EXEG 1706: A Novel Antimicrobial Agent Against Methicillin-Resistant Staphylococcus aureus

1. MIC* mg/ml 16 16 8 128 8 64 64 8 16 8 8 16 8 8 8 4 4 8 8 16 16 16 4 16 2 Vancomycin S S S S S S S S S S S S S S S S S S S S S S S S S ≤8 Oxacillin R R R R R R S S S S S S R R R R R R R R R R R R S ≤2 or 0.5 Clindamycin R S S R R R S S S S S S S S R S R S S R S R R S R ≤0.5 Quinolone R S S S R S S S S S S S S S S S S S S R R R R R S ≤1 or 2 Augmentin R S R S R S S S S S S S S R R S R S S S R S R R S ≤4/2 Organism MRSA MRSA MRSA S. epidermidis S. haemolyticus S. epidermidis S. haemolyticus MSSA MSSA MSSA MSSA MSSA MRSA heteroresistant MRSA heteroresistant MRSA quinolone susceptible MRSA quinolone susceptible MRSA quinolone susceptible MRSA quinolone susceptible MRSA quinolone susceptible MRSA quinolone resistant MRSA quinolone resistant MRSA quinolone resistant MRSA quinolone resistant MRSA quinolone resistant S. saprophyticus 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 MIC correlates for “S” determinants: MRSA = methicillin resistant S. aureus MSSA = methicillin susceptible S. aureus Quinolone = ciprofloxacin, ofloxacin, levofloxacin or gatifloxacin Code: “S” “R” Susceptible Resistant EXEG 1706: A Novel Antimicrobial Agent Against Methicillin-Resistant Staphylococcus aureus N. Pick1, RL. Goode2, D. Arad2,Y. Av-Gay1 1Division of Infectious Diseases, UBC, Vancouver, BC, Canada; 2eXegenics Inc., Dallas, TX, USA #735 2733 Heather St., VGH Vancouver, BC V5Z 3J5, Canada Tel: 604-875-4111 Extension: 63914 Fax: 604-875-4013 Background Staphylococcus resistance to current antibiotic treatment is accelerating, with methicillin-resistant Staphylococcus aureus (MRSA) being a leading hospital acquired infectious agent, causing up to 60% of all Staphylococcal infections. Current treatments (vancomycin) is insufficient due to parenteral administration and toxic side effects. Resistance to linezolide (oxazolidinones) is already emerging. We have developed a novel series of compounds, exemplified by EXEG 1706, that exhibit potential effectiveness against Staphylococci including MRSA, with pharmaceutically in vitro acceptable activity, solubility and safety. Figure 3. Trypan blue analysis of toxicity vs. time of 400 µg/ml EXEG 1706 with THP-1 macrophages. Toxicity was maximal at 4 hours. No additional toxicity was observed up to 24 hours. Effect of EXEG 1706 treatment on undifferentiated THP-1 cells, measured by PI exclusion staining - Flow Cytometry Methods Standard disk diffusion assays were done first on several compounds (EXEG 1706 = 317). Micro dilution assays were performed in duplicates only on EXEG 1706 to determine minimal inhibitory concentration (MIC) against different bacterial species according to NCCLS guidelines (M7-T2). Toxicity assays were performed on human macrophage cell line THP-1 monocytes or after differentiation to macrophages. Different concentrations of the drug were added to same amount of THP-1 cells and toxicity was plotted versus different time points using Trypan blue. In addition, Propidium Iodine (PI) exclusion staining assays were performed in 96 wells plates and read by flow cytometry or fluorescence-activated cell sorting (FACS). EXPO 32 software was used to analyze the data. Microdilution MIC of EXEG 1706 on Different Staphylococci. MIC against MSSA and MRSA, including quinolone resistant and heteroresistant strains ranged from 2-16 µg/ml. EXEG 1706 was examined against variety of Staphylococci in an external reference laboratory: Richard Venezia, 35 S. Drive, NY 12159. Dose curve analysis of EXEG 1706 against Staphylococci FACS toxicity results on undifferentiated THP-1 cells. LD 50 is 100 mg/ml. Toxicity is over 5 times the MIC. Sensitivity of different Staphylococci to EXEG 1706.Staphylococci aureus and haemolyticus VR seem to be sensitive linearly. Staphylococci epidermidis is the most resistant of Staphylococci. Conclusion EXEG 1706 is a representative of the bromo-tyrosine novel class of antimicrobial compounds. It is active against a variety of Gram-positive bacteria. It is highly active at microgram levels against Staphylococci, including MSSAand MRSA. MIC against MSSA and MRSA including quinolone resistant andheteroresistant strains ranged from 2-16 mg/ml. Its in vitro toxicity profile shows more than 25 times the MIC. Toxicity against monocytes was 4 times more than toxicity against differentiated macrophages. Results A series of bromo-tyrosine compounds were screened, showing that EXEG 1706 was the most active against MSSA and MRSA. Streptococcus pneumoniae-clinical isolate-VGH, Vancouver, Canada Staphylococcus aureus Vancomycin resistant (VR) Staphylococcus aureus MSSA Staphylococcus aureus MRSA Toxicity of EXEG 1706 Figure 2. Narrow range concentration toxicity analysis of EXEG 1706 for 24h with THP-1 macrophages. LD 50 is 400 µg/ml. Figure 1. Toxicity vs. concentration over 24h incubation of EXEG 1706 with differentiated THP-1 macrophages. No toxicity was observed up to 100 µg/ml. Acknowledgements We would like to thank Dr. Zakaria Hmama and Scott Cameron for their help with the macrophage toxicity assays. Results of toxicity profile on differentiated THP-1 cells. LD 50 is 400mg/ml. No significant toxicity was seen up to 100 mg/ml. Staphylococcus haemolyticus VR Staphylococcus aureus MSSA Staphylococcusaureus VR Mycobacterium bovis BCG