Transcript ICAAC Poster September 2003

#735
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
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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.
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
0.150
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
Vancomycin Clindamycin Quinolone
S
R
R
S
S
S
S
S
S
S
R
S
S
R
R
S
R
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
S
R
S
S
S
S
S
R
S
S
S
S
S
S
S
S
R
R
S
S
R
S
R
R
S
R
R
S
S
R
S
R
S
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
0.125
OD 605nm
Background
MIC*
mg/ml
16
16
8
128
8
64
64
8
16
8
8
16
8
8
8
4
4
8
8
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4
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2
2733 Heather St., VGH
Vancouver, BC
V5Z 3J5, Canada
Tel: 604-875-4111
Extension: 63914
Fax: 604-875-4013
0.100
0.075
0.050
0.025
0.000
1
2
4
8
16
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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.
Time (h)
Effect of EXEG 1706 treatment on undifferentiated THP-1 cells,
measured by PI exclusion staining - Flow Cytometry
MIC correlates for “S” determinants:
≤8
≤2 or 0.5
Code: “S”
“R”
MRSA = methicillin resistant S. aureus
MSSA = methicillin susceptible S. aureus
Quinolone = ciprofloxacin, ofloxacin, levofloxacin or gatifloxacin
Methods
Staphylococcus
aureus MRSA
Staphylococcus aureus
Vancomycin resistant
(VR)
Susceptible
Resistant
Control
50 mg/ml
100 mg/ml
200 mg/ml
3.0
S. aureus VR
2.5
S. haemolyticus VR
2.0
FACS toxicity results on undifferentiated THP-1 cells. LD 50 is 100
mg/ml. Toxicity is over 5 times the MIC.
S. epidermidis
1.5
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.
0.5
A series of bromo-tyrosine compounds were screened, showing that
EXEG 1706 was the most active against MSSA and MRSA.
≤4/2
Dose curve analysis of EXEG 1706 against Staphylococci
1.0
Results
≤1 or 2
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.
OD 600nm
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 (M7T2).
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.
Staphylococcus
aureus MSSA
≤0.5
0.0
0.001
0.010
0.100
1.000
10.000
Conclusion
EXEG 1706 is a representative of the bromo-tyrosine novel class of
antimicrobial compounds.
mg/ml
Streptococcus
pneumoniae-clinical
isolate-VGH,
Vancouver, Canada
It is active against a variety of Gram-positive bacteria.
Toxicity of EXEG 1706
It is highly active at microgram levels against Staphylococci, including
MSSA and MRSA.
1.75
0.200
1.50
0.150
OD 605nm
OD 605nm
1.25
1.00
0.75
MIC against MSSA and MRSA including quinolone resistant and
heteroresistant strains ranged from 2-16 mg/ml.
0.100
LD 50
0.50
0.050
Its in vitro toxicity profile shows more than 25 times the MIC.
0.25
DISK ASSAYS
0.00
10.0
100.0
1000.0
Concentration of EXEG 1706 (mg/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.
Staphylococcus
aureus MSSA
Staphylococcus
haemolyticus VR
Staphylococcus
aureus VR
Mycobacterium
bovis BCG
0.000
50 100
200
300
400
500
600
700
800
Concentration of EXEG 1706 (mg/ml)
Figure 2. Narrow range concentration
toxicity analysis of EXEG 1706 for 24h with
THP-1 macrophages. LD 50 is 400 µg/ml.
Results of toxicity profile on differentiated THP-1 cells. LD 50 is
400mg/ml. No significant toxicity was seen up to 100 mg/ml.
Toxicity against monocytes was 4 times more than toxicity against
differentiated macrophages.
Acknowledgements
We would like to thank Dr. Zakaria Hmama and Scott Cameron for their
help with the macrophage toxicity assays.