Keywords: CB-183,315; Clostridium difficile; susceptibility; intestinal anaerobes

AAC Accepts, published online ahead of print on 19 December 2011 Antimicrob. Agents Chemother. doi:10.1128/aac.05655-11 Copyright 2011, American Society for Microbiology. All Rights Reserved. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 In vitro activity of CB-183,315, vancomycin and metronidazole against 556 strains of Clostridium difficile, 445 other intestinal anaerobes and 56 Enterobacteriaceae species 1 Diane M. Citron*, 1 Kerin L. Tyrrell, 1 C. Vreni Merriam and 1,2 Ellie J.C. Goldstein 1 R.M. Alden Research Lab, Culver City, CA 2 Geffen School of Medicine, UCLA, Los Angeles, CA Keywords: CB-183,315; Clostridium difficile; susceptibility; intestinal anaerobes Running title: CB-183,315 against C. difficile and intestinal organisms Corresponding author: Diane M. Citron R.M. Alden Research Lab 6133 Bristol Parkway Suite 175 Culver City, CA 90230 P 310-641-8340 F-310-641-8840 d.m.citron@att.net www.rmaldenresearch.com Abstract 1

30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 MICs for CB-183,315, a novel lipopeptide antibiotic, vancomycin and metronidazole were determined for intestinal anaerobes and Enterobacteriaceae. The MIC 90 for gram-negative anaerobes was >8192, 8192 and 4 µg/ml for CB-183,315, vancomycin and metronidazole, respectively. Against Enterobacteriaceae, MIC 90s were >8192 µg/ml, 1024 µg/ml and1024 µg/ml, respectively. CB-183,315 MIC 90 for C. difficile was 0.5 µg/ml. Its lack of activity against normal fecal organisms makes it a promising new agent for treating C. difficile. Clostridium difficile infection (CDI) is the most common cause of healthcare related diarrhea, usually associated with prior exposure to antimicrobial agents. The current theory is that the 2

53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 antibiotics disrupt the normal fecal microbiota, changing its complexity and diversity as a primary event, with the subsequent acquisition of toxigenic C. difficile as a secondary event in the development of the disease (6,11,14). CDI has increased in prevalence and severity during the last decade, resulting in increased mortality and complications, recurrent disease and prolonged hospital stay (7,17). Non-healthcare related cases of CDI are now being reported (7,16). Treatment options have been limited by unsatisfactory efficacy of current therapeutic agents, high recurrence rates of disease, disruption of normal intestinal microbiota, and colonization by VRE (1,2,15). Although a recently introduced macrocyclic antibiotic, fidaxomicin, shows equivalent primary cure rates with reduced recurrence rates compared to vancomycin (15) it shows equivalence against REA type BI strain associated disease. CB-183,315 is a novel lipopeptide antibiotic with gram-positive activity and is bactericidal against C. difficile. To study the spectrum of its activity, including effect on the normal components of fecal microbiota, we determined MICs of CB-183,315 and vancomycin against C. difficile and other intestinal anaerobes, plus E. coli, Klebsiella spp. and Enterobacter spp. For comparison, metronidazole was tested against the gram-negative anaerobes and the Enterobacteriaceae spp. The C. difficile strains were recent clinical isolates (2005 2008) recovered from stools of patients with CDI. Other isolates were from stool or infections containing organisms presumed to be of intestinal origin, identified by standard methods (13) and occasionally by 16S rdna sequence analysis (20), and stored in 20% skim milk at -70 o C. MIC values were determined by the agar dilution method according to CLSI procedures (4). Vancomycin and metronidazole laboratory standard powders were obtained from Sigma (St. 3

75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 Louis, MO) and CB-183,315 was provided by Cubist Pharmaceuticals, Inc. (Lexington, MA). All assay media for testing CB-183,315 were supplemented with a final concentration of 50 mg/l calcium, where Ca ++ concentrations were confirmed by Laboratory Specialists, Inc. (Westlake, Ohio). The Escherichia coli, Klebsiella pneumoniae and Enterobacter spp. strains were tested using Mueller Hinton agar, and incubated at 37 o C in ambient air, except for metronidazole, which was incubated in the anaerobic chamber (4). Quality control strains included Clostridium difficile ATCC 700057, Bacteroides fragilis ATCC 25285, Staphylococcus aureus ATCC 29213 and E. coli ATCC 25922 and were included each day of testing of the relevant set of test organisms. Time-kill studies were carried out on one restriction endonuclease analysis (REA) BI type (NAP1, ribotype 027) strain and one REA type Y (NAP4, ribotype 014) strain (3). CB-183,315 and vancomycin were prepared at 2, 4, and 8 times their MICs for each strain in supplemented Brucella broth. A drug-free growth control tube was included. Tubes were inoculated with ~ 10 6 CFU/ml, placed on a shaker and assayed at 0, 2, 4, 8, and 24h of incubation at 37 o C. Table 1 shows the range, MIC 50 and MIC 90 for the major groups of organisms. The CB-183,315 MIC range for C. difficile was 0.06 2.0 µg/ml with MIC 90 of 0.5 µg/ml; other gram-positive strains were inhibited by 0.03 16 µg/ml. The MIC 90 for CB-183,315 against the Bacteroides fragilis group, Prevotella spp., gram-negative cocci (Veillonella spp. and Acidaminococcus sp.), 4

98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 as well as E. coli, Enterobacter spp., and Klebsiella spp. was greater than 8192 µg/ml. Against fusobacteria, and Porphyromonas spp., the MIC 90 was 8192 µg/ml and 2048 µg/ml, respectively. In contrast, vancomycin had equally high MICs of >8192 µg/ml only against the fusobacteria and the gram-negative cocci; the B. fragilis group was inhibited by vancomycin at MIC 90 of 128 µg/ml (range 32-256 µg/ml), Porphyromonas by 4 µg/ml, and Prevotella spp. by 256 µg/ml of vancomycin. Vancomycin MICs ranged from 64 1024 µg/ml for E. coli and 256-1024 µg/ml for Enterobacter and Klebsiella species. Metronidazole was active against the gram-negative anaerobes at 0.06 to 8 µg/ml, but showed poor activity against the Enterobacteriaceae, with MICs ranging from 32 to1024 µg/ml. The time-kill studies showed that CB-183,315 at 4 and 8 times the MIC resulted in a 3 log 10 reduction in colony count after 24h incubation against both REA types tested. Vancomycin produced a similar result.. CB-183,315 reaches concentrations of 6494 + 3104 μg/g of feces after a 1g bid dose at day 5 of a 14 day course (Cubist Pharmaceuticals data on file) which is lower than the high MICs (>8192 µg/ml) demonstrated for Bacteroides and other groups of organisms of the intestinal anaerobes, thus CB-183,315 is likely to spare many of these important members of the normal microbiota. In contrast, vancomycin at a dose of 250 mg qid results in fecal levels that are generally above 2,000 μg/ml (12). Vancomycin thus has the potential for killing or inhibiting much of the normal aerobic and anaerobic fecal microbiota. Moreover, oral vancomycin was shown to decrease or suppress Bacteroides in volunteers while increasing occurrence of vancomycin-resistant enterococci (8,14). In contrast, fidaxomicin, a narrow spectrum 5

120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 macrocycle, displayed less impact on the Bacteroides populations in patients treated for CDI (14), which has been postulated as one reason for the associated lower relapse rate. Using temporal temperature gradient electrophoresis and fluorescent in situ hybridization and flow cytometry, Tannock et al.(19) compared the impact of fidaxomicin and vancomycin on seven phylogenetic groups, including clostridial clusters IV and XIVa, Bacteroides-Prevotella, Bifidobacterium, Atopobium, enterobacteria, and the Enterococcaceae-Lactobacillaceae group. Minimal changes were seen in these groups for the fidaxomicin treated patients while the vancomycin treated patients showed a marked decrease in Clostridium clusters IV and XIVa, Bacteroides and Bifidobacterium, and an increase in the Enterococcus-Lactobacillus and the enterobacteria groups. MICs for CB-183,315 against clostridia were similar to those reported for fidaxomicin (10), showing decreased activity against several species in Clostridium clusters XIVa, XVI, and XVIII, although unlike fidaxomicin, decreased activity of CB-183,315 was also present against several species in cluster I (5,9,18). MIC 90 for the C. clostridioforme group (cluster XIVa), was 16 µg/ml; for C. innocuum (cluster XVI) 4 µg/ml; for C. ramosum (Cluster XVIII) 8 µg/ml; and C. sphenoides, 1 strain, (cluster XIVa) 16 µg/ml. These findings suggest that CB-183,315 would show a similar lack of impact as fidaxomicin on the composition of the major groups of bowel microbiota. With its excellent activity against C. difficile and its narrower spectrum and lack of activity against other colonic aerobic and anaerobic microbiota, with the potential for fewer relapses, CB- 183,315 is a promising new drug treatment for CDI. 6

143 144 145 146 147 Acknowledgement This study was sponsored by a grant from Cubist Pharmaceuticals, Inc. We thank Eliza Leoncio for excellent technical assistance. Downloaded from http://aac.asm.org/ on October 1, 2018 by guest 7

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217 218 Table 1. In vitro activity of CB-183,315, Vancomycin and Metronidazole against Intestinal Organisms (μg/ml) CB-183,315 Vancomycin Metronidazole Group N Range MIC 50 MIC 90 Range MIC 50 MIC 90 Range MIC 50 MIC 90 Actinomyces species a 15 0.125-8.0 1.0 8.0 0.25 1.0 0.5 1.0 NT Bifidobacterium species b 14 0.06-2.0 0.5 2.0 0.25 2.0 0.5 1.0 NT Eggerthella lenta 17 1.0-16 4.0 8.0 1.0 2.0 2.0 2.0 NT Eubacterium limosum 13 0.06 1 0.25 0.5 1.0-2.0 2.0 2.0 NT Eubacterium group (other) c 35 0.06 8.0 0.25 2.0 0.25-32 1.0 2.0 NT Lactobacillus species, VAN-R d 20 0.5 2.0 1.0 2.0 >32 - >32 >32 >32 NT Lactobacillus species, VAN-S e 17 0.125 16.0 1.0 4.0 0.25 4.0 1.0 2.0 NT Propionibacterium species f 15 0.5-2.0 0.5 2.0 0.5 2.0 0.5 1.0 NT Clostridium clostridioforme NT grp g (CL. XIVa) 20 1.0-16.0 8.0 16.0 0.5 1.0 1.0 1.0 Clostridium difficile (Cl. XI) 556 0.06-2 0.5 0.5 0.25 4.0 1.0 2.0 NT Clostridium innocuum (Cl.XV) 22 1.0 4.0 2.0 4.0 8.0 16.0 8.0 16.0 NT Clostridium perfringens (Cl.I) 20 0.25-1.0 0.5 1.0 0.5-1 0.5 1 NT Clostridium ramosum (Cl. NT XVIII) 20 2.0-8.0 4.0 8.0 2-8 4 8 Clostridium species (other) h 39 0.125-16.0 1.0 16.0 0.25-32 1 2 NT Anaerobic gram-positive cocci i 49 <0.03-4.0 0.25 0.5 0.06-1 0.25 0.5 NT Bacteroides fragilis group j 21 8192->8192 8192 >8192 32-256 64 128 0.25-2 1 2 128- Fusobacterium species k 20 128->8192 512 8192 >8192 512 >8192 0.06-1.25.5 Porphyromonas species l 21 64-8192 1024 2048 1-32 4 4 0.06-1 0.25 1 Prevotella species m 20 8192->8192 8192 >8192 32-512 128 256 0.25-4 1 4 256- Gram-negative cocci n 21 8192->8192 >8192 >8192 >8192 2048 >8192 0.06-8 2 4 >8192 >8192 128- E. coli 18 4096->8192 64-256 128 256 1024 256 512 12

219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 >8192- >8192 >8192 Enterobacter species o 18 >8192 256-1024 512 1024 32-1024 512 1024 >8192 >8192 >8192 128- Klebsiella species p 20 256-1024 1024 1024 1024 512 1024 Cl, phylogenetic cluster a Actinomyces israelii (2), A. meyeri (2), A. naeslundii (1), A. neuii ss anitratus (2), A. neuii ss neuii (1), A. odontolyticus (2), A. turicensis (2), and A. viscosus (3). b Bifidobacterium adolescentis (2), B. bifidum (1), B. breve (4), B. catenulatum (2), B. dentium (1), B. longum (2), B. pseudocatenulatum (1), and B. urinalis (1). c Collinsella aerofaciens (6), Eubacterium biforme (1), E. contortum (1), E. cylindroides (1), E. timidum (3), Pseudoramibacter alactolyticum (4) and Eubacterium species (19). d Lactobacillus brevis (1), L. casei (7), L. fermentum (2), L. rhamnosus (6), and Lactobacillus GG (4). e Atopobium parvulum (1), Lactobacillus acidophilus (2), L. catenaformis (3), L. crispatus (1), L. gasseri (3), L. iners (1), L. jensenii (4), and L. leichmannii (2 ) f Propionibacterium acnes (7), P. avidum (4), and P. granulosum (4). g Clostridium aldenense (4), C. bolteae (5), C. citroniae (3), C. clostridioforme (2), and C. hathewayi (6). h Clostridium bifermentans (1), C. butyricum (5), C. cadaveris (3), C. cochlearium (2), C. fallax (1), C. glycolicum (3), C. hastiforme (1), C. hylemonae (1), C. indolis (1), C. leptum (2), C. nexile (1), C. paraputrificum (4), C. sartagoforme (1), C. scindens (1), C. sordelli (2), C. sphenoides (1), C. sporogenes (2), C. subterminale (1), C. symbiosum (4), and C. tertium (2). 13

240 241 242 243 244 245 246 247 248 249 250 251 252 253 i Anaerococcus lactolyticus (2), A. octavius (1), A. prevotii (6), A. tetradius (5), A. vaginalis (4) ; Finegoldia magna (7) ; Peptoniphilus asaccharolyticus (5), P. harei (2), P. indolicus (2), P. ivorii (3), and P. lacrimalis (2); Peptostreptococcus anaerobius (5) and Parvimonas micra (5). j Bacteroides caccae (1), B. distasonis (1), B. fragilis (11), B. nordii (1), B. ovatus (3), B. thetaiotaomicron (3), B. vulgatus (1). k Fusobacterium gonidiaformans (1), F. mortiferum-varium group (6), F. necrophorum (2), F. nucleatum (10), and F. russii (1). l Porphyromonas asaccharolytica (9), P. gingivalis (4), P. somerae (6), P. uenonis (2). m Prevotella bivia (10), P. disiens (3), P. intermedia/nigrescens (1), P. loeschii (3), and P. melaninogenica (3). n Acidaminococcus fermentans (8), Veillonella species (13). o Enterobacter aerogenes (6), Enterobacter cloacae (11), Enterobacter sp. (1) p Klebsiella oxytoca (3), Klebsiella pneumoniae (17) 14