe der Medizinischen Fakultät der Universität Bern, where all the experiments were performed. One day before an experiment, rabbits were anesthetized with intramuscular injections of ketamine (30 mg/kg) and xylazine (15 mg/kg) and fitted with prostheses on their calvarium to facilitate subsequent placement within a stereotactic frame. On the day of the experiment, rabbits received 1.75 grams/kg ethylcarbamate (urethane) by subcutaneous injection and then 10 mg/kg pentobarbital by intravenous (IV) injection to induce deep anesthesia. The animals were fixed in a stereotactic frame, and a 3.5 inch (25G) spinal needle was introduced into the cisterna magna. Following the withdrawal of 0.2 ml of cerebrospinal fluid (CSF), meningitis was induced by injection of the test bacteria (1x10 5 colony forming units (CFU) in 0.2 ml of saline solution) into the subarachnoid space. After inoculation the animals were brought back to the cages for the night. The next day the rabbits were again fitted in the frames using the techniques and anesthesia described above. A catheter was fixed in the femoral artery for serum sampling, and a spinal needle was fixed again in the subarachnoid space. Intravenous
5 109 110 111 112 113 114 115 116 antibiotic administration was initiated 8 hours after inoculation and evaluations were performed over a period of 8 hours. Ceftaroline fosamil was injected intravenously (IV) at 40 mg/kg at hours 0 and 4 in order to mimic approximately kinetics in humans. The comparator regimen, cefepime 100 mg/kg was injected at hours 0 and 4, as a standard dose previously used in this model and corresponding to high doses in humans (11, 12). CSF (0.2 ml) was sampled at 0, 1, 2, 4, 6, and 8 hours after infection in all groups. Blood was sampled at 0.25, 0.5, 1, 2, 3, 4, 4.25, 4.5, 5, 6, 7 and 8 hours only in the ceftaroline group. Each group included untreated control rabbits. 117 118 119 120 121 122 123 124 125 126 127 128 129 Determination of antibiotic levels and CFU-titers Drug concentrations in serum and CSF were determined by diffusion microbioassays using agar plates containing Bacillus subtilis (sus-1-a) at 10 6 CFU/0.1 ml, (Raven Biological Laboratories, Inc.; Omaha, NE) (13, 14). The limit of detection was 0.2 mg/l for ceftaroline and 0.5 mg/l for cefepime. The intra- and interday variability was less than 10 %. CFU per ml was determined by serial dilution of CSF and plating on agar plates with incubation overnight at 37 C. The limit of bacterial detection of this assay was estimated to be 50 CFU/ml (1.7 log 10 ). Penetration into the CSF was determined by comparing areas under the concentration versus time curves (AUC) for serum and CSF, using the GraphPad Prism Software (GraphPad Software Inc., San Diego, CA). 130 131 132 133 134 135 136 137 138 Statistical analysis The Student t-test and one-way analysis of variance (Tukey-Cramer multiple comparisons test) were used for parametric data. Comparison of positive and negative cultures was analyzed by the two-tailed Fisher exact test. A P-value of <0.05 was considered significant. The efficacy of the different regimens is presented as log 10 CFU/ml/hour and as log 10 CFU/ml/8hours. A value of 1.7 log 10 CFU/ml was assigned to the first sterile CSF sample and a value of 0 assigned to each subsequent sterile CSF sample.
6 139 140 141 142 143 144 145 MICs of the test strains Prior to beginning studies to assess the pharmacokinetics (PK) of ceftaroline in the rabbit meningitis model, ceftaroline and cefepime MICs were determined for the test organisms. The MICs of ceftaroline against K. pneumoniae and E. coli were 1 mg/l and 0.06 mg/l, respectively; cefepime exhibited MICs of 0.25 mg/l and 0.12 mg/l, respectively, against these strains 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 Results and Discussion This study in rabbits showed that 15 minutes after an injection of ceftaroline fosamil 40 mg/kg, serum levels of ceftaroline in rabbits with inflamed meninges peaked around 40 mg/l and decreased rapidly to 4 mg/l four hours later. 15 minutes after a second injection, ceftaroline levels in serum reached approximately 44 mg/l decreasing to 2 mg/l at the end of the experimental period, i.e. after 8 hours. In the CSF, ceftaroline levels increased rapidly to 3.6 mg/l after one hour and decreased slowly to 2 mg/l before the second injection at hour 4. After the second injection, ceftaroline levels in the CSF peaked around 6.5 mg/l and decreased slowly to 2 mg/l at the end of the treatment period (see Figure 1). In healthy rabbits with uninflamed meninges, ceftaroline CSF levels were negligible (see Figure 2). Following IV injection of cefepime 100 mg/kg, levels in the CSF of rabbits with inflamed meninges were two- to three- fold higher than corresponding levels following injection of ceftaroline fosamil 40 mg/kg (Figure 3). Based on CSF and serum AUCs, the penetration of ceftaroline through inflamed meninges was approximately 15%, whereas penetration through uninflamed meninges was only about 3%. The PK parameters of ceftaroline into the CSF are summarized in Table 1. The PK parameters of cefepime have been described in previous studies (2, 4). 166 167 168 The MIC of ceftaroline was lower than that of cefepime for E. coli (0.06 mg/l for ceftaroline versus 0.12 mg/l for cefepime). However, the MIC of ceftaroline for K. pneumoniae was
7 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 four-fold higher than of that cefepime (1 mg/l for ceftaroline versus 0.25 mg/l for cefepime). The efficacies of the different regimens in vivo are summarized in Table 2. Against K. pneumoniae, both regimens exhibited bactericidal activity, although the killing rates of ceftaroline per hour (P<.0012) and at the end of the experimental period (P<.0007) were significantly superior compared with cefepime. Ceftaroline treatment resulted in a 5.6 log 10 reduction in viable counts over the 8-hour study compared with a 3.5 log 10 reduction for cefepime, despite the higher MIC of ceftaroline compared with that of cefepime for the K. pneumoniae isolate studied. Similar results were observed with E. coli, where both treatment regimens were bactericidal, and the antibacterial activity of ceftaroline was significantly superior compared with cefepime (P<.0015: killing rate/hour and P<.0016: killing rate/8 hours, respectively). The higher activity of ceftaroline against E. coli may be partially explained by the lower MIC of ceftaroline but the reason of the superiority of ceftaroline against K. pneumoniae remains unclear. In summary, ceftaroline fosamil was efficacious against the two Gram-negative pathogens tested in this experimental model of meningitis, with a bactericidal activity superior to cefepime,. It is conceivable that higher doses of ceftaroline fosamil could further increase the antibacterial activity in this experimental model. In both experimental settings, higher CSF levels did not correlate with higher killing rates. The penetration of ceftaroline through inflamed meninges of 15% of serum levels over the studied dosing interval is sufficient to provide bactericidal CSF levels for isolates with these MICs. The penetration of ceftaroline through uninflamed meninges (approximately 3%) can be considered as negligible. These results suggest that further studies in this animal model about the efficacy of ceftaroline fosamil in meningeal infections, including assessments of efficacy in infections caused by other common pathogens (e.g., S. pneumoniae), and studies to assess the effect of additional dosing regimens against isolates with higher MICs are warranted.
8 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 5. References 1. Durand ML, Calderwood SB, Weber DJ, et al. Acute bacterial meningitis in adults. A review of 493 episodes. N Engl J Med. 1993;328(1):21-8. 2. Cherubin CE, Marr JS, Sierra MF, Becker S. Listeria and gram-negative bacillary meningitis in New York City, 1972-1979. Frequent causes of meningitis in adults. Am J Med. 1981;71(2):199-209. 3. Mangi RJ, Quintiliani R, Andriole VT. Gram-negative bacillary meningitis. Am J Med. 1975;59(6):829-36. 4. Fang CT, Chen YC, Chang SC, Sau WY, Luh KT. Klebsiella pneumoniae meningitis: timing of antimicrobial therapy and prognosis. Qjm. 2000;93(1):45-53. 5. Fang CT, Chang SC, Hsueh PR, Chen YC, Sau WY, Luh KT. Microbiologic features of adult community-acquired bacterial meningitis in Taiwan. J Formos Med Assoc. 2000;99(4):300-4. 6. Kim KS. Strategy of Escherichia coli for crossing the blood-brain barrier. J Infect Dis. 2002;186 Suppl 2:S220-4. 7. Flamm RK, Sader HS, Farrell DJ, Jones RN. Ceftaroline potency among 9 US Census regions: report from the 2010 AWARE Program. Clin Infect Dis. 2012;55 Suppl 3:S194-205. 8. Mushtaq S, Warner M, Ge Y, Kaniga K, Livermore DM. In vitro activity of ceftaroline (PPI- 0903M, T-91825) against bacteria with defined resistance mechanisms and phenotypes. J Antimicrob Chemother. 2007;60(2):300-11. 9. Flamm RK, Sader HS, Farrell DJ, Jones RN. Summary of ceftaroline activity against pathogens in the United States, 2010: report from the Assessing Worldwide Antimicrobial Resistance Evaluation (AWARE) surveillance program. Antimicrob Agents Chemother. 2012;56(6):2933-40. 10. Dacey RG, Sande MA. Effect of probenecid on cerebrospinal fluid concentrations of penicillin and cephalosporin derivatives. Antimicrob Agents Chemother. 1974;6(4):437-41. 11. Cottagnoud P, Gerber CM, Cottagnoud M, Tauber MG. Gentamicin increases the efficacy of vancomycin against penicillin-resistant pneumococci in the rabbit meningitis model. Antimicrob Agents Chemother. 2002;46(1):188-90. 12. Gerber CM, Cottagnoud M, Neftel K, Tauber MG, Cottagnoud P. Evaluation of cefepime alone and in combination with vancomycin against penicillin-resistant pneumococci in the rabbit meningitis model and in vitro. J Antimicrob Chemother. 2000;45(1):63-8. 13. Blaser J, Vergeres P, Widmer AF, Zimmerli W. In vivo verification of in vitro model of antibiotic treatment of device-related infection. Antimicrob Agents Chemother. 1995;39(5):1134-9. 14. Louie A, Kaw P, Liu W, Jumbe N, Miller MH, Drusano GL. Pharmacodynamics of daptomycin in a murine thigh model of Staphylococcus aureus infection. Antimicrob Agents Chemother. 2001;45(3):845-51. Figure 1 246
9 Ceftaroline Concentration (mg/l) 60 60 CSF Penetration : 15 % 50 50 Serum 40 40 30 30 20 20 10 CSF 10 0 0 0 2 4 6 8 10 Time (hours) 247 248 249 250 251 252 253 254 255 256 257 258 259 260 Figure 2 261
10 Ceftaroline Concentration (mg/l) 14 14 12 12 10 10 8 8 6 6 4 4 2 inflamed 2 0 uninflamed 0-2 -2 0 2 4 6 8 10 Time (hours) 262 263 264 265 266 267 268 269 270 271 272 273 274 275
11 276 Figure 3 277 CSF Concentration (mg/l) 25 20 15 10 5 0 0 1 2 3 4 5 6 7 8 9 Time (hours) = second dose 278 279 280 281 282
12 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 Table 1 Pharmacokinetics of ceftaroline into inflamed and uninflamed meninges in a rabbit model following iv injections of ceftaroline fosamil 40 mg/kg at 0 and 4 hours. AUC 0-8 Serum (mg.h/l) AUC 0-8 CSF (mg.h/l) Penetration (%) Cmax Serum (mg/l) Cmax CSF (mg/l) Inflamed meninges 111 + 18 16.7 + 2.8 15.1 + 9.7 46.6 + 9.7 5.6 + 3.6 Uninflamed meninges 113 + 12 3.58 + 1.8 3.17 + 1.29 57 + 11 0.88 + 0.45 Table 2 Efficacy of ceftarolinefosamil compared with cefepime against a K. pneumoniae and an E. coli strain in experimental meningitis, expressed as killing rates per hour and after 8 hours. Klebsiella pneumoniae Antibiotic Inoculum at 0h (log 10 CFU/ml) Killing/hour ( log 10 CFU/ml/hr) Killing/8 hours ( log 10 CFU/ml/8hr) Controls 6.13 + 0.24 +0.12 + 0.02 +1.08 + 0.22 (5 rabbits) Ceftaroline 6.18 + 0.70-0.70 + 0.17 1-5.61 + 1.08 2 (10 rabbits) Cefepime (10 rabbits) 5.82 + 0.88-0.48 + 0.09 1-3.54 + 0.94 2 1: ceftaroline fosamil versus cefepime: P<0.0012, highly significant 2: ceftaroline fosamilversus cefepime: P<0.0007, highly significant Escherichia coli Antibiotic Inoculum at 0h (log 10 CFU/ml) Killing/hour ( log 10 CFU/ml) Killing/8 hours ( log 10 CFU/8ml) Controls 6.01 + 0.28 +0.11 + 0.05 +0.83 + 0.23 (5 rabbits) Ceftaroline 6.42 + 0.69-0.77 + 0.15 1-5.65 + 1.31 2 (10 rabbits) Cefepime (10 rabbits) 6.01 + 0.25-0.45 + 0.14 1-3.67 + 1.08 2 1: ceftaroline fosamil versus cefepime: P<0.0015, highly significant 2: ceftaroline fosamil versus cefepime: P<0.0016, highly significant
13 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 Legends Figure 1 Serum and cerebrospinal fluid (CSF) levels in inflamed meninges following IV injections of ceftaroline fosamil 40 mg/kg at 0 and 4 hours. The filled squares ( ) represent serum levels and the empty squares ( ) CSF levels. Results are expressed as means + a standard deviation. Figure 2 Ceftaroline cerebrospinal fluid (CSF) levels in inflamed (filled squares ) versus non inflamed meninges (empty squares ) following IV injections of ceftaroline fosamil 40 mg/kg at 0 and 4 hours. Results are expressed as means + a standard deviation.. Figure 3 Cerebrospinal fluid (CSF) levels in inflamed meninges following IV injections of cefepime 100 mg/kg at 0 and 4 hours. Results are expressed as means + a standard deviation. Acknowledgments This study was supported by Forest Laboratories, Inc.