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.