1. Methods Revised Abstract Methods Results TP-271 is a Potent, Broad-Spectrum Fluorocycline with Activity Against Community-Acquired Bacterial Respiratory and Biothreat Pathogens T. GROSSMAN 1, C.FYFE 1, W. O’BRIEN 1, M. HACKEL 2, J. SUTCLIFFE 1 1 Tetraphase Pharmaceuticals, Inc., Watertown, MA, 2 International Health Management Associates, Inc. 52 nd Annual ICAAC 9 - 12 September, 2012 San Francisco, CA Conclusions Poster F-1525 Background: TP-271 is a novel, fully synthetic fluorocycline antibiotic in preclinical development for IV/oral treatment of respiratory infections caused by susceptible and multidrug-resistant (MDR) public health and biothreat pathogens. Method: In vitro susceptibility testing against recent isolates was done according to CLSI guidelines. Tetracycline-specific resistance and ESβL genes were detected by PCR. Results: TP-271 was potent against Streptococcus spp., Staphylococcus aureus, Haemophilus influenzae, and Moraxella catarrhalis isolates and Gram-positive pathogens vancomycin-resistant Enterococcus faecium and Enterococcus faecalis (see Tables). The MIC 50/90 values of TP-271 against ESβL + Escherichia coli (n=34) and Klebsiella pneumoniae (n=48) were 1/ 2, and 1/ 4  g/ml, respectively. Against other Enterobacteriaceae, the MIC 50 and MIC 90 values of TP-271 were 0.25 – 4 and 1-8 µg/ml, respectively. The MIC 50/90 values against Acinetobacter baumannii (n=54), Acinetobacter lwoffi (n=29), and Stenotrophomonas maltophiliia (n=35) were 0.13/1, 0.063/0.5, 0.25/2 µg/ml, respectively. The MIC 50/90 values against biothreat pathogens Yersinia pestis (n=30), Bacillus anthracis (n=30), Francisella tularensis (n=27), Burkholderia mallei (n=30) and Burkholderia pseudomallei (n=30) were 0.12/0.25, ≤0.008/≤0.008, 0.25/0.5, 0.06/0.12, and 1/4 µg/ml, respectively. Conclusions: TP-271 is active against community-acquired respiratory and biothreat pathogens, and problematic multidrug-resistant (MDR) pathogens. Susceptibility testing. All minimal inhibitory concentration (MIC) assays were performed as per CLSI guidelines [1]. Testing of public health pathogens was performed at Tetraphase Pharmaceuticals, Inc. and International Health Management Associates (IHMA) using recent clinical isolates obtained from Eurofins Medinet and IHMA. Testing of biothreat agents was performed at United States Army Medical Research Institute for Infectious Diseases (USAMRIID). Genotypic characterization of ESβL-producing isolates was done by standard PCR methodology and sequence confirmation. 1 Clinical and Laboratory Standards Institute (CLSI). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—Nineth Edition. CLSI document M07-A9.. Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2012. Results TP-271, a novel fully synthetic tetracycline, exhibits excellent potency against key MDR public health respiratory pathogens, including S. pneumoniae, MRSA, S. pyogenes, H. influenzae and M. catarrhalis. TP-271 also exhibits potent activity against five important biothreat agents: Y. pestis, B. anthracis, F. tularensis, B. mallei, and B. pseudomallei, supporting its potential for the treatment of biothreat infections. The broad spectrum potency of TP-271 is demonstrated by good activity against difficult-to-treat MDR Gram- negatives and Gram-positive pathogens including VRE. TP-271 Table 2. Determination of MIC 50 and MIC 90 values for Gram-positive & respiratory pathogens Table 1. Determination of MIC 50 and MIC 90 values for biothreat pathogens Contact: Leland Webster Tetraphase Pharmaceuticals lwebster@tphase.com Table 3. Determination of MIC 50 and MIC 90 values for Gram-negative pathogens Organism MIC 50 /MIC 90 (µg/ml) (range) NTP-271TetracyclineTigecyclineMacrolide a Fluoroquinolone b LinezolidVancomycin Streptococcus pneumoniae 137 ≤0.016/≤0.0161/>32 c ≤0.016/≤0.0164/>321/11/1 c 0.25/0.5 c ≤0.016-≤0.016≤0.016->32≤0.016-≤0.016≤0.016->320.25-320.13-2≤0.016-0.5 S. pneumoniae penicillin-R d 58 ≤0.016/≤0.01632/>32≤0.016/≤0.016>32/>321/1 0.25/0.5 ≤0.016-≤0.0160.031->32≤0.016-≤0.016≤0.016->320.5-10.5-20.25-0.5 S. pneumoniae macrolide-R 58 ≤0.016/≤0.01632/>32≤0.016/≤0.016>32/>321/1 0.25/0.5 ≤0.016-≤0.0160.13->32≤0.016-≤0.0161->320.25-320.25-20.13-0.5 Streptococcus pyogenes 64 ≤0.016/≤0.0160.25/>32≤0.016/≤0.0160.063/40.5/11/20.5/0.5 ≤0.016-≤0.0160.13->32≤0.016-0.063≤0.016->320.25-20.5-20.25-0.5 Streptococcus agalactiae 30 ≤0.031/0.06332/320.031/0.031≤0.13/>41/11/2≤0.5/≤0.5 ≤0.031-0.063≤2->32≤0.016-0.063≤0.13->40.5-21-2≤0.5-≤0.5 Streptococcus anginosus 73 ≤0.031/≤0.03116/32≤0.016/0.031≤0.12/>4 m 0.5/11/2≤0.5/1 ≤0.03-0.063≤2->32≤0.016-0.25≤0.12->4≤0.12-1≤0.25-2≤0.5-1 Staphylococcus aureus 155 0.06/0.25≤2/320.12/0.25>4/>4 2/41/1 ≤0.03-10.063->32≤0.016-0.50.25->4≤0.13->40.5-64≤0.5-8 S. aureus (MRSA) 124 0.063/0.13≤2-320.13/0.25>4/>4 2/41/1 ≤0.016-10.063->32≤0.016-0.50.25->4≤0.13->41-64≤0.5-8 S. aureus (MRSA) PVL+ 25 0.063/0.12≤2/≤20.12/0.12>4/>42/>42/21/1 0.063-0.12≤2-160.063-0.251->4≤0.13->41-4≤0.5-1 S. aureus (MSSA) 31 0.12/0.25≤2/≤20.12/0.251/>40.25/0.52/41/1 ≤0.031-0.25≤2-320.03-0.250.5->4≤0.13->40.5-4≤0.5-1 Staphylococcus epidermidis 62 0.063/0.5≤2/>320.25/0.5>4/>40.25/>42/2 ≤0.03-1≤2->320.06-1≤0.13->4 1-4≤0.5-2 S. epidermidis methicillin-S 32 0.063/0.25≤2/320.12/0.250.5/>40.25/0.52/2 ≤0.03-0.5≤2->320.06-0.5≤0.13->4 1-4≤0.5-2 S. epidermidis methicillin-R 30 0.25/0.5≤2/>320.5/0.5>4/>4 2/2 0.06-1≤2->320.06-10.25->4≤0.13->41-2 Enterococcus faecalis 113 ≤0.031/0.063>32/>320.12/0.25>4/>4 g 2/>42/22/>16 ≤0.031-0.50.13->32≤0.016-10.5->41->4≤0.25-32≤0.5->16 E. faecalis (VRE) 37 ≤0.031/0.06332/>320.12/0.25>4/>4 h >4/>42/2>16/>16 ≤0.03-0.25≤2->32 0.03-14->41->4≤0.25-4>16->16 E. faecalis (VSE) 68 ≤0.031/0.063>32/>320.12/0.25>4/>4 i 2/>42/41/2 ≤0.03-0.50.13->32≤0.016-10.5->41->40.5-321-4 Enterococcus faecium 81 ≤0.031/≤0.031≤2/>320.06/0.25≤0.13/>4 j >4/>42/4>16/>16 ≤0.031-0.063≤2->32≤0.03-14->40.25->40.5->4≤0.5->16 E. faecium (VRE) 45 ≤0.031/≤0.03116/>320.06/0.25>4/>4 k >4/>42/4>16/>16 ≤0.031-0.031≤2->32≤0.03-0.50.25->4>4->40.5->416->16 E. faecium (VSE) 36 ≤0.031/≤0.031≤2/>320.06/0.25>4/>4 l >4/>42/40.5/1 ≤0.03-0.06≤2->32≤0.03-1≤0.13->40.25->41-40.5-4 Haemophilus influenzae 65 0.031/0.130.5/40.063/0.258/8≤0.016/0.0318/16>32/>32 e ≤0.016-0.250.13-16≤0.016-0.50.063-16≤0.016-0.134-3216->32 Moraxella catarrhalis 57 ≤0.016/≤0.0160.5/32≤0.016/0.0310.063/0.250.031/0.0638/8>32/>32 f ≤0.016-0.0310.13->32≤0.016-0.13≤0.016-40.031-0.132-3216->32 a erythromycin, azithromycin or clarithromycin; b ciprofloxacin or levofloxacin; c 126 S. pneumoniae isolates; d penicillin MIC ≥2 µg/ml; e 51 H. influenzae isolates; f 43 M. catarhalis isolates; g 74 E. faecalis isolates; h 30 E. faecalis isolates; i 36 E. faecalis isolates; j 67 E. faecalis isolates; k 32 E. faecium isolates; l 35 E. faecium isolates; m 51 S. anginosus isolates Results OrganismN MIC 50 /MIC 90 (µg/ml) (range) TP-271DoxycyclineCiprofloxacin Yersinia pestis 30 0.12/0.250.5/10.03/0.06 0.015-0.50.06-20.015-0.06 Bacillus anthracis 30 ≤0.008/≤0.008 0.12/0.25 ≤0.008-≤0.008≤0.008-0.0150.03-0.25 Francisella tularensis 27 0.25/0.50.12/0.250.06/0.5 0.03-20.015-10.015-8 TP-271DoxycyclineAzithromycin Burkholderia mallei 30 0.06/0.120.03/0.121/2 ≤0.008-1 0.25-2 TP-271DoxycyclineCeftazidime Burkholderia pseudomallei 30 1/41/82/2 0.25-80.06-160.25-32 These studies were funded in part by NIAID Partnership Grant # 1R01AI093484 – 01 and NIAID Contract #: HHSN272201100028C awarded to CUBRC and Tetraphase Pharmaceuticals Organism MIC 50 /MIC 90 (  g/ml) (range) NTP-271TetracyclineTigecyclineCarbapenem a Fluoroquinolone b 3rd Gen Ceph c Pipericilin/ Tazobactam Aminoglycoside d Klebsiella pneumoniae ESBL 48 1/48/>320.5/10.13/>3232/>32>32/>32>128/>12816/>32 0.25-321->320.25-160.031->32 2->320.5->1280.5->32 Klebsiella oxytoca 41 0.25/2≤4/>320.5/1≤1/≤1≤0.25/>4≤2/>322/64≤2/>32 0.031-8≤4->320.063-4≤1-1≤0.25->4≤2->32≤1->64≤2->32 Klebsiella oxytoca ESBL 11 0.5/2>32/>320.25/0.50.063/0.250.5/>32>32/>328/>32>32/>32 0.031-80.5->320.063-10.031-10.031->324->320.5->32 Escherichia coli ESBL 34 1/2>32/>320.13/0.50.031/0.1316/>32>32/>328/>1284/>32 0.031-42->320.063-1≤0.016-10.031->320.5->321->1281->32 Enterobacter cloacae 44 1/4≤4/>321/2≤1/>8≤0.25/>4≤2/>328/>64≤2/>16 0.25-16≤4->320.25-8≤1->8≤0.25->4≤2->32≤1->64≤2->16 Enterobacter spp. AmpC + 15 1/88/>321/4>8/>8>4/>4>32/>32>64/>6416/>16 0.25-16≤4 - >320.5-42->8≤0.25 - >4>32->3216->64≤2 - >16 Enterobacter aerogenes 30 0.5/1≤4/160.5/2≤1/≤1≤0.25/1≤2/>324/>64≤2/≤2 0.25-4≤4->320.25-2≤1-≤1≤0.25->4≤2->322->64≤2-8 Citrobacter freundii 30 0.25/1≤4/≤40.5/1≤1/≤1≤0.25/22/>322/>64≤2/≤2 0.12-2≤4->320.25-2≤1-8≤0.25-4≤2->32≤1->64≤2->16 Proteus mirabilis 20 4/8>32/>324/88/80.13/320.031/0.0310.5/12/8 2-832->322-84-160.063-32≤0.016-0.130.25-20.5->32 Proteus vulgaris 30 2/48/322/4≤1/≤1≤0.25/≤0.25≤2/>32≤1/≤1≤2/≤2 0.25-4≤4->320.5-4≤1-≤1≤0.25-4≤2->32≤1->64≤2->16 Serratia marcescens 30 2/232/>321/2≤1/≤1≤0.25/0.5≤2/≤22/16≤2/≤2 0.5-8≤4->320.5-4≤1-≤1≤0.25-2≤2->32≤1->64≤2->16 Salmonella spp.35 0.5/2≤4/>320.5/2≤1/≤1≤0.25/0.5≤2/22/16≤2/≤2 0.12-16≤4->320.12-2≤1-≤1≤0.25->4≤2->32≤1-64≤2->16 Shigella spp.18 0.13/0.5≤4/>320.5/1≤1/≤1≤0.25/≤0.25≤2/≤22/8≤2/≤2 ≤0.031-2≤4->320.12-2≤1-≤1≤0.25->4≤2->32≤1->64≤2->16 Morganella morganii 30 4/832/>322/8≤1/≤1≤0.25/>44/16≤1/32≤2/>16 1-16≤4->321-8≤1-2≤0.25->4≤2->32≤1->64≤2->16 Providencia stuartii 30 4/8>32/>324/8≤1/≤14/>4≤2/>164/644/>16 0.13->16≤4>320.13-16≤1-≤1≤0.25->4≤2->16≤1-64≤2->16 Pseudomonas aerguinosa 35 32/>32>32/>3216/321/322/>328/>3216/>1288/>32 4->3232->321->320.5-320.25->321->322->1280.5->32 Acinetobacter baumannii 54 0.13/116/>320.5/44/>328/32 N/D e N/D 32/>32 ≤0.016-20.25->32≤0.016-80.13->320.063->321->32 Acinetobacter lwoffii 30 0.063/0.12≤4/≤40.25/1≤1/≤1≤0.25/≤0.258/>16≤1/16≤2/≤2 ≤0.031-0.5≤4-16≤0.031-2≤1->8≤0.25-4≤2->16≤1->64≤2->16 Stenotrophomonas maltophilia 35 0.25/216/320.5/4>32/>321/16>32/>3264/>128>32/>32 ≤0.016-320.5->320.031-42->320.13-328->328->1281->32 a imipenem or meropenem; b levofloxacin or ciprofloxacin; c ceftriaxone, cefotaxime or ceftazidime; d gentamicin or tobramycin; e not done