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DISK ASSAYS
0.001 0.010 0.100 1.000 10.0000.0
0.5
1.0
1.5
2.0
2.5
3.0
S. aureus VR
S. haemolyticus VR
S. epidermidis
g/ml
OD
600
nm
Concentration of EXEG 1706 (g/ml)10.0 100.0 1000.0
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
OD
605
nm
50 100 200 300 400 500 600 700 8000.000
0.050
0.100
0.150
0.200
Concentration of EXEG 1706 (g/ml)
OD
605
nm
LD 50
1 2 4 8 16 320.000
0.025
0.050
0.075
0.100
0.125
0.150
OD
605
nm
Time (h)
200 g/ml100 g/ml50 g/mlControl
EXEG 1706: A Novel Antimicrobial Agent Against Methicillin-Resistant Staphylococcus aureusN. Pick1, RL. Goode2, D. Arad2, Y. Av-Gay1
1Division of Infectious Diseases, UBC, Vancouver, BC, Canada; 2eXegenics Inc., Dallas, TX, USA
BackgroundStaphylococcus 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.
Toxicity of EXEG 1706
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.
Figure 2. Narrow range concentration toxicity analysis of EXEG 1706 for 24h with THP-1 macrophages. LD 50 is 400 µg/ml.
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
ConclusionEXEG 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 MSSA and MRSA.
MIC against MSSA and MRSA including quinolone resistant and heteroresistant strains ranged from 2-16 g/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.
FACS toxicity results on undifferentiated THP-1 cells. LD 50 is 100 g/ml. Toxicity is over 5 times the MIC.
MethodsStandard 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.
ResultsA series of bromo-tyrosine compounds were screened, showing that EXEG 1706 was the most active against MSSA and MRSA.
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.
OrganismMRSAMRSAMRSAS. epidermidisS. haemolyticusS. epidermidisS. haemolyticusMSSAMSSAMSSAMSSAMSSAMRSA heteroresistantMRSA heteroresistantMRSA quinolone susceptibleMRSA quinolone susceptibleMRSA quinolone susceptibleMRSA quinolone susceptibleMRSA quinolone susceptibleMRSA quinolone resistantMRSA quinolone resistantMRSA quinolone resistantMRSA quinolone resistantMRSA quinolone resistantS. saprophyticus
123456789
10111213141516171819202122232425
MIC*g/ml
16168
1288
64648
1688
168884488
1616164
162
OxacillinRRRRRRSSSSSSRRRRRRRRRRRRS
≤2 or 0.5
VancomycinSSSSSSSSSSSSSSSSSSSSSSSSS
≤8
ClindamycinRSSRRRSSSSSSSSRSRSSRSRRSR
≤0.5
QuinoloneRSSSRSSSSSSSSSSSSSSRRRRRS
≤1 or 2
AugmentinRSRSRSSSSSSSSRRSRSSSRSRRS
≤4/2
MIC correlates for “S” determinants:
Code: “S”“R”
SusceptibleResistant
MRSA = methicillin resistant S. aureusMSSA = methicillin susceptible S. aureusQuinolone = ciprofloxacin, ofloxacin, levofloxacin or gatifloxacin
#735 2733 Heather St., VGH Vancouver, BCV5Z 3J5, CanadaTel: 604-875-4111 Extension: 63914Fax: 604-875-4013
Results of toxicity profile on differentiated THP-1 cells. LD 50 is 400g/ml. No significant toxicity was seen up to 100 g/ml.
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.
Dose curve analysis of EXEG 1706 against Staphylococci
Staphylococcus aureus MSSA
Staphylococcus aureus MRSA
Staphylococcus aureus Vancomycin resistant
(VR)
Streptococcus pneumoniae-clinical
isolate-VGH, Vancouver, Canada
Staphylococcus aureus MSSA
Staphylococcus haemolyticus VR
Staphylococcus aureus VR
Mycobacterium bovis BCG
AcknowledgementsWe would like to thank Dr. Zakaria Hmama and Scott Cameron for their help with the macrophage toxicity assays.
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