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POSTER NUMBER Activity of Brilacidin (PMX-30063) Against

E-1474 Drug Resistant Staphylococci 170 N. Radnor Chester Rd., Suite 300 52nd Interscience Conference on Radnor, PA 19087-5221 USA and Chemotherapy (ICAAC) 1 1 2 2 3 3 4,5 4,5 R. Scott , B. Korczak , D. Jorgensen , S. Hawser , M. Hackel , S. Bouchillon , Y. Xiong , A. Bayer www.polymedix.com September 9-12 , 2012 Tel:(484) 598-2400 San Francisco, CA, USA 1Polymedix, Radnor, PA; 2IHMA Europe Sàrl, Epalinges, CH; 3IHMA, Inc., Schaumburg, IL, USA; 4LA Biomed. Res. Inst., Torrance, CA; 5Geffen Sch. of Med. at UCLA, Los Angeles, CA

ABSTRACT RESULTS: RESULTS: CLINICAL ISOLATE SCREEN Brilacidin vs. ISOGENIC STRAIN PAIRS OF S R Background: Brilacidin (PMX-30063), a small nonpeptidic mimic of host defense MDR S. aureus and MDR CNS DAP and DAP MSSA and MRSA proteins recently completed a Phase 2 for treatment of Staphylococcus- associated ABSSSI. Brilacidin is rapidly bactericidal against Gram-positive and Gram- negative pathogens. In the current studies, brilacidin was assessed against multi-drug resistant (MDR) S. aureus and coagulase-negative staphylococci (CNS) and six Table 1: Comparison of brilacidin activity vs. MDR S. aureus (n = 103) Table 4: Susceptibility (MIC and MBC) of the isogenic strain pairs vs. brilacidin S R

Isogenic Strain Pairs

isogenic strain pairs of -sensitive (DAP ) and -resistant (DAP ) MSSA and S R [DAP I DAP ]

MRSA. DAPS I DAPR DAPS I DAPR DAPS I DAPR DAPS I DAPR DAPS I DAPR DAPS I DAPR Clav Brilacidin and comparators were tested against 206 MDR staphylococci (103 / Methods: 214 215 11/11 2145 616 701 211 212 282 283 300 755 MDR S. aureus, 103 MDR CoNS). All single patient isolates were from 2010-2011 with Daptomycin Brilacidin Amox MIC/MBC the exception of 19 isolates collected by the Network on Resistance in 1/1 1/1 1/1 1/1 1/1 1/1 1/1 2/2 1/1 2/2 1/1 1/1 1 >8 >4 >32 1 >4 2 8 0.06 0.12 1 (ug/ml) Staphylococcus aureus (NARSA) which were comprised of 10 vancomycin-resistant, 7 MIC50 2 >8 >4 >32 1 >4 4 >8 0.25 0.25 4 vancomycin-intermediate and 2 linezolid resistant isolates. MICs were determined by MIC90 broth microdilution according to CLSI guidelines. Time-kill studies were done under Mode 1 >8 >4 >32 1 >4 2 >8 0.06 0.12 1 CLSI conditions using polypropylene plasticware. Mean* 1.183 7.429 >4 31.572 0.880 3.313 2.139 4.512 0.088 0.110 1.136 Figure 1: Killing kinetics of brilacidin at 2x, 5x and 10x the MIC Range 1 - 2 2 - >8 4 ->4 16 ->32 0.5 ->2 0.25 ->4 1 - >8 0.03 ->8 ≤0.03 - 4 0.03 – 0.5 0.5 ->8 Results: MIC90 values for MDR staphylococci are shown in the Table below. Brilacidin S R *Geometric Mean; All values expressed as µg/ml; All clinical isolates were collected from 2010 – 2011. Ayala Strain 214 (DAP-S) vs 215 (DAP-R) Strain 11/11 (DAP-S) vs REF2145 (DAP-R) MICs for the DAP and DAP isogenic MRSA strain pairs were equivalent and killing 10.00 10.00 kinetics were rapid at 2x MICs and nearly identical over a 24 hour test period. 9.00 9.00 8.00 214 8.00 11/11

11/11(2X) 7.00 214(2X) 7.00 MIC90 (µg/ml) 214(5X) 11/11(5X) 6.00 Table 2: Comparison of brilacidin activity vs. MDR CoNS (n = 103) 214(10X) 6.00 11/11(10X) 5.00 5.00 2145 CFU/ml Antibiotic MDR S. aureus MDR CoNS NARSA 215 CFU/ml

10

10 2145(2X)

215(2X) 4.00 4.00

215(5X) 2145(5X) log Brilacidin 2 1 2 Log 3.00 3.00

215(10X) 2145(10X) Ceftriaxone >32 >32 >32 2.00 2.00 Clav

/ 1.00 1.00 Daptomycin 1 1 2 0 2 4 6 8 10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24 Time (h) Time (h) Cefoxitin Ceftriaxone Daptomycin Levofloxacin Linezolid Meropenem Minocycline Tigecycline Vancomycin Levofloxacin >4 >4 >4 Brilacidin Amox

Linezolid 4 2 >8 MIC50 1 8 >4 >32 1 >4 1 8 0.25 0.25 2 Strain 616 (DAP-S) vs 701 (DAP-R) Strain 211 (DAP-S) vs 212 (DAP-R) Meropenem >8 >8 >8 1 >8 >4 >32 1 >4 2 >8 0.5 0.5 2 10.00 MIC90 10.00 Minocycline 0.25 0.5 4 Mode 1 >8 >4 >32 1 >4 1 >8 0.06 0.25 2 9.00 9.00 616 8.00 8.00 211 616(2X)

211(2X) Tigecycline 0.25 0.5 0.25 Mean* 0.886 3.665 3.868 24.947 0.790 3.334 1.354 4.045 0.171 0.225 1.508 7.00 7.00 616(5X) 211(5X) 6.00 616(10X) Vancomycin 4 2 >8 Range 0.5 - 2 0.25 - >8 2 ->4 2 ->32 0.25 - 2 0.12 ->4 0.5 - 4 0.03 ->8 ≤0.03 - 4 0.06 – 2 0.5 -4 6.00 211(10X)

CFU/ml 701 CFU/ml 5.00 5.00 212 10

701(2X) 10 *Geometric Mean; All values expressed as µg/ml; All clinical isolates were collected from 2010 – 2011; CoNS: 4.00 4.00 212(2X) : Brilacidin exhibited good activity against all MDR S. aureus, MDR CNS 701(5X)

Conclusions Log

Log 212(5X) coagulase-negative S. aureus 3.00 701(10X) 3.00 212(10X) and 19 NARSA isolates with an MIC range of 0.5 – 2 for the 225 isolates and MIC90 of 1 2.00 2.00 – 2 µg/ml. Excellent bactericidal activity of brilacidin was demonstrated against 6 1.00 1.00 S R 0 2 4 6 8 10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24 isogenic strain pairs of DAP and DAP MSSA and MRSA. These results combined with Time (h) Time (h) a previous screen of 131 MRSA isolates show that brilacidin is highly active in vitro Table 3: Comparison of brilacidin activity against sub-group isolates against MDR staphylococci (e.g. resistant to , daptomycin, levofloxacin, with specific resistance phenotypes (n = 19)

Strain 282 (DAP-S) vs 283 (DAP-R) Strain 300 (DAP-R) vs 755 (DAP-S)

meropenem and vancomycin).

10.00 10.00

Phenotype 9.00 9.00 282 Clav 8.00 8.00 300

(NARSA /

282(2X)

300(2X) 7.00 7.00 isolates) 282(5X) 300(5X) 6.00 282(10X) 6.00 300(10X) CFU/ml CFU/ml Cefoxitin Ceftriaxone Daptomycin Levofloxacin Linezolid Meropenem Minocycline Tigecycline Vancomycin Brilacidin Amox

5.00 283 5.00 755 10 INTRODUCTION 10 755(2X) 1 >8 >4 >32 2 >4 1 >8 0.06 0.12 2 4.00 283(2X) 4.00

VISA Log Log 283(5X) 755(5X) 3.00 3.00 283(10X) 755(10X) VISA 1 8 >4 >32 2 >4 1 8 1 0.12 2 2.00 2.00 R 1.00 1.00 Host defense proteins (HDPs) are an essential component of the innate immune VISA/Dap 1 8 >4 >32 >2 >4 2 8 ≤0.03 0.06 4 0 2 4 6 8 10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24 Time (h) Time (h) system and display broad-spectrum action against bacteria, yeast, and fungus by VISA 1 >8 >4 >32 2 >4 2 >8 0.06 0.12 4 specifically disrupting their membranes rather than binding to specific molecular VISA 1 >8 >4 >32 2 >4 2 >8 0.12 0.25 4 Methods. MIC and time kill studies were done under CLSI conditions using polypropylene targets. Importantly, this mechanism is associated with a lower risk for the VISA 1 >8 >4 >32 2 >4 1 >8 ≤0.03 0.06 2 plasticware. All strain pairs are MRSA except 616/701 and 300/755 which are MSSA. development of resistance. It appears that HDPs are ideal therapeutic agents to R LZD 2 >8 >4 >32 1 >4 >8 >8 4 0.25 1 DAPS: daptomycin susceptible; DAPR: daptomycin resistant. treat infections. However, significant problems with stability, tissue penetration and R LZD 2 >8 >4 >32 1 >4 >8 >8 4 0.12 1 toxicity have hampered their clinical progress. PolyMedix has developed a series of R LZD 2 >8 >4 >32 0.5 >4 8 8 0.12 0.12 1 Results. small nonpeptidic mimics of the HDPs which have robust activity against bacteria VRSA 2 >8 >4 >32 1 >4 2 >8 0.5 0.25 >8 Table 4. Brilacidin was bactericidal vs. all strains of S. aureus. The MICs and MBCs for and markedly lower toxicity. The approach is to capture the structural and biological VRSA 1 >8 >4 >32 1 >4 2 >8 0.06 0.06 >8 brilacidin versus the DAPS and DAPR MSSA and MRSA isogenic strain pairs were within 1 properties of the HDPs on a fully synthetic, nonpeptidic framework that would be less VRSA 1 >8 >4 >32 0.5 >4 2 >8 2 0.12 >8 doubling dilution of each other, indicating that mechanisms associated with daptomycin expensive to produce, have better tissue distribution, and be much easier to fine- VRSA 2 >8 >4 >32 0.5 >4 2 >8 2 0.06 >8 resistance had little influence on susceptibility to brilacidin. tune structurally to improve activity and minimize toxicity. VRSA 1 >8 >4 >32 1 >4 2 >8 0.12 0.12 >8 VRSA 2 >8 >4 >32 1 >4 2 8 0.12 0.06 >8 Figure 1. To examine this question more rigorously, time-kill studies were conducted to VRSA 1 2 >4 >32 0.5 >4 2 0.5 0.25 0.25 >8 measure the kinetics of bactericidal activity of brilacidin against the DAPS and DAPR VRSA 1 >8 >4 >32 1 >4 2 8 0.12 0.12 >8 isogenic strain pairs. Although there may be a small reduction in the killing kinetics with the VRSA 1 >8 >4 >32 0.5 >4 2 >8 ≤0.03 0.06 >8 R S DAP strain 283 vs. the isogenic DAP strain 282, for all other isogenic strain pairs, the VRSA 1 >8 >4 >32 1 >4 2 >8 0.06 0.06 >8 kinetics of killing were nearly identical at each concentration. Results from the MIC and NARSA: Network on Antibiotic Resistance in Staphylococcus aureus ; All MIC values expressed as µg/ml time-kill assays indicate the daptomycin resistance phenotype has no meaningful impact on the antimicrobial activity of brilacidin. Methods. Minimum inhibitory concentration (MIC) endpoints were determined by broth microdilution according to CLSI guidelines (CLSI, M100-22, 2011; M7-A9, SUMMARY 2012). Panels were prepared at IHMA using CAMHB and used the same day. Colonies were taken directly from a second-pass culture plate and prepared to a  Brilacidin was potently active against all MDR S. aureus and MDR CoNS clinical isolates suspension equivalent of the 0.5 McFarland standard using normal saline. The panels in a panel collected from 2010 to 2011 were incubated at 35 °C for 16 to 20 hours before reading the MIC endpoints. Quality control (QC) testing was performed each day of testing as specified by the CLSI  The antimicrobial activity of brilacidin, as judged by MIC90 values, was not affected by We propose that these HDP mimics provide the basis for developing highly effective using S. aureus ATCC 29213 and E. faecalis ATCC 29212. resistance to /, cefoxitin, ceftriaxone, levofloxacin, and anti-bacterial therapeutics to treat drug-resistant infections that minimize the potential meropenem for the appearance of resistance. The most advanced compound, brilacidin, is in Results. Brilacidin exhibited good activity against all isolates tested in the study, with Phase 2 clinical study for ABSSSI. MICs ranging from 0.5 – 2 µg/ml.  As judged by MIC values vs. a sub-group of clinical isolates, resistance to daptomycin, linezolid and/or vancomycin did not appear to influence the antimicrobial activity of Table 1. Against MDR S. aureus isolates, brilacidin exhibited MIC50 and MIC90 of 1 brilacidin and 2 µg/ml, respectively, and the MIC range was 1 – 2 µg/ml. The activity of brilacidin against MDR S. aureus was not affected by resistance or MDR phenotypes  Daptomycin resistance in 6 isogenic strain pairs of MSSA and MRSA had no significant with respect to amoxicillin/clavulanic acid, cefoxitin, ceftriaxone, levofloxacin, and impact on the antimicrobial activities of brilacidin in susceptibility (MIC) or time-kill meropenem. assays.

Table 2. Against MDR CoNS, brilacidin exhibited MIC50 and MIC90 of 1 and 1 µg/ml, CONCLUSIONS respectively, and the MIC range was 0.5 – 2 µg/ml. The activity of brilacidin against MDR coagulase-negative staphylococci was not affected by resistance or MDR In agreement with its novel mechanism of action, brilacidin is highly active against drug- phenotypes with respect to amoxicillin/clavulanic acid, cefoxitin, ceftriaxone, levofloxacin, and meropenem. resistant and multi-drug-resistant strains of S. aureus and coagulase-negative S. aureus (CoNS)

Table 3. A sub-group of isolates exhibiting specific resistance to daptomycin, linezolid and/or vancomycin was also tested. Brilacidin exhibited MICs of 1 to 2 µg/ml ACKNOWLEDGEMENTS AND COI DISCLOSURES irrespective of the resistance phenotype, suggesting that resistance to daptomycin, linezolid and vancomycin did not affect the activity of brilacidin. Some of the isolates in the present study were from NARSA, who we thank for having provided such isolates. R. Scott, B. Korczak and D. Jorgensen are employees of PolyMedix, Inc.

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