September 10, 2007

MICROBIOTIX

A product-focused, small molecule, anti-infective drug discovery company

CONFIDENTIAL

September 10, 2007

The development of novel broad-spectrum anti-bacterials for

intracellular BW threats

September 10, 2007

AGENDA

Terry Bowlin, Ph.D. – Introduction/Welcome

John Williams, Ph.D. – Chemistry

Michelle Butler, Ph.D. – Microbiology/Cytotoxicity

Donald Moir, Ph.D. – Mechanism

Terry Bowlin, Ph.D. – Animal Studies

Jennifer Brooks – Development Plan

Terry Bowlin, Ph.D. – Goals/Milestones

September 10, 2007

AIMS Aim 1. Demonstrate potent, selective inhibitory activity of one or more bis-

(imidazolinylindole) compounds in animal models of infection (year 1). Milestone: Identify an inhibitor exhibiting in vivo efficacy (ED50<30 mg/kg) against >2 category A or B pathogens and minimum toxicity (MTD>300 mg/kg).

Aim 2. Establish the mechanism of action of the bis-(imidazolinylindole) class of compounds (year 1). Milestone: Defined mechanism of action and target which are common to multiple bacterial BW species but distinctly different in mammalian cells

Aim 3. Demonstrate structure-activity relationships for the potency and selectivity of the bis-(imidazolinylindole) class of compounds (year 2). Milestone: Identify key structural features for potency and selectivity; provide back-up compounds with MIC in serum <1 µg/ml with a selectivity index (CC50/MIC) >100.

Aim 4. Conduct IND-enabling pharmacokinetic, toxicology and safety pharmacology studies (year 2). Milestone: Complete two species GLP toxicology & safety pharmacology studies for the optimal bis-(imidazolinylindole) compound suitable for IND submission.

Aim 5. Prepare and file an IND application for a broad spectrum anti-bacterial active against intracellular BW threats (end of year 2). Milestone: IND approval for clinical Phase I human safety evaluation.

September 10, 2007

CHEMISTRY John Williams, Ph.D.

September 10, 2007

Synthesis of MBX 1066

5 Steps overall

NC NO2

CHO

OHCNO2 O2N

CNNC

NHNC N

H CN

NSC 317,881

NH

NH

HN

NN

NH

NCpiperidinesulfolane150 °C

HNO3

H2SO4

P(OEt)3reflux

NH2H2N

P2S5120 °C

MBX 1066

NH

NH

HN

NN

NH•2 TFA

September 10, 2007

Synthesis of MBX 1090

NC NO2 O

OEt

O

NC

NO2

NH

COOEt

NCNH

CHO

NC

NC

NHNC

NH CN

NSC 317,880

NH

NHN

NHHN

N

•2 TFANH

NHN

NHHN

N

MBX 1090

HNO3

H2SO4

NaOEtDEO

EtOH

ZnAcOH

LiBH4

THF

Ti0

DME

reflux

H2NNH2 P2S5

120 °C

TFA

7 Steps overall

September 10, 2007

Synthesis of MBX 1113

NCN

BrBoc

NH

CN

Br

Br NO2 Br NO2

OEt

O

O NH

COOEt

Br

NH

CONH2

Br

1) KOH/EtOH/H2O2) SOCl23) NH4OH (aq.)

NaOEtDEO

EtOH

Zn

AcOH

POCl3

reflux

Boc2ODMAP

THF

September 10, 2007

Synthesis of MBX 1113

8 Steps overall

NCN

BrBoc

NHNC

NH

CN

NNCN CNBoc

Boc

NSC 330,687(MBX 1113)

NB(OH)2

NCBoc

+

NH N

H

N

NHHN

N

Pd(PPh3)4Na2CO3

toluene/EtOH/H2Oreflux

200 °C

P2S5120 °C

H2NNH2

September 10, 2007

Synthesis of MBX 1128

CHO

OH

CHO

O COOEt OCOOH

OCONH2

OCN

OCN

Br

OHC

OCN

BrCH2COOEtK2CO3

DMF

KOHEtOH

reflux

1) SOCl22) NH4OH (aq.)

POCl3

reflux

NBS(BzO)2

CCl4reflux

TEANODMSO/H2O70 °C

September 10, 2007

Synthesis of MBX 1128

Br

NO2Br

OCN

OHC OCN

NO2Br

NHBr

O

CNNBr

O

CNBoc

P(OEt)3reflux

Boc2ODMAP

THF

PPh3+Br-

NO2Br K2CO3THF18-C-6

PPh3

toluenereflux

September 10, 2007

Synthesis of MBX 1128

13 Steps overall

NBrO

CNBoc

NNCNBoc O

Boc CN

NH N

HO

NH

NN

NH

NSC 369,718(MBX 1128)

NNCBoc

B(OH)2

NHNC

NH

OCN

Pd(PPh3)4Na2CO3

toluene/EtOH/H2Oreflux

+

200 °C

H2NNH2

P2S5120 °C

September 10, 2007

Analogs of MBX 1066

NH

NH

HN

NN

NH MBX 1066

NH

NH

NH2

NH

NH2

NOH

NH2

NNH2

NH

OEt

NH2

N

NH2

N

NH2

N

NH2

N

N

OH

N

O

HN

N

HN

N

HN

N

OH

HN

N

Amidine Analogs Alkylated Amidines Ring-Expanded

September 10, 2007

MICROBIOLOGY/CYTOTOXICITY Michelle Butler, Ph.D.

September 10, 2007

Aim 1/2 Microbiology Studies

Microbiology -- Original 4 compounds plus MBX 1066 analogs

MICs against standard Gram-pos. and Gram-neg. lab strains

MICs against category A or B bioterrorism pathogens

Cytotoxicity (CC50) of compound

September 10, 2007

Average MIC (µg/mL)

Bacterial StrainMBX 1066

(NSC-317881)MBX 1090

(NSC-317880)MBX 1113

(NSC-330687)MBX 1128

(NSC-369718)

Bacillus subtilis BD54 0.117 0.156 0.156 0.068

B. cereus ATCC 4342 0.078 0.156 0.156 0.521

B. thuringiensis ATCC 10792 0.078 0.313 0.235 0.182

B. anthracis Sterne 0.235 0.313 0.156 1.25

B. anthracis Ames ANR (pXO1-, pXO2-) 0.098 0.313 0.313 36.3

B. megaterium ATCC 12872 0.078 0.156 0.078 0.176

B. licheniformis ATCC 14580 0.059 0.313 0.156 0.117

Staphylococcus aureus ATCC 25923 0.117 0.625 0.313 0.283

S. aureus (Smith) ATCC 13709 0.078 0.313 0.156 0.078

Meth-res S. aureus (MRSA) 1094, clinical 0.137 0.625 0.313 0.508

S. aureus MT23142 NorA++ 0.039 0.313 0.235 0.088

Enterococcus faecalis ATCC 29212 0.137 0.313 0.313 0.107

Vanc-res E. faecalis (VRE) ATCC 51575 0.117 0.625 0.469 0.107

E. faecium ATCC 19434 0.059 0.156 0.274 0.088

VRE faecium B42762, clinical 0.039 0.313 0.156 0.068

MBX BLS1/2 MIC Data

September 10, 2007

Average MIC (µg/mL)

Bacterial StrainMBX 1066

(NSC-317881)MBX 1090

(NSC-317880)MBX 1113

(NSC-330687)MBX 1128

(NSC-369718)

Escherichia coli J53, lab strain 0.391 0.625 0.313 53.3

E. coli XL1Blue, lab strain 0.078 0.156 0.156 1.8

E. coli 700 TolC+ 1.25 0.625 0.313 80

E. coli 701 TolC- 0.156 0.156 0.156 21.3

Klebsiella pneumoniae 5657, clinical 0.235 0.580 0.352 16.3

Pseudomonas aeruginosa PAO1 7.5 25 25 >80

P. aeruginosa PAO1 ΔmexAB-oprM 1.15 >20 ND ND

P. aeruginosa 27853 2.5 12.5 1.09 >80

Burkholderia thailandensis E264 6.25 >80 35 >80

Stenotrophomonas maltophilia ATCC 13637 0.176 0.625 0.313 11.3

Estimated Maximum Solubility in PBS (µg/mL)

MBX 1066 MBX 1090 MBX 1113 MBX 1128

40-80 80-160 20-40 20-40

MBX BLS1/2 MIC Data (continued)

September 10, 2007

Average MIC (g/mL)

Bacterial Strain Test Site MBX 1066 MBX 1090 MBX 1113 MBX 1128

P. aeruginosa PAO1 (control) Calgary 8 5.3 >8 >8*

S. aureus (Smith) ATCC 13709 (control)

Calgary 1.125 2 0.75 >8*

Burkholderia pseudomallei 1026b Calgary 0.65 3.2 >8 >8*

Burkholderia mallei GB3 Calgary 1 2 0.67 >8*

Bacillus anthracis Ames USAMRIID 0.067 0.099 0.11 0.145

Burkholderia mallei ATCC 23344 USAMRIID 0.42 1.6 1.8 >9.7

Burkholderia pseudomallei DD503 USAMRIID 1.7 3.1 1.8 >9.7

Francisella tularensis Schu4 USAMRIID ND 1.56 0.92 4.9

Yersinia pestis CO92 USAMRIID 3.4 >12.5 >7.4 >9.7

U. Calgary and USAMRIID BSL3 Data for MBX Compounds

*Value determined only once.

September 10, 2007

Average MIC (µg/mL)

Bacterial StrainMBX 1066

(NSC-317,881)MBX 1142 MBX 1143 MBX 1162

Bacillus subtilis BD54 0.117 0.068 0.034 0.063

B. cereus ATCC 4342 0.078 0.107 0.039 0.054

B. thuringiensis ATCC 10792 0.078 0.156 0.078 0.117

B. anthracis Sterne 0.235 0.215 0.078 0.088

B. anthracis Ames ANR (pXO1-, pXO2-) 0.098 0.156 0.039 0.063

B. megaterium ATCC 12872 0.078 0.098 0.034 0.037

B. licheniformis ATCC 14580 0.059 0.156 0.049 0.088

Staphylococcus aureus ATCC 25923 0.117 0.274 0.117 0.156

S. aureus (Smith) ATCC 13709 0.078 0.215 0.078 0.102

Methicillin-res. S. aureus (MRSA) 1094, clinical 0.137 0.293 0.156 0.127

S. aureus MT23142 NorA ++ 0.039 0.178 0.078 0.166

Enterococcus faecalis ATCC 29212 0.137 0.176 0.078 0.156

Vanc-resistant E. faecalis (VRE) ATCC 51575 0.117 0.182 0.156 0.137

E. faecium ATCC 19434 0.059 0.235 0.137 0.186

VRE faecium B42762, clinical 0.039 0.235 0.156 0.137

BLS1/2 MIC Data for MBX 1066 Analogs

September 10, 2007

Average MIC (µg/mL)

Bacterial StrainMBX 1066

(NSC-317881)MBX 1142 MBX 1143 MBX 1162

Escherichia coli J53, lab strain 0.391 0.43 0.195 0.274

E. coli XL1Blue, lab strain 0.078 0.215 0.098 0.254

E. coli 701 TolC- 0.156 0.254 0.156 0.137

Klebsiella pneumoniae 5657, clinical 0.235 0.254 0.137 0.146

Pseudomonas aeruginosa PAO1 7.5 0.938 0.235 0.293

P. aeruginosa PAO1 ΔmexAB-oprM 1.15 0.313 0.156 0.254

P. aeruginosa 27853 2.5 0.781 0.215 0.254

Burkholderia thailandensis E264 6.25 22.5 0.352 0.352

Stenotrophomonas maltophilia ATCC 13637

0.176 0.156 0.078 0.156

Estimated Maximum Solubility in PBS (µg/mL)

MBX 1066 MBX 1142 MBX 1143 MBX 1162

40-80 ≥160 ND 40-80

BLS1/2 MIC Data for MBX 1066 Analogs (continued)

September 10, 2007

Average MIC (g/mL)

Bacterial Strain Test Site MBX 1066 MBX 1142 MBX 1143 MBX 1162

P. aeruginosa PAO1 (control) Calgary 8 4 1 2

S. aureus (Smith) ATCC 13709 (control)

Calgary 1.125 4 0.5 0.75

Burkholderia pseudomallei 1026b Calgary 0.65 1 1 0.375

Burkholderia mallei GB3 Calgary 1 ND ND ND

Burkholderia mallei ATCC 23344 USAMRIID 0.42 1.8 1.8 0.6

Burkholderia pseudomallei DD503 USAMRIID 1.7 1.8 0.6 ND

Francisella tularensis Schu4 USAMRIID ND 1.8 ND 1.8

Yersinia pestis CO92 USAMRIID 3.4 3.5 ND 3.5

U. Calgary and USAMRIID BSL3 Data for MBX 1066 Analogs

September 10, 2007

CC50 of MBX 1066 and its analogs on HeLa cells

CompoundsCC50

(µg/mL)MIC S. aureus 25923

(µg/mL)Selectivity Index

(in vitro)

MBX 1066 >20 0.117 >170

MBX 1090 10 0.625 16

MBX 1113 3 0.313 9.6

MBX 1128 17 0.283 60

MBX 1142 14 0.274 51

MBX 1143 13 0.117 111

MBX 1162 4 0.156 26

HB-EMAU 35 5 7

September 10, 2007

MICROBIOLOGY SUMMARY• Accomplishments:

• All four of the original lead compounds have been remade and retested in an independent laboratory with similar antibacterial potencies, especially with relevant BSL3 strains

• MBX 1066 displays the most favorable in vitro selectivity index with low mammalian cell cytotoxicity

• 14 analogs of MBX 1066 have been tested to date and several maintain activity against the Gram-positive strains while displaying greater potency against Gram-negative strains

• Future work:

• We will continue to acquire and test other relevant bacterial strains against the current compounds and new series as they are synthesized

September 10, 2007

MECHANISMDonald Moir, Ph.D.

September 10, 2007

AIM 2

Establish the mechanism of action of the bis-(imidazolinylindole) class of compounds (year 1).

Milestone: Defined mechanism of action and target which are common to multiple bacterial BW species but distinctly

different in mammalian cells

September 10, 2007

Antibacterial Mechanism of bis-(imidazolinylindole) compounds • Favorable in vitro therapeutic index (CC50/MIC) indicates selectivity for bacteria

• Rapid bactericidality implicates DNA, RNA, cell wall or membrane targets• DNA synthesis is the most sensitive of the macromolecular pathways to MBX 1066

(effects observed at >10x MIC)• The bis-(imidazolinylindole) compounds interact with DNA

• Fluorescence enhancement in the presence of DNA (Max1/2~0.4 μM)

• Inhibition of ReplixTM (IC50 ~2 μM) & replicative helicase (IC50~1 μM)

• ~2x preference for AT-rich B. anthracis DNA vs. calf thymus DNA• Target appears to be cytoplasmic

• Fluorescence enhancement of compound observed within bacterial cells • MIC is significantly lower in efflux mutant of P. aeruginosa

• Very low frequency of mutation to resistance• Minimal effects on cell membranes

• No lysis of membranes• No perturbation of the membrane potential near the MIC for some compounds

• Working Hypothesis: The bis-(imidazolinylindole) compounds enter bacterial cells, bind preferentially to AT-rich DNA, and inhibit one or more DNA replication functions

September 10, 2007

0

2

4

6

8

10

12

0 10 20 30

Lo

g C

FU

/mL

Time (hours)

MBX Compounds vs. S. aureus in a Time Kill Assay at 4x MIC

Control

MBX 1066

MBX 1090

MBX 1142

MBX 1162

0

2

4

6

8

10

12

0 1 2 3 4

Log

CFU

/mL

Time (hours)

MBX Compounds vs. S. aureus in a Time Kill Assay at 4x MIC

Control

MBX 1066

MBX 1090

MBX 1142

MBX 1162

Rapid Bactericidal Activity of MBX 1066, 1090, 1142 and 1162

Compound MBX 1066 MBX 1090 MBX 1142 MBX 1162

Time to reach cidal effect (hours)

2 4 1.5 1

September 10, 2007

Macromolecular Synthesis Assays in S. aureus — MBX 1066

DNA synthesis is the most sensitive macromolecular pathway to MBX 1066 treatment – effects are observed at >10 μg/ml

MBX-1066-40xMBX-1066-20x

MBX-1066-10xMBX-1066-5x

0

20

40

60

80

100

120

DNA RNAProtein Cell

wallLipid

% o

f C

on

tro

l

Macromolecule

MBX-1066 (5x, 10x, 20x, 40x MIC)

CiprofloxacinRifampicin-10x

Chloram-10xVancomycin-10xIrgasan-2x

0

20

40

60

80

100

120

140

DN

A

RN

A

Pro

tein

Ce

ll w

all

Lip

id

% o

f C

on

tro

l

Macromolecule

Rifampicin (RNA), Chloramphenicol (protein), Ciprofloxacin (DNA), Vancomycin (cell wall) and Irgasan (lipid)

September 10, 2007

Conclusion: Half-maximal DNA interaction by MBX 1066 occurs at about 0.4 μM (~0.3 μg/ml)

Fluorescence Enhancement of MBX 1066 in the Presence of DNA – Concentration Dependence

September 10, 2007

MBX 1066, 1090 and 1113 are Potent Inhibitors of Replix™, a Permeable Cell DNA Replication Assay

IC50 µM (µg/mL) Against Permeable Bacteria

Compound B. subtilis B. anthracis

MBX 1066 2.2 (1.5) 4.1 (2.8)

MBX 1090 4.8 (3.0) 7.7 (4.8)

MBX 1113 2.6 (0.95) 6.1 (2.2)

HB-EMAU (pos. ctl.) 1.1 (0.35) 2.0 (0.63)

September 10, 2007

0.00

20.00

40.00

60.00

80.00

100.00

120.00

0 10 20 30 40 50 60 70 80

Concn (uM)

% I

nhib

itio

n

0.00

20.00

40.00

60.00

80.00

100.00

120.00

0 10 20 30 40 50 60 70 80

M02

E10

N18

F21

MBX1066

MBX1090

Log. (E10)

Log. (M02)

Log. (N18)

Log. (F21)

Log. (MBX1066)

Log. (MBX1090)Conclusion: MBX 1066 & 1090 are very potent B. anthracis helicase inhibitors with

IC50’s of <1 μM (<0.6 μg/ml)

Helicase Inhibition by MBX 1066 & 1090 as Measured by 32P-Based Unwinding Assay – Comparison to Other Helicase Inhibitors

September 10, 2007

DNA Interaction with MBX 1066 & Hoechst 33258 in the Presence of Increasing Concentrations of Calf Thymus or B. anthracis

Genomic DNA

MBX-1066 fluorescent enhancement from B. anthracis or calf thymus genomic

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

0.001 0.01 0.1 1 10 100

[uM] nt bp's

rati

o o

f 1-

(fre

edru

g/d

rug

+D

NA

)

1066+B.anthracis DNA1066+Calf Thymus DNA

Hoechst fluorescent enhancement from B.anthracis or Calf thymus

-0.2

0

0.2

0.4

0.6

0.8

1

0.001 0.01 0.1 1 10 100

[uM] nt bp's

rati

o o

f 1-

(fre

edru

g/d

rug

+D

NA

)

33258+B.anthracis DNA

33258+Calf thymus DNA

Conclusion: Affinity of both MBX 1066 and Hoechst 33258 for AT-rich B. anthracis DNA is ~2-fold stronger than for calf

thymus DNA

Average A+T content: 64% for B. anthracis DNA vs. 58% for calf thymus DNAc

September 10, 2007

In situ Fluorescence of MBX 1066 in S. aureus cells is Consistent with Cell Penetration & DNA Binding

None 1 X MBX 1066 4 X MBX 1066 1 X MBX 1090 4 X MBX 1090

4 X MBX 1113

DIC

DAPI

DIC

DAPI

Intracellular fluorescence readily detected at 1X MICConsistent with DNA-dependent fluorescence enhancement

1 X MBX 1066

Contrast enhanced10X zoom

cytoplasmiclocalization

September 10, 2007

Average MIC (µg/mL)

Bacterial StrainMBX 1066

(NSC-317881)MBX 1142 MBX 1143 MBX 1162

Pseudomonas aeruginosa PAO1 7.5 0.938 0.235 0.293

P. aeruginosa PAO1 ΔmexAB-oprM 1.15 0.313 0.156 0.254

MBX MIC Data for MBX 1066 & AnalogsIsogenic P. aeruginosa Strains +/- a Major Efflux Pump

Conclusion: MIC of MBX 1066 is significantly improved by loss of major efflux pump; analogs may be better at escaping

efflux

September 10, 2007

Mutation to Resistance to MBX 1066 is Rare in S. aureus NCTC-8325 Serial Passage

A B C D E F G H

Hig

he

st S

ub

leth

al

Co

nce

ntr

ati

on

(F

old

MIC

)

128643216

8421

0.50.25

0.125MBX 1066

1 5 10 15 20

Time (days)

MBX 1090

128643216

8421

0.50.25

0.125

1 5 10 15 20

Time (days)

MBX 1113

1 5 10 15 20

Time (days)

128643216

8421

0.50.25

0.125

S. aureus NCTC 8325

Resistant mutants-16X MIC

September 10, 2007

MBX 1090 Resistant Mutants are not Cross-Resistant to MBX 1066

Clone MBX-1066 MBX-1090 MBX-1113A1 1 32 4A2 1 64 4A3 1 32 4C1 2 32 4C2 2 32 4C3 2 32 4C4 2 32 4G1 1 32 4WT 2 2 2

RESISTANCE (FOLD MIC)

MICs vs MBX 1090, MBX 1066, and MBX 1113

No cross resistance to MBX 1066, suggesting different MOAs for MBX 1090 and MBX 1066

September 10, 2007

Bacterial membrane perturbation assay using DiSC3(5)

DiSC3(5)

Ex-622Em-670

e- transportQUENCHQUENCH

2H+

2H+

Ex-622Em-670

Membrane disrupter

Membrane potential

perturbation

No membrane potential No membrane potential perturbation by compoundperturbation by compound

Membrane potential perturbation by compound

September 10, 2007

Summary of Membrane effects of bis-(imidazolinylindole) Compounds in DiSC3(5) assay

Conclusion: MBX 1066 & 1128 do not perturb membrane potential at concentrations near the MIC

September 10, 2007

0

100

200

300

400

500

600

700

800

No

cm

pd

CC

CP

Va

n-3

2X

11

62-

0.2

5X

11

62-

1X

11

62-

4X

11

62-

32

X

10

66-

0.2

5X

10

66-

1X

10

66-

4X

10

66-

32

X

RF

U

DiSC3(5) Membrane Perturbation Assay of MBX 1066 & Analog MBX 1162

Results of DiSC3(5) assay 10 min after compound addition

Conclusion: MBX 1066 & 1162 do not perturb membrane potential at concentrations near the MIC

September 10, 2007

MBX 1066 & 1090 do not disrupt HeLa cell membranes

0

5

10

15

20

64X

MIC

16X

MIC

1X

MIC

64X

MIC

16X

MIC

1X

MIC

No

an

tib

ioti

c

To

talL

ysi

s

RF

U x

103

MBX-1066 VAN

0

5

10

15

20

64X

MIC

16X

MIC

1X

MIC

64X

MIC

16X

MIC

1X

MIC

No

an

tib

ioti

c

To

talL

ysi

s

RF

U x

103

MBX-1066 VAN

• Monolayers of HeLa cells were exposed to MBX 1066 and a control antibiotic (vancomycin) for 1 h.

• Activity of the cytoplasmic enzyme lactate dehydrogenase (LDH) released into the media was measured after 30 min.

• Similar results obtained with MBX 1090 and MBX 1113

September 10, 2007

Favorable Features of MBX 1066 Antibacterial Mechanism

• In vitro therapeutic index (CC50/MIC >170) is favorable for MBX 1066

• MBX 1066 is rapidly bactericidal • DNA synthesis is the most sensitive macromolecular pathway to MBX 1066

(effects observed at >10x MIC)• Interacts with DNA

• MBX 1066 fluorescence increase in the presence of DNA (Max1/2~0.4 μM)

• Inhibits ReplixTM (IC50 ~2 μM) & replicative helicase (IC50~1 μM)

• ~2x preference for AT-rich B. anthracis DNA vs. calf thymus DNA• Target appears to be intracellular

• Fluorescence enhancement observed within bacterial cells • MIC is significantly lower in efflux mutant of P. aeruginosa

• Very low frequency of mutation to resistance• Minimal effects on cell membranes

• MBX 1066 does not lyse membranes or perturb the membrane potential at <4x MIC

• Conclusion: MBX 1066 is less cytotoxic, exhibits fewer membrane effects, and is less susceptible to mutation to resistance than are MBX 1090, 1113, or 1128

September 10, 2007

Future Mechanism Studies Perform genetic expression profile analysis. Expression profiling in the presence of various concentrations of bis(imidazolinylindole) compounds to identify genes up- and down-regulated in response to compound treatment

Perform target under-expression hypersensitivity and over-expression resistance assays. For implicated single gene targets, construct and test strains over- and under-expressing those putative targets to confirm MOA in the cell

Map loci responsible for resistance. Select resistant strains and map resulting mutations to identify genes which can confer resistance

September 10, 2007

ANIMAL STUDIESTerry Bowlin, Ph.D.

September 10, 2007

Testing of antimicrobials in Ames challenge model

0

20

40

60

80

100

0 50 100 150 200 250

Time (Hrs)

% m

ice

surv

ival

control

PW 317881

MBX 1090

MBX 1113

In Vivo Testing of Lead Antimicrobial Compounds in B. anthracis

Note: MBX 1066 protected 5/5 mice

for 14 days in a previous Ames

challenge experiment

September 10, 2007

In Vivo Testing Against F. Tularensis

F . T u la r e n s is m o d e l

0

2 0

4 0

6 0

8 0

1 0 0

0 2 5 5 0 7 5 1 0 0 1 2 5 1 5 0

h o u r s p o s t c h a l la n g e

% m

ice

su

rviv

al

c o n t r o l

M B X 1 0 9 0

M B X 1 1 1 3

M B X 1 1 4 2

M B X 1 1 6 2

September 10, 2007

In Vivo Testing in Y. Pestis Murine Model

Y.Pestis survival study

0

20

40

60

80

100

0 5 10 15 20 25 30

Days post-infection

% m

ice

surv

ival

control

MBX 1066

MBX 1142

MBX 1162

September 10, 2007

Efficacy of MBX 1162 in a murine IP/IP B. pseudomallei infection model

Group n Treatment Dose, mg/kg#

surviving at 48 hr

% survival

1 5 D/po - 0 02 5 Tetracycline 10 5 100

3 5 MBX 1162 10 5 100

Three groups of 5 Balb/C mice (female, 20-22g) were inoculated intraperitoneally with 106 cells of Burkholderia pseudomallei strain

1026b. Mice were treated intraperitoneally ten minutes post infection with tetracycline (10 mg/kg), MBX 1162, or vehicle alone

September 10, 2007

Efficacies of MBX compounds in a murine IP/IP S. aureus infection model

survivors

Group n treatment Dose, mg/kg 8 hr 18 hr 24 hr 48 hr%

survival

1 10 D/po - 7 1 1 1 102 10 Dapto 10 9 9 9 9 90

3 10 MBX 1066 1 10 10 10 10 100

4 10 MBX 1066 10 10 10 10 10 100

5 10 MBX 1090 1 10 10 10 10 100

6 10 MBX 1090 10 10 10 10 10 100

7 6 D/po - 5 1 1 1 16.7

8 6 Dapto 10 6 6 6 6 100

9 10 MBX 1113 1 10 10 10 10 100

10 10 MBX 1113 10 10 10 10 10 100

11 10 MBX 1128 1 10 10 10 10 100

12 10 MBX 1128 10 10 10 10 10 100

September 10, 2007

survivors

Group n treatment Dose, mg/kg 8 hr 18 hr 24 hr 48 hr%

survival

1 10DMA/D5W,

pH 3.52- 2 2 2 2 20

2 10 Dapto 10 10 10 10 10 100

3 10 MBX 1066 10 9 8 8 8 80

4 10 MBX 1090 10 10 9 9 9 90

5 2 MBX 1113 10 2 mice died immediately after injection

5’ 8 MBX 1113 1 2 1 1 1 12.5

6 1 MBX 1128 10 1 mouse died immediately after injection

6' 9 MBX 1128 1 5 2 0 0 0

Efficacies of MBX compounds in a murine IP/IV S. aureus infection model

September 10, 2007

NCI ID Dose (mg/kg/ injection)

Sched. Route #Mice #Surviving on Day 5

NSC 317880 100 Q04DX003 i.p. 6 6(MBX 1090) 200 Q04DX003 i.p. 6 6

400 Q04DX003 i.p. 6 6NSC 317881 100 Q04DX003 i.p. 6 6(MBX 1066) 200 Q04DX003 i.p. 6 6

400 Q04DX003 i.p. 6 6NSC 330687 25 Q04DX003 i.p. 6 6(MBX 1113) 50 Q04DX003 i.p. 6 6

100 Q04DX003 i.p. 6 5200 Q04DX003 i.p. 6 5400 Q04DX003 i.p. 6 2

NSC 369718 50 Q01DX005 i.p. 6 6(MBX 1128) 100 Q01DX005 i.p. 6 6

200 Q01DX005 i.p. 6 6

Toxicity Determination in Mice

September 10, 2007

MBX 1066 Product Development

Jennifer BrooksRegulatory Affairs Manager

Microbiotix, Inc.

September 10, 2007

MBX 1066 Development

GoalDevelop a new chemical class of therapeutic agents for use against intracellular bacterial biowarfare threats.

IndicationTreatment of exposure to Bacillus anthracis: inhalational anthrax (post-exposure); to reduce the incidence or progression of disease following exposure to aerosolized B. anthracis.

Route Intramuscular

Dosing Regimen TBD based on additional pharmacokinetic data

September 10, 2007

Preclinical Development Plan

X In vitro pharmacology

PK – rodent (IV/IM)

PK – primate (IV/IM)

Toxicology – rodent (IV)

Toxicology – primate (IVIM)

Efficacy – primate (IM) – concurrent with Phase 1

Genetic toxicology

Plasma protein binding

Plasma compatibility

Safety pharmacology

September 10, 2007

Efficacy Study Design Rhesus monkeys

N = 10 per group

Control group

Active control group

30 day study

70 day observation

PK samples

Endpoints will include: survival, bacteremia, microbial burden, histopathology

September 10, 2007

Clinical Studies Phase 1

Concurrent with primate efficacy study Safety Pharmacokinetics

Phase 2/3 depending on feedback from FDA Alternate indications

Approval/Marketing Advisory committee likely ?Restricted distribution (eg, military only)

September 10, 2007

CMC Development PlanChemical characterization • Solubility• Hygroscopicity• Xray crystallography• Etc.

Formulation development

Source materials/excipients

Analytical method development and testing• In-process• Release (water, impurities, residual solvents, etc.)

Stability testing• Routine (including accelerated)• Other (photostability, etc.)

Identify and qualify manufacturers

Develop preliminary specifications

September 10, 2007

Regulatory Next steps

Pre-IND meeting early 2008

Preliminary toxicology data PK data

Conduct remaining IND-enabling studies

IND August 2008

Anticipate approval under Subpart H

Accelerated approval

Surrogate endpoint – efficacy in primates; PK and microbiology data

September 10, 2007

MBX 1066 SUMMARY Very potent broad spectrum agent that is active against

Gram-positive and Gram-negative bacteria

Rapidly bactericidal

MOA consistent with DNA binding/helicase inhibition

No resistance observed so far

Effective in murine models against Gram-positive and Gram-negative bacteria, with ED50<10mg/kg

Well tolerated, with murine MTD >400mg/kg

Easy and inexpensive to synthesize

Next step: IND enabling GLP toxicology

September 10, 2007

TIMELINE

September 10, 2007

TIMELINE (continued)Year 1 Year 2

Task 1Q 2Q 3Q 4Q 1Q 2Q 3Q 4Q

September 10, 2007

TIMELINE (continued)Year 1 Year 2

Task 1Q 2Q 3Q 4Q 1Q 2Q 3Q 4Q