November 7, 2008 MICROBIOTIX A small molecule, anti-infective drug discovery company CONFIDENTIAL.

November 7, 2008

MICROBIOTIX

A small molecule, anti-infective drug discovery company

CONFIDENTIAL

November 7, 2008

The development of novel broad-spectrum anti-bacterials for

intracellular BW threats

DTRA01-06-BAA-01Project Title

November 7, 2008

Introduction – Goals of Program

DTRA Mission: Protect the warfighter from conventional or genetically engineered biological threats

Program Mission: Discover and develop broad spectrum anti-bacterials for military use against category A&B biowarfare pathogens

Microbiotix Contract Objective : “Develop a new class of therapeutic agents, the bis-(imidazolinylindole) series discovered in preliminary studies, for use against intracellular bacterial warfare threats”

November 7, 2008

Introduction – Project Strategy

Microbiotix originally structured this extremely rapid anti-bacterial development program to provide the greatest chance of success within the two year time-frame.

The program was initially designed using the best case scenario with no complicating issues anticipated, based upon the data available at contract initiation.

November 7, 2008

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.

November 7, 2008

Synthesis of bis-(imidazolinylindole) compounds

November 7, 2008

Synthesis of Lead Compounds for Anti-Infective Studies

Structure NSC # MBX # Synthesis

317,8811066 (2 TFA)1336 (free base)

5 steps

317,880 1090 (2 TFA) 7 steps

330,687 1113 (free base) 8 steps

369,718 1128 (free base) 13 steps

NH

N

NH

NH

N

HN

NH

N

NH

NH

N

HN

NH N

H

N

NHHN

N

NH N

HO

N

NH

NH

N

November 7, 2008

Representative Synthesis: MBX 1066

NC NO2

CHO

OHCNO2 O2N

CNNC

NHNC N

H CN

MBX 1336

NH

NH

HN

NN

NH

NCpiperidinesulfolane150 °C

HNO3

H2SO4

P(OEt)3reflux

NH2H2N

P2S5120 °C

MBX 1066

NH

NH

HN

NN

NH•2 TFA

TFA

November 7, 2008

SAR Example: MBX 1066/1162 Tether Modifications

NEtOOC COOEt

NOHC CHO

N

NO2

O2N

NC

CN

NHNC N

H CNNNH

NH

N

HN

NN

NHn n

1) LAH THF

2) DMP CH3CN

NO2NC

piperidine130 °C

P(OEt)3reflux

P2S5diamine

120 °C

November 7, 2008

Lead and Backup Compounds and Salt Forms

NH

NH

NH

NH

N

NH HN

N N

NH HN

N

MBX 1336

MBX 1066 (•2 TFA)

MBX 1143

MBX 1162 (•2 TFA)

NH

NHN

NHHN

N

MBX 1090 (•2 TFA)

November 7, 2008

IND-Enabling Studies: Scale-up of Lead Compound Precursors

Structure Precursor to:AmountSynthesized

MBX 1336 (free base of MBX 1066)

>100 g (several batches)

MBX 1090 15 g

MBX 1113 5 g

MBX 1128 650 mg

NH

NH

NC CN

NHNC

NH

CN

NH

NCNH

CN

NH

NCNH

OCN

November 7, 2008

IND-Enabling Studies: Scale-up of Lead Compounds

NH

N

NH

NH

N

HN

NH

NH

HN

NN

NH

MBX 1336>100 g

(non-GMP)

MBX 1143>100 g

(non-GMP)

November 7, 2008

IND-Enabling Studies: Radiolabeled Drug

NH

NH

HN

NN

NHMBX 1143*

45 mCi (~1 mCi/mmol)

**

November 7, 2008

Chemistry Summary

Established synthetic route for original 4 compounds

Small scale synthesis of ~100 analogs in SAR program to improve potency, solubility, and minimize cytotoxicity

MBX 1066 analog MBX 1162 identified as a potential backup

Scaled-up synthesis of three compounds Performed salt selection studies Synthesized radiolabeled MBX 1162 free base

(MBX 1143)

November 7, 2008

In vitro activity (MICs) against category A and B

pathogens

November 7, 2008

Average MIC (g/mL)

Bacterial Strain Test Site MBX 1066 MBX 1090 MBX 1113 MBX 1128 MBX 1162

Burkholderia pseudomallei 1026b

Calgary 0.65 3.2 >8 >8 0.375

Burkholderia mallei GB3 Calgary 1 2 0.7 >8 0.125

Burkholderia mallei ATCC 23344

USAMRIID 0.42 1.6 1.8 >9.7 0.6

Burkholderia pseudomallei DD503

USAMRIID 1.7 3.1 1.8 >9.7 0.9

Francisella tularensis Schu4 USAMRIID 1.7 1.6 0.9 4.9 1.8

Yersinia pestis CO92 USAMRIID 3.4 >12.5 >7.4 >9.7 3.5

Bacillus anthracis Ames USAMRIID 0.07 0.10 0.11 0.15 0.4

Bacillus anthracis Ames 105-6 (Cipro MIC > 100)

USAMRIID 0.20 0.37 0.22 4.8 0.07

MBX Compounds Have Potent in vitro Activities Against Category A & B Biowarfare Agents

November 7, 2008

MIC90 Values for 20 Strains each of B. pseudomallei and B. mallei

Strain (n) Compound MIC90 (µg/mL) MIC50 (µg/mL) Range

Burkholderia pseudomallei (20) Tetracycline 1 0.5 0.25 - 2

MBX 1066 2 1 0.5 - >8

MBX 1090 >8 8 1 - >8

MBX 1162 1 0.5 0.25 - 1

Burkholderia mallei (20) Tetracycline 0.125 0.06 0.03 – 0.25

MBX 1066 0.125 0.125 0.06 – 0.25

MBX 1090 0.25 0.25 0.125 - 1

MBX 1162 0.125 0.06 0.06 – 0.25

November 7, 2008

MIC90 values Against Multiple Isolates of

Gram-positive and Gram-negative

Species

November 7, 2008

Gram-Positive (Staphylococci) Laboratory/Clinical StrainsGr+ Bacterial Species Type # of Isolates Compound MIC90 (µg/mL) MIC50 (µg/mL) Range (µg/mL)

Staphylococcus aureus MSSA 27 MBX 1066 0.25 0.12 0.004-0.5

MBX 1162 0.5 0.12 0.008-0.5

Linezolid 4 2 2-4

Vancomycin 1 0.5 0.5-2

Daptomycin 0.5 0.5 0.25-1

S. aureus MRSA 12 MBX 1066 0.12 0.06 0.06-0.12

MBX 1162 0.12 0.06 0.03-0.12

Linezolid 4 2 2-4


Daptomycin 0.25 0.25 0.12-0.5

Staphylococcus epidermidis MSSE 27 MBX 1066 0.03 0.008 0.004-0.06

MBX 1162 0.06 0.03 0.008-0.06

Linezolid 2 1 0.5-2

Vancomycin 2 1 1-4

Daptomycin 1 0.5 0.5-1

S. epidermidis MRSE 12 MBX 1066 0.03 0.015 0.004-0.03

MBX 1162 0.06 0.015 0.008-0.06

Linezolid 2 1 1-2

Vancomycin 2 2 1-2


November 7, 2008

Gram-Positive (Enterococci) Laboratory/Clinical StrainsGr+ Bacterial Species Type # of Isolates Compound MIC90 (µg/mL) MIC50 (µg/mL) Range (µg/mL)

Enterococcus faecalis VSE 27 MBX 1066 0.06 0.06 0.004-0.12

MBX 1162 0.06 0.06 0.004-0.25

Linezolid 2 2 0.5-2

Vancomycin 2 1 0.5-2

Daptomycin 2 1 0.03-4

E. faecalis VRE 12 MBX 1066 0.06 0.03 0.015-0.06

MBX 1162 0.03 0.015 0.008-0.03

Linezolid 1 1 0.5-2

Vancomycin >64 >64 >64


Enterococcus faecium VSE 27 MBX 1066 0.015 0.004 0.002-0.03

MBX 1162 0.015 0.004 0.002-0.03

Linezolid 4 2 2-4


Daptomycin 4 4 1-8

E. faecium VRE 12 MBX 1066 0.004 0.004 0.002-0.008

MBX 1162 0.004 0.004 0.004-0.008

Linezolid 2 2 1-2

Vancomycin >64 >64 64->64

Daptomycin 4 2 1-4

November 7, 2008

Gram-Positive (Streptococci) Laboratory/Clinical Strains

Gr+ Bacterial Species Type # of Isolates Compound MIC90 (µg/mL) MIC50 (µg/mL) Range (µg/mL)

Streptococcus pneumoniae PSSP 27 MBX 1066 0.03 0.015 0.008-0.12

MBX 1162 0.03 0.03 0.015-0.03

Linezolid 2 1 0.5-2

Vancomycin 0.25 0.25 0.12-0.25

Daptomycin 0.25 0.06 <0.03-0.5

S. pneumoniae PRSP 12 MBX 1066 0.06 0.03 0.03-0.06

MBX 1162 0.06 0.03 0.015-0.06

Linezolid 1 1 0.5-1

Vancomycin 0.25 0.25 0.25-0.5

Daptomycin 0.12 0.06 <0.03-0.12

Streptococcus agalactiae 12 MBX 1066 0.06 0.06 0.03-0.12

MBX 1162 0.06 0.06 0.06-0.12

Linezolid 2 2 1-2

Vancomycin 0.5 0.5 0.5-1


Streptococcus pyogenes 12 MBX 1066 0.03 0.03 0.03

MBX 1162 0.03 0.03 0.03

Linezolid 2 1 1-2

Vancomycin 1 1 0.5-1


November 7, 2008

Gram-Negative (Nonfermentors) Laboratory/Clinical StrainsGr- Bacterial Species Type # of Isolates Compound MIC90 (µg/mL) MIC50 (µg/mL) Range (µg/mL)

A. baumannii 27 MBX 1066 >16 2 0.06->16

MBX 1162 0.5 0.25 0.12-4

Imipenem 1 0.12 0.06-8

Tigecycline 1 0.5 0.06-4

Ciprofloxacin 2 0.5 0.015->8

A. baumannii MDR 13 MBX 1066 >16 >16 1->16

MBX 1162 4 2 0.12-4

Imipenem >32 4 0.06->32

Tigecycline 4 2 0.25->32

Ciprofloxacin >8 >8 0.12->8

P. aeruginosa 27 MBX 1066 >16 >16 0.06->16

MBX 1162 1 0.25 0.03->16

Imipenem >8 1 0.5->8

Tigecycline >8 8 1->8

Ciprofloxacin >2 0.25 0.12->2

B. cepacia 11 MBX 1066 0.06 <0.015 <0.015-4

MBX 1162 0.12 0.06 0.03-0.25

Imipenem >8 4 4->8

Tigecycline 4 2 1-4

Ciprofloxacin 2 2 0.5-2

November 7, 2008

Gram-Negative (Enterobacteriaceae) Laboratory/Clinical StrainsGr- Bacterial Species Type # of Isolates Compound MIC90 (µg/mL) MIC50 (µg/mL) Range (µg/mL)

Escherichia coli 27 MBX 1066 0.5 0.12 0.03-0.12

MBX 1162 0.25 0.12 0.06-0.25

Imipenem 0.25 0.25 0.06-0.5

Tigecycline 0.25 0.12 0.12-0.25


Klebsiella pneumoniae 27 MBX 1066 8 2 0.25->16

MBX 1162 0.5 0.25 0.12-1

Imipenem 16 0.25 0.06-32



K. pneumoniae ESBL 12 MBX 1066 >16 1 0.5->16

MBX 1162 0.5 0.12 0.06-0.5

Imipenem 1 0.25 0.12-2


Ciprofloxacin >8 >8 0.06->8

Serratia marcescens 12 MBX 1066 2 1 0.06-2

MBX 1162 0.25 0.12 0.12-0.5

Imipenem >8 4 2->8

Tigecycline 1 1 0.5-2

Ciprofloxacin 1 0.25 0.06->2

November 7, 2008

Gram-Negative (Atypical) and Gram-positive (Anaerobe) Laboratory/Clinical Strains

Gr- Bacterial Species Type # of Isolates Compound MIC90 (µg/mL) MIC50 (µg/mL) Range (µg/mL)

H. influenzae 12 MBX 1066 >16 4 1->16

MBX 1162 4 1 0.5-4

Levofloxacin 0.06 0.015 0.008-1

Azithromycin 2 1 0.5-2

Cefotaxime >4 1 0.03->4

Amox/Clav 8/4 1/0.5 0.5/0.25-16/8

Clostridium difficile 16 MBX 1066 0.12 0.06 0.03-0.25

MBX 1162 0.06 0.06 0.03-0.12

Clindamycin >8 4 0.25->8

Imipenem 4 4 0.5->8

Metronidazole 0.5 0.12 0.06->8

November 7, 2008

MBX compounds are rapidly bactericidal

0

2

4

6

8

10

12

0 10 20 30

Lo

g C

FU

/mL

Time (hours)

MBX Compounds vs. Y. pestis in a Time Kill Assay at 4x MIC

Control

MBX 1066

MBX 1090

MBX 1142

MBX 1162 0123456789

0 10 20 30

Lo

g C

FU

/mL

Time (hours)

MBX Compounds vs. B. anthracis in a Time Kill Assay at 4x MIC

Control

MBX 1066

MBX 1090

MBX 1142

MBX 1162

Time (hr) to cidal effect

Species / Compound MBX-1066 MBX-1090 MBX-1142 MBX-1162

Y. pestis ≤1 ≤1 ≤1 ≤1

B. anthracis 6 ≤1 4 ≤1

November 7, 2008

Mammalian cytotoxicity values consistent with favorable selectivity indices

CompoundsHeLa Cell CC50

(µg/mL)MIC S. aureus 25923

(µg/mL)Selectivity Index

(in vitro)

MBX 1066 32.5 0.12 270

MBX 1090 10 0.63 16

MBX 1113 3 0.31 9.6

MBX 1128 17 0.28 60

MBX 1142 14 0.27 51

MBX 1143 13 0.12 111

MBX 1162 4 0.16 26

MBX 1195 15 45 0.33

MBX 1196 15 0.16 96

HB-EMAU 35 5 7

Method: Human HeLa cells were exposed for 72 hours to serial dilutions of compounds, then assessed for cell viability using an MTT assay

November 7, 2008

Microbiology Summary

Potent in vitro activity against category A or B bioterrorism pathogens

Potent in vitro activity against a broad-spectrum of Gram-pos. and Gram-neg. strains, including clinical isolates and multiple-drug resistant strains

Potency vs. >10 isolates/species (MIC90 values)

Rapidly bactericidal mechanism of action

Low 3-day cytotoxicity (CC50) of compounds

November 7, 2008

In vivo Potency in Murine Infection

Models

November 7, 2008

Survivors

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

1 10 DMA/D5W - 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

7 10 MBX 1162 10 10 10 10 10 100

8 10 MBX 1162 1 9 6 6 6 60

MBX compounds are efficacious in a murine S. aureus infection model (i.p./i.v.)

Infection: S.aureus (4X108 cfu, Smith strain) injected i.p. Treatment: Compound (10 or 1 mg/kg in 10% DMA/D5W) given IV 15 min. post-infection

November 7, 2008

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

MBX compounds are efficacious in a Yersinia pestis infection model when administered single-dose i.p.

Infection: Y. pestis (100 cfu, CO92 strain) given i.pTreatment: Compound (1.5 mg/kg /injection in 1.5 % DMSO in water) given i.p. qid starting at 6 hours post-infection and ending 5 days post-infection

November 7, 2008

MBX compounds are efficacious in a Yersinia pestis infection model when administered i.p. or i.m.

Infection: Y. pestis (100 cfu, CO92 strain) given i.pTreatment: Compound (2 mg/kg /injection in 1.5 % DMSO in water) given i.p. or i.m. qid starting at 6 hours post-infection and ending 5 days post-infection

0

20

40

60

80

100

0 3 6 9 12 15 18

% s

urv

iva

l

Days post infection

Y.Pestis model

control (n=10)

MBX 1066 IP (n=10)

MBX 1066 IM (n=10)

MBX 1162 IP (n=10)

MBX 1162 IM (n=10)

November 7, 2008

MBX compounds are efficacious in a murine Burkholderia pseudomallei infection model (i.p./i.p.)

Infection: B. pseudomallei (1x 106 cfu, 1026b strain) given by the i.p. route (n = 5)Treatment: Compound (10 mg/kg/injection in 10% DMSO/PBS) given i.p. once at 1 hour post-infection

0

20

40

60

80

100

120

0 1 2 3 4

% S

urv

iva

l

Days Post-infection

B. pseudomallei i.p. Treatment

Vehicle control

Tetracycline

MBX 1090

MBX 1066

MBX 1162

November 7, 2008

MBX 1090 and 1162 are somewhat efficacious in a Burkholderia mallei murine infection model (intranasal/i.v.)

0

20

40

60

80

100

120

0 1 2 3 4 5

% S

urv

iva

l

Days Post-challenge

Burkholderia mallei IV Treatment

Control

Tetracycline

MBX 1090

MBX 1162

Infection: B. mallei (1x 106 cfu, GB5 strain) given by the intranasal route (n = 5)Treatment: Compound (10 mg/kg/injection in 10% DMSO/PBS) given IV once at 1 hour post-infection

*MBX 1066 was not tested due to the deaths observed in the B. pseudomallei model.

November 7, 2008

MBX compounds were not effective in a multiple-dose F. tularensis infection model (i.p./i.p.)

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

Infection: F. tularensis (Schu4 strain) given i.pTreatment: Compound (1.0, 1.5, 1.5, 0.5 mg/kg/injection for MBX 1090, 1142, 1162 and 1113, respectively) given i.p. qid starting at 6 hours post-infection and ending 5 days post-infection

November 7, 2008

Infection: B. anthracis Ames spores (500 cfu) given IP

Treatment: MBX 1066 (10 mg/kg/inj. dissolved in aq. 4% DMSO) given IP qid starting at the indicated post-infection time and ending after 5 days

0

20

40

60

80

100

120

0 5 10 15 20 25 30

% S

urv

iva

l

Days Post-Infection

Efficacy of MBX 1066 Given at Varying Times Post-infection

Control

6 hrs

12 hrs

18 hrs

24 hrs

Demonstrated efficacy with MBX-1066 in a single-dose Bacillus anthracis infection model (i.p./i.p.)

November 7, 2008

Efficacy in a single-dose Bacillus anthracis infection model (i.p./i.v.)

Infection: B. anthracis (860 cfu, Ames strain) given by the i.p. route (n = 10)Treatment: Compound (10 mg/kg for MBX 1066 and 1162, 5 mg/kg for MBX 1090 in 10% DMA/D5W) given IV at 6 hours post-infection; only 1 treatment

0

25

50

75

100

0 5 10 15

% s

urv

iva

l

Days post challenge

B. anthracis Single IV treatment

control

MBX 1066

MBX 1090

MBX 1162

November 7, 2008

Efficacy in a multiple-dose Bacillus anthracis murine infection model (i.p./i.v.)

Infection: B. anthracis (860 cfu, Ames strain) given by the i.p. route (n = 10)Treatment: Compound (10 mg/kg for MBX 1066 and 1162; 5 mg/kg for MBX 1090 in 10% DMA/D5W) given IV at 6 hours post-infection; 5 mg/kg for MBX 1066 and 1162; 2 mg/kg for MBX 1090 given IV at 18 and 42 hours post-infection—a total of 3 treatments

0

25

50

75

100

0 5 10 15 20

% s

urv

iva

l

Days post challenge

B. anthracis Triple IV treatment

control

MBX 1066

MBX 1090

MBX 1162

November 7, 2008

Summary of MBX-1066 & 1162 Efficacy in Animal Models of Infection

Both are active against S. aureus in 1-10 mg/kg range, single-dose i.p. or i.v., with no toxicity observed

Both prolonged survival inY. pestis infection model at 6-8 mg/kg/day x5d, multiple-dose i.p. and i.m.

MBX-1162 is active against B. pseudomallei at 10 mg/kg, single-dose i.p.

Slight prolongation of survival in B. mallei intranasal infection model at 10 mg/kg, single-dose i.v.

Not active against F. tularensis under conditions examined

Both are active against B. anthracis in 10-40 mg/kg range i.v. or i.p.

November 7, 2008

Mechanism of action studies of the bis-

(imidazolinylindole) class of compounds

November 7, 2008

Macromolecular Synthesis Assays

•None of the MMS pathways affected at killing dose (5x MIC)•Target not identified by MMS studies•DNA synthesis is inhibited at >10X MIC (secondary effect)

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)

S. aureus

MBX-1066 Controls

November 7, 2008

Membrane perturbation assays

0

1

2

3

4

5

6

7

Flo

ure

scen

ce r

atio

(re

d/g

reen

)

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

MBX-1066 does not perturb bacterial or mammalian cellular membranes at bactericidal concentrations

Bacterial membrane perturbationDiOC(2)/FACS

Mammalian membrane lysisLDH release assay

November 7, 2008

Map loci responsible for bis-(imidazolinylindole) resistance

Resistant mutants-16X MIC

MBX-1066 resistance is not detectable

Serial passage of S. aureus NCTC-8325 in subinhibitory compound concentrations to select resistance mutants

MBX-1162 resistance is not detectable

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 201 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

A B C D E F G H

0.125

0.25

0.5

1

2

4

8

16

32

64

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Hig

he

st S

ub

leth

al

Co

nc

en

tra

tio

n

(Fo

ld M

IC)

MBX-1066 MBX-1090 MBX-1162

Passage Number Passage Number Passage Number

November 7, 2008

MBX-1090 resistance maps to mepR, regulator of a drug-sodium antiporter

328800 329600 330400 331200

SAOUHSC_00314mepR

MarR-like repressor

SAOUHSC_00315mepA

Multi Antimicrobial Extrusion (MATE)

drug/sodium antiporter

SAOUHSC_00316mepB

Unknown function

WT

1090R mutants (A1, B1, and C1)

mepR mepA mepB

X

OFF

ON

Compound NCTC 8325 (WT) 1090R d20A1MBX-1066 0.5 0.5MBX-1090 1 8MBX-1113 1 2MBX-1128 1 1MBX-1162 0.5 0.5MBX-1195 16 32MBX-1196 1 1MBX-1335 0.0625 0.0625distamycin 50 200

MIC (µg/ml)

No cross resistance vs. other bis-(imidazolinylindole) compounds

Model confirmed by extensive genetic and transcription profiling analyses

•MBX-1090 is a MepA substrate•Other bis-(imidazolinylindole) compounds are NOT MepA substrates

November 7, 2008

Average MIC (µg/mL)

Bacterial Strain MBX 1066 MBX 1090 MBX 1113 MBX 1128 MBX 1162

E. coli 700 TolC+ (efflux proficient) 1.3 0.63 0.31 80 0.16

E. coli 701 TolC- (efflux deficient) 0.16 0.16 0.16 21 0.14

Pseudomonas aeruginosa PAO1 (efflux proficient)

7.5 3.1 25 >80 0.29

P. aeruginosa PAO1 ΔmexAB-oprM (efflux deficient)

1.15 3.1 1.3 >80 0.25

Compound MBX-1162 is not susceptible to bacterial efflux

November 7, 2008

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

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

Affinity of MBX 1066 for AT-rich B. anthracis DNA is ~2-fold stronger than for calf thymus DNA

DNA Interaction with MBX-1066 in the Presence of Increasing Concentrations of Calf Thymus or B. anthracis Genomic DNA

Analysis of DNA binding activity of bis-(imidazolinylindole) compounds

November 7, 2008

In situ fluorescence of MBX-1066 in S. aureus cells is consistent with DNA binding at 1X MIC

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

November 7, 2008

5’-CGXXXXC3’-GCXXXXGA

A AAA

Fluorescent displacement assayMBX-1162

Preference for A/T rich sequencesHighest affinity for AATT

136 possible sequences

Relative affinity for AATTScatchard plot (Kapp)

Dr. Eric Long (IUPUI)

DNA sequence preference for MBX-1162 binding and affinity constant

Slope = Kapp

November 7, 2008

y = 130.74x-1.836

R² = 0.5601

0.1

1

10

100

1 10 100K

app

x 1

06

(M-1

)CC50 (µg/ml)

y = 0.0632x-1.988

R² = 0.38

0.1

1

10

100

0.1 1

Kap

px

10

6(M

-1)

MIC (µg/ml) in 20 µg reserpine/ml

Absence of correlation between DNA binding and biological activity

antibacterial cytotoxicity

MIC and cytotoxicity correlate poorly with DNA binding

Compound 0 μg reserpine/ml 20 μg reserpine/ml CC50 (μg/ml) Kapp x 106 (M-1)

MBX-1066 0.25 0.125 32.5 0.5MBX-1090 1 0.5 10 0.25MBX-1195 16 2 4 ND (too weak)MBX-1196 1 0.25 15 1MBX-1162 0.5 0.125 4 31

MIC (μg/ml)

1162

1196

1066

1090

November 7, 2008

Profiling the changes in gene expression in response to MBX-1066 and -1090 for MOA (in progress)

Grow S. aureus NCTC 8325 in presence of

MBX-1066, MBX-1090 and a compendium of

antibiotics that affect RNA/DNA synthesis at 1-

2X MIC for 1 doubling time (3 h in MHB) in

triplicate

Harvest cells and prepare RNA.

Microarray analyses at NimbleGen

Identify genes up- and down-regulated by

MBX-1066 and -1090. Compare profile to

other antibiotics using statistical methods.

antibiotic concentration1 control NA2 1090Rd20A1 NA3 MBX 1066 1X MIC4 MBX 1090 1X MIC5 Distamycin 2X MIC6 Novobiocin 2X MIC7 Trimethoprim 1X MIC8 Nalidixic Acid 1X MIC9 Ciprofloxacin 2X MIC10 actinomycin D 1X MIC11 Rifampicin 1X MIC12 Phleomycin 2X MIC

Samples prepared for profiling

November 7, 2008

Genes affected by MBX-1066 and -1090

MBX-1066691 MBX-1090

13174

Numbers of genes Up- and Down-regulated (4X, 90% confidence)

Up=333Down=358conserved hypothetical protein = 325

Up=44Down=87conserved hypothetical protein = 65

peptide ABC transporter, ATP-binding protein, putative 5.511 upsuperoxide dismutase, putative 5.103 upchaperonin, 10 kDa, GroES, putative 9.694 upprophage genes 5-23 upphosphoribosylformylglycinamidine synthase, PurS protein 4.687 upphosphoribosylformylglycinamidine synthase II 4.924 up

capsular polysaccharide synthesis enzyme Cap5B 9.603 downacetyl-CoA acetyltransferase, putative 5.995 downiron (chelated) ABC transporter, permease protein, putative 9.980 downclumping factor 4.173 down

MBX-1090 selected genes

November 7, 2008

+4.8 +15.8

DIS

T-R

EP

106

6-R

EP

109

0-R

EP

NA

-RE

P

TM

P-R

EP

CIP

-RE

P

PH

LE

O-R

EP

RIF

-RE

P

NO

VO

-RE

P

AC

TD

-RE

P

Hierarchical clustering: Intersection MBX-1090 and 1066 genes 4X UP and DOWN (74)

MBX-1066 and MBX-1090 clusters with Distamycin (DNA minor groove binder)

November 7, 2008

Biolog-MOA using Phenotypic Arrays

1. Measure magnitude of synergy/antagonism (SAVs) of experimental compound against a panel of 60 antibiotics with known MOA using PM technology

2. Generate matrix of SAVs that describes the chemical interaction between the chemicals in the PM plate and the added inhibitors being tested.

3. Use statistical clustering program to group antibiotics based on SAVs. Antibiotics with like MOAs cluster together.

November 7, 2008

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.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

94

130

109

125

110

145

57

65

202

67

55

212

206

196

8

231

282

280

244

245

217

152

224

2

101

384

457

179

179

179

226

163

163

348

132

240

74

458

270

52

139

515

516

10

81

313

351

361

4

211

Spiramycin

Tylosin

Erythromycin

Erythromycin

Erythromycin

Josamycin

Dirithromycin

Dirithromycin

Roxithromycin

Clindamycin

Lincomycin

Cadmium Chloride

Tyrothricin

Monensin

Antimony (III) Chloride

Coumarin

MBX-1066

MBX-1090

2,4-Dintrophenol

CCCP

Pentachlorophenol

Salicylanilide

Sodium Azide

1,10-Phenanthroline

Hexachlorophene

Macrolide, 16 ring

Ketolide

Macrolide, 14 ring

Macrolide, 14 ring

Macrolide, 14 ring

Macrolide, 16 ring

Macrolide, 14 ring .

Macrolide, 14 ring .

Macrolide

Lincosamine

Lincosamine

toxic cation

Polyether

toxic cation

AminoCoumerins

phenol

Phenanthroline

Ribosome 50S, 23S RNA,.


Ribosome, 50S, 23S RNA,.







Ribosome, 50S, Peptidyltr.

Ribosome, 50S, Peptidyltr.

Cation toxicity

membrane, cyclic peptide

Ionophore, K?

Cation toxicity

DNA Topoisimerase II (D.

Uncoupler, Respiration

Uncoupler, Respiration

membrane permeability, .


respiration, uncoupler

Chelator, Fe (Zinc?)


Phenotypic Microarray-Clustering results

MBX-1066 and MBX-1090 cluster with toxic cations—suggests non-specific MOA

November 7, 2008

MOA Summary

• Activity against DNA-dependent macromolecular synthesis• Inhibition of DNA and RNA synthesis at >10x MIC in cell-based MMS assay • Inhibition of replicative helicase (IC50~1 μM; 4X MIC)

• Inhibition of ReplixTM (IC50 ~2 μM; 8X MIC)

• Above activities are not potent enough to correlate with antibacterial activity

• Minimal effects on bacterial and mammalian cell membranes• Extremely low frequency of resistant mutants• The bis-(imidazolinylindole) compounds interact with DNA• Fluorescence enhancement in the presence of DNA (Max1/2~0.4 μM)

• Intracellular fluorescence observed at 1X MIC• AATT is optimal binding site• DNA binding affinity correlates poorly with antibacterial activity & cytotoxicity

• Profiling studies suggest non-specific or multiple MOAs

November 7, 2008

Selection of Lead and Backup Compounds• MBX 1066 was selected as a preclinical candidate

• Potent vs. category A and B biothreat agents

• Broad-spectrum antibacterial activity

• Undetectable frequency of mutation to resistance

• Maximal in vitro selectivity index

• Rapid bactericidal activity

• Low cost of goods; ease of synthesis scale-up; short synthetic route

• Prolongs survival in multiple animal models of infection

• MBX 1162 (analog of MBX 1066) was selected as a backup candidate by SAR

• Similar benefits as MBX 1066

• Greater potency vs. Gram-neg. while retaining Gram-pos. potency

• Broader spectrum of activity than MBX 1066

• Increased solubility

November 7, 2008

IND Enabling Studies

November 7, 2008

Nomenclature

MBX-1066 (TFA salt of MBX-1336) MBX-1162 (TFA salt of MBX-1143) MBX-1336 (free base of MBX-1066) MBX-1143 (free base of MBX-1162)

November 7, 2008

Target Product Profile Indication: Treatment and prevention of infections from biowarfare agents Mechanism of Action: Broad-spectrum antibacterial activity against intracellular biowarfare

agents. Safety Profile: The benefits of treatment outweigh the risks. MIC: <1 µg/ml Clinical Efficacy: Must be effective in primate efficacy model. Resistance: Compounds with new mechanisms of resistance or no resistance will be

favored. Route of Administration: Intramuscular may be more field-deployable; intravenous will be

used initially (bolus ideal; up to 1 hour infusion acceptable). Dosing Regimen: Ideally one time dose; for multiple dosing 1 -2 times daily, no more than

3-4 times daily. Dosage Form: Low volume parenteral compatible with standard intravenous solutions Monitoring Requirements: Monitoring of serum/plasma drug concentrations should not be

required. No clinically significant adverse reactions observed in the efficacious dose range. Product Stability: Drug product should be stable for at least 2 years. Product Storage Conditions: The drug product ideally should be stored at room

temperature. Refrigerated or frozen drug product may be acceptable.

November 7, 2008

Preclinical Studies

Pilot Rat Toxicology Genetic Toxicology

AMES CHO Rat Micronucleus

Rat Single Dose PK Rat Single Dose Acute Toxicity (Bolus) Rat Single Dose Acute Toxicity (Infusion) Rat ADME - ongoing Dog Dose Escalation (Infusion) – ongoing

November 7, 2008

Pilot Rat Toxicology Study Single Dose Toxicity (MBX-1066, MBX-1162)

Bolus administration (5, 10, 15 mg/kg) to male rats Doses limited by solubility of MBX-1066 Toxicity seen with higher vehicle concentrations (20 %

DMA in D5W); modified to 10% DMA in D5W MBX-1066 MTD: 5 mg/kg MBX-1162 MTD: 15 mg/kg

Conclusions: Solubility limited; unclear if formulation is contributing to toxicity

November 7, 2008

Resolution of Formulation Issues Six month extension requested due to compound formulation

issues which included low solubility, use of organic solvent, and potential vehicle toxicity

Hired contractor to develop a new suitable clinical formulation Formulation issue resolution included a change to the free base

form and new formulation of the lead compound Microbiotix set-up pilot GMP manufacturing and preliminary stability

studies with the lead compound, MBX-1336 (free base form of MBX-1066). A pilot batch of MBX-1336 was made in April 2008. A non-GMP batch of MBX-1143 (back-up compound) was made.

November 7, 2008

Plan as of May 2008Results of June-initiated toxicology and

pharmacokinetic studies will: Confirm lead compound Trigger cGMP manufacturing Trigger remaining IND-enabling preclinical

toxicology studies Trigger request for pre-IND meeting with

FDA

November 7, 2008

Genetic Toxicology Ames Testing (MBX-1066, MBX-1162)

Completed in March 2008 Neither compound induced mutations

CHO Study (MBX-1143) Completed in August 2008 Did not induce chromosomal aberrations

Rat Micronucleus Study (MBX-1143) Completed in August 2008 Did not increase incidence of micronucleated polychromatic

erythrocytes Conclusion: There were no issues in the genetic toxicology

studies. Results support proceeding with additional studies.

November 7, 2008

Single Dose Rat Pharmacokinetic Study

Bolus injection (MBX-1336, MBX-1143) IV, IM, IP administration at 1 and 10 mg/kg 11/12 rats in MBX-1336 IV 10 mg/kg group died

within 1 minute of administration (6M, 5F) No toxic effects observed for MBX-1143 PK parameters

MBX-1336 IM and IP had BLQ plasma levels See other data next slide

November 7, 2008

PK ParametersCompound Route Dose

(mg/kg)Sex Tmax

(hr)t1/2 (hr) Cmax

(ng/mL)AUClast (hr*ng/mL)

AUC0-∞

(hr*ng/mL)

MBX-1336 IV 1 M 0 41.03 6581 869 1165

MBX-1336 IV 1 F 0 NR 9565 892 918

MBX-1336 IV 10 M NR NR NR NR NR

MBX-1336 IV 10 F 0 2.46 3516 1104 1122

MBX-1143 IV 1 M 0 1.58 13594 1674 1696

MBX-1143 IV 1 F 0 2.35 14240 1692 1698

MBX-1143 IV 10 M 0 2.65 162795 16930 16976

MBX-1143 IV 10 F 0 5.65 146200 16349 16418

MBX-1143 IP 1 M 1 5.4 52.3 435 461

MBX-1143 IP 1 F 1.5 2.79 46.3 278 296

MBX-1143 IP 10 M 6 10.47 321 4364 5789

MBX-1143 IP 10 F 6 8.85 380 4560 5663

MBX-1143 IM 1 M 4 22.45 14.5 258 490

MBX-1143 IM 1 F 6 16 17.4 346 540

MBX-1143 IM 10 M 12 NR 33.2 631 NR

MBX-1143 IM 10 F 12 NR 37.0 740 NR

November 7, 2008

Single Dose Rat Pharmacokinetic Study

Results / Conclusions MBX-1336 caused toxicity at the high end of the

pharmacologic range (10 mg/kg) Plasma levels provided insight into pharmacology data

MBX-1336 does not reach the plasma when given i.p. or i.m. MBX-1143 does reach the plasma when administered i.p., i.m., or

i.v. MBX-1143 PK data was dose-proportional (with

exception of i.m.) MBX-1143 was selected as the new lead candidate

November 7, 2008

Single Dose Rat Acute Toxicity MBX-1143 (free base of MBX-1162)

Bolus Study Design

6 groups of 6M, 6F 10, 30 , 20, 15 and 5 mg/kg and vehicle control Dose followed by 14 day observation period

November 7, 2008

FindingsDose (mg/kg; n=6/sex/group)

0 5 10 15 20 30M F M F M F M F M F M F

Unscheduled Deaths* 0 (1) 0 (1) 0 0 0 0 (2) 3 1 (3) 6 1 (2) 6 6

Clinical Signs + + + + + + ++ ++ ++ ++ +++ +++

Body Weight (thru Day 11) ↑↑ ↑↑ ↑ ↑↑ ↑ ↑↑ ↓ ↑ ↓ ↓ ↓↓ ↓↓

Food Consumption (Days -1 to4 compared to control)

- - N/A N/A ↓ ↓ ↓ ↓ ↓↓ ↓ ↓↓ ↓

Clinical Pathology - - + + + + + + N/A + N/A N/A

Gross Pathology - - = = + + + + + + + +

Organ Weights - - + + + + + + + + N/A N/A

*Number of deaths is expressed as non-procedure related deaths (total deaths).

Single Dose Rat MBX-1143 (free base of MBX-1162) Acute Toxicity – Bolus

November 7, 2008


Summary of Results 5 mg/kg: minor clinical signs – minor changes in clinical

pathology 10 mg/kg: some clinical signs – changes in clinical

pathology; decreased body weight & food consumption 15 mg/kg: 3M and 1F died/sac’d 20 mg/kg: 6M and 1F died/sac’d 30 mg/kg: 6M and 6F died/sac’d

November 7, 2008


Results / Conclusions NOAEL <5 mg/kg MBX-1143 was toxic to animals that received >15 mg/kg

Number of deaths per group increased with dose Males appeared to be more sensitive

Two phases of toxicity resulting in death Acute within 1 hr of dosing Sub-acute within 9-13 days of dosing

Unclear if the toxicity was related to Cmax or AUC.

November 7, 2008


Infusion Study Design

1 hour infusion 3 dose groups (10, 30 and 50 mg/kg) 2 TK groups (low and high dose) 1 vehicle control group

November 7, 2008

FindingsDose (mg/kg; n=6/sex/group, except control n=2/sex)

0 10 30 50M F M F M F M F

Unscheduled Deaths* 0 0 0 0 6/6 6/6 6/6 6/6

Clinical Signs - - ↓ feces

↓ feces + + + +

Body Weight (thru Day 11) ↑↑ ↑↑ ↑ ↑ ↓↓ ↓↓ ↓↓ ↓↓

Food Consumption (Days -1 to 3 compared to control) - - = = ↓ = ↓↓ ↓↓

Clinical Pathology - - + + N/A N/A N/A N/A

Gross Pathology - - + + + + + +

Organ Weights - - + + N/A N/A N/A N/A

Single Dose Rat MBX-1143 (free base of MBX-1162) Acute Toxicity - Infusion

November 7, 2008

Single Dose Rat MBX-1143 (free base of MBX-1162) Acute Toxicity –

Infusion Summary of Results

10 mg/kg: some clinical signs – decreased feces (2/6 animals)

30 mg/kg: 6M and 6F died/sac’d; all died on day 10-11 50 mg/kg: 6M and 6F died/sac’d; males, day 1-8;

females, day 9-10

November 7, 2008


Infusion Results / Conclusions

NOAEL <10 mg/kg MBX-1143 was lethal to animals that received >30 mg/kg

at 30 mg/kg sub-acute tox in both sexes at 50 mg/kg males suffered acute to sub-acute tox and females

suffered sub-acute tox MBX-1143 was toxic by infusion at doses of 30 mg/kg.

We were unsure if this was species-specific toxicity, so we decided to evaluate the compound in dogs.

November 7, 2008

Dose Escalation MBX-1143 (free base of MBX-1162) Dog Study

Study Design Group 1 (1M, 1F): 0.3 mg/kg; 3 mg/kg Group 2 (1M, 1F): 1 mg/kg; 10 mg/kg 4 days between each dose level (groups

alternate) Confirmation group

November 7, 2008

Results 0.3 mg/kg: No issues during 1 week follow-up 1 mg/kg: No issues during 1 week follow-up 3 mg/kg: No issues in 4 days following dosing so 10 mg/kg

group dosed; Male found dead 9 days after 3 mg/kg dose;

Female sac’d 10 days after last dose 10 mg/kg: Male sac’d moribund 2 days after dosing; Female

found dead 5 days after dosing


November 7, 2008

Results Male received 0.3 and 3.0 mg/kg; found dead 9 d after the 3

mg/kg dose Female received 0.3 and 3.0 mg/kg; sac’d 10 d after 3 mg/kg

dose Male received 1 and 10 mg/kg; sac’d 2 d after 10 mg/kg dose Female received 1 and 10 mg/kg; found dead 10 d after 10

mg/kg dose


November 7, 2008

Conclusions: Severe toxicity observed in both groups

Two phases of toxicity resulting in death Acute within 2 days of dosing Sub-acute within 5-10 days of dosing


November 7, 2008

There is a toxicity issue preventing further development Lethal at 10-15 mg/kg in rat Lethal at 0.3-3.3 mg/kg in dog

The efficacious dose is 1-10 mg/kg There is not an acceptable margin of safety for continued

development As a result, we will not continue additional animal studies and

we will not meet the IND milestone

Summary of MBX-1143 Toxicity Studies

November 7, 2008

Further development of backups – e.g., MBX-1090 Topical treatment for wounds – e.g., MDR A.

baumannii Alternative indications – device-related infections

Future Alternatives

November 7, 2008

Future SAR Plans: Analogs of MBX 1090

NH

NHN

NHHN

N

MBX 1090 (•2 TFA)

NH

NH

N

NH HN

N

NH

NHN

NHHN

N

NH

N

NH

NH

HN

N

X

X

X

X

X

X

X

XN

NHHN

N

n n

n n

nn

nn

n

November 7, 2008 MICROBIOTIX A small molecule, anti-infective drug discovery company CONFIDENTIAL.

Documents

synthesis of lead compounds

project title slide

synthesis of bis

salt forms slide

free base of mbx

synthesized mbx

batches mbx

backup compounds