ANTI-ANAEROBIC ACTIVITIES OF SULOPENEM COMPARED TO SIX OTHER. Departments of Pathology, Hershey Medical Center, Hershey, PA 17033

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1 AAC Accepts, published online ahead of print on 17 February 2009 Antimicrob. Agents Chemother. doi: /aac Copyright 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. ANTI-ANAEROBIC ACTIVITIES OF SULOPENEM COMPARED TO SIX OTHER AGENTS LOIS M. EDNIE PETER C. APPELBAUM * Departments of Pathology, Hershey Medical Center, Hershey, PA Running title: Sulopenem anti-anaerobic activity Correspondence to: Dr. Peter C. Appelbaum Department of Pathology Hershey Medical Center P.O Box 850 Hershey, PA Telephone no.: (717) Telefax no.: (717) pappelbaum@psu.edu

2 2 ABSTRACT. Agar dilution MIC methodology was used to compare the activity of sulopenem with that of amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin/tazobactam, imipenem, clindamycin and metronidazole against 431 anaerobes. Overall, MIC 50 / 90 values (µg/ml) were: sulopenem, 0.25/1.0 amoxicillin/clavulanate, 0.5/2.0; ampicillin/sulbactam, 0.5/4.0; piperacillin/tazobactam, 0.25/8.0; imipenem, 0.06/1.0; clindamycin, 0.25/16.0; metronidazole, 1.0/4.0. Downloaded from on May 12, 2018 by guest

3 3 Anaerobes are becoming increasingly resistant to ß-lactams due to ß-lactamase production and other mechanisms. Although ß-lactamase production, and concomitant resistance to ß-lactams, is the norm amongst the Bacteroides fragilis group, other anaerobic Gram-negative bacilli in the genus Prevotella, Porphyromonas and Fusobacterium have increasingly become ß-lactamase positive. ß-lactamase production also has been described in clostridia. Metronidazole resistance in organisms other than non-sporeforming Gram-positive bacilli has been described, as has clindamycinresistance in anaerobic Gram-negative bacilli (1-5). Although most anaerobes are susceptible to carbapenems, resistance has occurred. Quinolone resistance has developed, importantly in Clostridium difficile, necessitating development of other agents to treat pseudomembranous colitis (6,8-10,12,14,16). CP-65,207, whose in vitro activity was published in 1989 (13) is a diastereomeric mixture of two isomers, the active component of the two being sulopenem. Sulopenem development was put on hold in the 1990s as discovery/development efforts were focused on Gram-positive organisms. There is now an urgent need for drugs against Gram-negative strains and sulopenem has re-entered clinical development (M. Huband, personal communication). PF is a novel oral prodrug of sulopenem (11). The oral prodrug approach makes this compound unique amongst the penems and carbapenems. PF and sulopenem both entered phase II studies December Structures of sulopenem and its oral prodrug PF are shown in Figures 1 and 2 respectively. This study examined the activity of sulopenem compared with that of

4 4 amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin/tazobactam, imipenem, clindamycin and metronidazole against 431 anaerobes. All anaerobes were clinical strains identified by standard procedures (16) and kept frozen in double strength skim milk (dehydrated skim milk: BD, Sparks, MD) at -70 o C until use. Prior to testing, strains were subcultured twice onto enriched Brucella agar plates (7). Sulbactam and sulopenem susceptibility powders were obtained from Pfizer Central Research, Groton, CT, and other drugs from respective manufacturers. ß-lactamase testing was by the nitrocefin disk method (Cefinase: BD, Sparks, MD). Agar dilution susceptibility testing was according to the latest method recommended by the Clinical and Laboratory Standards Institute (CLSI)(7), using Brucella agar with 5% sterile laked sheep blood. Clavulanate and sulbactam were combined with amoxicillin and ampicillin, respectively, in 1:2 ratios, and tazobactam with piperacillin at a fixed concentration of 4.0 µg/ml. Breakpoint values, where CLSI approved, were as follows: amoxicillin/clavulanate, 4.0 µg/ml; ampicillin/sulbactam, 8.0 µg/ml; piperacillin/tazobactam, 32.0 µg/ml; imipenem, 4.0 µg/ml; clindamycin, 2.0 µg/ml; metronidazole, 8.0 µg/ml. Quality control strains recommended by CLSI (Bacteroides fragilis ATCC 25285, Bacteroides thetaiotaomicron ATCC 29741, and Eggerthella lenta ATCC 43055) were included with each run. Sulopenem MIC ranges for these strains (µg/ml) were , 0.25, and 1.0, respectively. Amongst the anaerobic Gram-negative bacilli tested, 95/101 (94%) of B. fragilis group, 57/100 (57%) of Prevotella/Porphyromonas and 3/60 (5%) of fusobacteria produced ß-lactamase. Results of MIC testing are presented in Table 1. Overall, MIC 50 / 90 values (µg/ml) were: sulopenem, 0.25/1.0; amoxicillin/clavulanate, 0.5/2.0;

5 5 ampicillin/sulbactam, 0.5/4.0; piperacillin/tazobactam, 0.25/8.0; imipenem, 0.06/1.0; clindamycin, 0.25/16.0; metronidazole, 1.0/4.0. With the exception of 6 strains of lactobacilli, sulopenem and imipenem both had MICs <4.0 µg/ml against all strains tested in this study. Clindamycin resistance was found in all of the Bacteroides species tested as well as 5 of the 15 different species of the Prevotella and Porphyromonas tested. Eighteen of the 21 strains of Fusbacterium varium showed resistance to clindamycin while all other fusobacteria were clindamycin susceptible. For all anaerobic Gram-positive cocci tested, clindamycin resistance was observed in only one strain of Finegoldia magna and one Peptoniphilus asaccharolyticus. All groups of anaerobic non-spore-forming Gram-positive rods except Propionibacterium acnes had some degree of clindamycin resistance. All Clostridium tertium strains as well as some strains in 5 of the 11 other Clostridium species were clindamycin resistant. Most Gram-positive non-sporeforming rods (with the exception of Eggerthella) were metronidazole-resistant while all anaerobic Gram-negative rods, Gram-positive cocci, and clostridia were metronidazole susceptible. Pipercillin/tazobactam was active against all strains tested with MICs of 16 µg/ml. Resistance to amoxicillin/clavulanate was found in 4 of 7 species of Bacteroides as well as 2 strains of Fusobacterium mortiferum. All other strains of fusobacteria as well as all strains of Prevotella, Porphyromonas, Clostridium, and Gram-positive non-sporeforming rods were amoxicillin/clavulanate susceptible. Two of the 10 Peptostreptococcus anaerobius strains showed amoxicillin/clavulanate resistance, while all other Gram-positive cocci were susceptible. Ampicillin/sulbactam was active against all strains of Clostridium, anaerobic Gram-positive nonsporeforming rods, Prevotella and Porphyromonas tested. Gram-negative rods showing

6 6 ampicillin/sulbactam resistance included some strains in 3 of the 7 Bacteroides species tested and 2 strains of Fusobacterium mortiferum, while other species of fusobacteria were susceptible. While 413 of 431 strains (95.8%) tested had sulopenem MICs 2 µg/ml, 18 strains (4.2%) had MICs of 4.0 µg/ml. These included 7 Lactobacillus species (6 strains >8.0 µg/ml, 1 strain 4.0 µg/ml), 10 strains of Clostridium (4 C. difficile, 1 C. innocuum, and 5 unspeciated)(mics all 4.0 µg/ml), and 1 Peptostreptococcus anaerobius (MIC 4.0 µg/ml). Until pharmacokinetic/pharmacodynamic breakpoints for sulopenem can be established, the clinical significance of these relatively higher sulopenem MICs cannot be established. Gootz and co-workers (13) in their 1989 study on CP-65,207, reported on its activity against 84 anaearobes, many of which have since been reclassified. Excluding Peptococcus spp which are no longer included amongst clinically significant anaerobic Gram-positive cocci in standard manuals (16), results were similar to those obtained in our study. In a study of the comparative activity of sulopenem against a collection of recently isolated Gram-positive and -negative aerobic organisms, sulopenem MIC 90 s ranged between µg/ml against all clinically significant bacterial species tested. This high in vitro potency was also confirmed by in vitro time-kill studies (15). Our study adds anaerobes to the activity spectrum for this drug and suggests a potential place for sulopenem in treatment of mixed anaerobic infections. Pharmacokinetic/ pharmacodynamic and experimental animal studies are necessary to further delineate the clinical role of these compounds in treatment of anaerobic infections This study was supported by a grant from Pfizer Central Research, Groton, CT. and was presented at the 48 th Interscience Conference on Antimicrobial Agents and

7 7 Chemotherapy (abstract. F1-347).

8 8 Figure 1. Chemical structure of sulopenem OH S S N S + O - O OH O Sulopenem (CP ) Figure 2. Chemical structure of PF , oral prodrug of sulopenem OH S O S S N O O O O O

9 9 REFERENCES 1. Appelbaum, P.C., A. Philippon, M.R. Jacobs, S.K. Spangler, and L. Gutmann Characterization of ß-lactamases from non-bacteroides fragilis group Bacteroides spp. belonging to seven species and their role in ß-lactam resistance. Antimicrob. Agents Chemother. 34: Appelbaum, P.C., S.K. Spangler, and M.R. Jacobs Evaluation of two methods for rapid testing for beta-lactamase production in Bacteroides and Fusobacterium. Eur. J. Clin. Microbiol. Infect. Dis. 9: Appelbaum, P.C., S.K. Spangler, and M.R. Jacobs ß-Lactamase production and susceptibilities to amoxicillin, amoxicillin-clavulanate, ticarcillin, ticarcillinclavulanate, cefoxitin, imipenem, and metronidazole of 320 non-bacteroides fragilis Bacteroides and 129 fusobacteria from 28 U.S. centers. Antimicrob. Agents Chemother. 34: Appelbaum, P.C., S.K. Spangler, and M.R. Jacobs Susceptibility of 539 gram-positive and -negative anaerobes to new agents, including RP 59500, biapenem, trospectomycin and piperacillin/tazobactam. J. Antimicrob. Chemother. 32: Appelbaum, P.C., S.K. Spangler, G.A. Pankuch, A. Philippon, M.R. Jacobs, R. Shiman, E.J.C. Goldstein, and D. Citron Characterization of a ß-lactamase from Clostridium clostridioforme. J. Antimicrob. Chemother 33: Bauernfeind, A Comparison of the antibacterial activities of the quinolones Bay , gatifloxacin (AM 1155), trovafloxacin, clinafloxacin, levofloxacin and

10 10 ciprofloxacin. J. Antimicrob. Chemother. 40: Clinical Laboratory Standards Institute Methods for antimicrobial susceptibility testing of anaerobic bacteria; approved standard 7 th ed. M11-A7. CLSI, Wayne, PA. Wayne, PA. 8. Credito, K.L., and P.C. Appelbaum Activity of OPT-80, a novel macrocycle, compared with those of eight other agents against selected anaerobic species. Antimicrob. Agents Chemother. 48: Credito, K.L., L.M. Ednie, and P.C. Appelbaum Comparative antianaerobic activities of doripenem determined by MIC and time-kill analysis. Antimicrob. Agents Chemother. 52: Ednie, L.M., M.R. Jacobs, and P.C. Appelbaum Activities of gatifloxacin compared to those of seven other agents against anaerobic organisms. Antimicrob. Agents Chemother. 42: Forrest, A., A. Hazra, D. Girard, S. Finegan, J. O Donnell, and K. Soma Use of PK/PD analysis to select doses for PF with and without probenecid (P), to be studied in phase 2b trials Abstr. 48 th Intersci. Conf. on Antimicrob. Agents Chemother. (A-034). 12. Goldstein, E.J.C., and D.M. Citron Comparative activity of ciprofloxacin, ofloxacin, sparfloxacin, temafloxacin, CI-960, CI-990, and Win against anaerobic bacteria. Antimicrob. Agents Chemother. 36: Gootz, T., J. Retsema, A. Girard, E. Hamanaka, M. Anderson, and S. Sokolowski In vitro activity of CP-65,207, a new penem antimicrobial agent, in comparison with those of other agents. Antimicrob. Agents Chemother. 33: 1160-

11 Hoellman, D.B., L.M. Kelly, M.R. Jacobs, and P.C. Appelbaum Comparative antianaerobic activity of BMS Antimicrob. Agents Chemother. 45: Huband, M.D., Gootz, T.D., M.A. Cohen, L.M. Mullins, S.P. McCurdy, L.A. Brennan, J.M. Duignan, P.J. Pagano, and R.W. Murray In vitro antibacterial activity of sulopenem: a new oral penem antimicrobial versus recent bacterial isolates. Abstr. 48 th Intersci. Conf. on Antimicrob. Agents Chemother. (F1-344). 16. Jousimies-Somer, H.R., P. Summanen, D.M. Citron, E.J. Baron, H. Wexler, and S.M. Finegold SM Wadsworth-KTL anaerobic bacteriology manual. Star Publishing Co., Belmont, California. Downloaded from on May 12, 2018 by guest

12 9 TABLE 1. MICs (µg/ml) of agents Organism Drug MIC range MIC 50 MIC 90 Bacteroides fragilis (49/49) a Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin 0.03-> >32.0 Metronidazole Bacteroides thetaiotaomicron (10/10) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin 1.0-> >32.0 Metronidazole Bacteroides ovatus (10/10) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin 1.0-> >32.0 Metronidazole Bacteroides vulgatus (10/10) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin > Metronidazole Bacteroides distasonis (5/11) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin 0.03-> Metronidazole Bacteroides uniformis (6/6) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin 1.0-> Metronidazole

13 10 Bacteroides stercoris (5/5) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin 4-> Metronidazole Prevotella bivia (12/16) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin > >32.0 Metronidazole Prevotella buccae (8/16) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin Metronidazole Prevotella melaninogenica/denticola (12/17) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin Metronidazole Prevotella intermedia/nigrescens (8/13) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin Metronidazole Prevotella corporis (4/9) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin > Metronidazole

14 11 Prevotella disiens (6/7) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin Metronidazole Prevotella baroniae (0/3) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Clindamycin > Metronidazole Prevotella oralis group (1/3) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Clindamycin Metronidazole Prevotella veroralis (2/3) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Clindamycin Metronidazole Prevotella bergensis (2/2) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Clindamycin Metronidazole Prevotella salivae (1/2) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Clindamycin Metronidazole Prevotella loeschii (1/1) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Clindamycin

15 12 Metronidazole Prevotella sp (0/1) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Clindamycin Metronidazole Porphyromonas asaccharolytica (0/4) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Clindamycin > Metronidazole Porphyromonas gingivalis (0/3) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Clindamycin Metronidazole Fusobacterium nucleatum (1/20) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin Metronidazole Fusobacterium necrophorum (0/5) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin Metronidazole Fusobacterium mortiferum (2/14) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam 0.5-> >32.0 Piperacillin/tazobactam Clindamycin Metronidazole Fusobacterium varium (0/21) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin

16 13 Metronidazole Finegoldia magna (0/10) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin 0.06-> Metronidazole Micromonas micros (0/10) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam < Piperacillin/tazobactam Clindamycin Metronidazole Peptostreptococcus anaerobius (0/10) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin Metronidazole Peptoniphilus asaccharolyticus (0/7) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin 0.06-> Metronidazole Anaerococcus tetradius (0/10) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin Metronidazole Anaerococcus prevotii (0/3) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Clindamycin Metronidazole

17 14 Lactobacillus sp (0/10) Sulopenem 0.5->8.0 >8.0 >8.0 Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin Metronidazole >16.0 >16.0 >16.0 Bifidobacterium sp (0/10) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin > >32.0 Metronidazole 4->16.0 >16.0 >16.0 Propionibacterium acnes (0/18) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin Metronidazole >16.0 >16.0 >16.0 Actinomyces sp (0/5) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin Metronidazole 1.0->16.0 > Eggerthella lenta (0/17) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin 0.06-> Metronidazole Clostridium perfringens (0/19) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin 0.06-> Metronidazole

18 15 Clostridium difficile (0/10) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin 2.0-> >32.0 Metronidazole Clostridium sp (0/10) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin 1.0-> Metronidazole Clostridium tertium (0/5) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin Metronidazole Clostridium ramosum (0/4) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Clindamycin Metronidazole Clostridium cadaveris (0/3) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin Metronidazole Clostridium bifermentans (0/2) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin Metronidazole Clostridium innocuum (0/2) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin

19 16 Metronidazole Clostridium sordellii (0/2) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Clindamycin Metronidazole Clostridium butyricum (0/1) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin Metronidazole Clostridium clostridioforme (0/1) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin Metronidazole Clostridium hastiforme (0/1) Sulopenem Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam Piperacillin/tazobactam Clindamycin Metronidazole All strains (155/431) Sulopenem > Imipenem Amoxicillin/clavulanate Ampicillin/sulbactam > Piperacillin/tazobactam Clindamycin > Metronidazole > a No. ß-lactamase-positive/no. strains tested.