Transcript Document

MICROBIOTIX
A small molecule,
anti-infective drug
discovery company
November 7, 2008
CONFIDENTIAL
DTRA01-06-BAA-01
Project Title
The development of novel broadspectrum anti-bacterials for
intracellular BW threats
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
November 7, 2008
NSC #
MBX #
Synthesis
317,881
1066 (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
Representative Synthesis:
MBX 1066
November 7, 2008
SAR Example: MBX 1066/1162 Tether
Modifications
November 7, 2008
Lead and Backup Compounds and
Salt Forms
November 7, 2008
IND-Enabling Studies: Scale-up of
Lead Compound Precursors
Structure
November 7, 2008
Precursor to:
Amount
Synthesized
MBX 1336 (free
base of MBX
1066)
>100 g
(several batches)
MBX 1090
15 g
MBX 1113
5g
MBX 1128
650 mg
IND-Enabling Studies: Scale-up of
Lead Compounds
November 7, 2008
IND-Enabling Studies: Radiolabeled
Drug
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
MBX Compounds Have Potent in vitro Activities
Against Category A & B Biowarfare Agents
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
USAMRIID
0.42
1.6
1.8
>9.7
0.6
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
Burkholderia mallei ATCC
23344
Burkholderia pseudomallei
DD503
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
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
Burkholderia mallei
(20)
November 7, 2008
MIC90 values Against Multiple Isolates of
Gram-positive and Gram-negative
Species
November 7, 2008
Gram-Positive (Staphylococci) Laboratory/Clinical Strains
Gr+ 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
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
Vancomycin
1
0.5
0.25-1
Daptomycin
0.25
0.25
0.12-0.5
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
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
Daptomycin
1
0.5
0.5-1
S. aureus
Staphylococcus epidermidis
S. epidermidis
November 7, 2008
MRSA
MSSE
MRSE
12
27
12
Gram-Positive (Enterococci) Laboratory/Clinical Strains
Gr+ 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
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
Daptomycin
2
0.5
0.25-2
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
Vancomycin
1
0.5
0.5-4
Daptomycin
4
4
1-8
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
E. faecalis
Enterococcus faecium
E. faecium
November 7, 2008
VRE
VSE
VRE
12
27
12
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
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
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
Daptomycin
1
0.5
0.12-2
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
Daptomycin
2
0.5
0.03-2
S. pneumoniae
Streptococcus agalactiae
Streptococcus pyogenes
November 7, 2008
PRSP
12
12
12
Gram-Negative (Nonfermentors) Laboratory/Clinical Strains
Gr- Bacterial Species
Type
A. baumannii
A. baumannii
P. aeruginosa
B. cepacia
November 7, 2008
MDR
# of Isolates
Compound
MIC90 (µg/mL)
MIC50 (µg/mL)
Range (µg/mL)
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
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
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
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
13
27
11
Gram-Negative (Enterobacteriaceae) Laboratory/Clinical Strains
Gr- Bacterial Species
Type
Escherichia coli
Klebsiella pneumoniae
K. pneumoniae
Serratia marcescens
November 7, 2008
# of Isolates
Compound
MIC90 (µg/mL)
MIC50 (µg/mL)
Range (µg/mL)
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
Ciprofloxacin
>2
0.03
0.015->2
MBX 1066
8
2
0.25->16
MBX 1162
0.5
0.25
0.12-1
Imipenem
16
0.25
0.06-32
Tigecycline
2
0.5
0.25-2
Ciprofloxacin
>8
0.12
0.06->8
MBX 1066
>16
1
0.5->16
MBX 1162
0.5
0.12
0.06-0.5
Imipenem
1
0.25
0.12-2
Tigecycline
2
0.5
0.25-8
Ciprofloxacin
>8
>8
0.06->8
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
27
ESBL
12
12
Gram-Negative (Atypical) and Gram-positive (Anaerobe) Laboratory/Clinical Strains
Gr- Bacterial Species
H. influenzae
Clostridium difficile
November 7, 2008
Type
# of Isolates
Compound
MIC90 (µg/mL)
MIC50 (µg/mL)
Range (µg/mL)
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
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
16
MBX compounds are rapidly bactericidal
MBX Compounds vs. B. anthracis in a Time Kill
Assay at 4x MIC
MBX Compounds vs. Y. pestis in a Time Kill
Assay at 4x MIC
10
8
Control
6
MBX 1066
4
MBX 1090
2
MBX 1142
0
MBX 1162
0
10
20
Log CFU/mL
Log CFU/mL
12
9
8
7
6
5
4
3
2
1
0
30
Control
MBX 1066
MBX 1090
MBX 1142
MBX 1162
0
Time (hours)
10
20
30
Time (hours)
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
Compounds
HeLa 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
November 7, 2008
Method: Human
HeLa cells were
exposed for 72 hours
to serial dilutions of
compounds, then
assessed for cell
viability using an
MTT assay
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
MBX compounds are efficacious in a murine S.
aureus infection model (i.p./i.v.)
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
5’
8
MBX 1113
1
6
1
MBX 1128
10
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
November 7, 2008
2 mice died immediately after injection
2
1
1
1
12.5
1 mouse died immediately after injection
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
MBX compounds are efficacious in a Yersinia pestis
infection model when administered single-dose i.p.
Y.Pestis survival study
MBX 1066
100
% mice survival
control
MBX 1142
MBX 1162
80
60
40
20
0
0
5
10
15
20
Days post-infection
November 7, 2008
25
30
Infection: Y. pestis (100 cfu, CO92
strain) given i.p
Treatment: Compound (1.5 mg/kg
/injection in 1.5 % DMSO in water)
given i.p. qid starting at 6 hours postinfection and ending 5 days postinfection
MBX compounds are efficacious in a Yersinia pestis
infection model when administered i.p. or i.m.
Y.Pestis model
control (n=10)
100
MBX 1066 IP (n=10)
MBX 1066 IM (n=10)
80
MBX 1162 IP (n=10)
% survival
MBX 1162 IM (n=10)
60
40
20
0
0
3
November 7, 2008
6
9
12
Days post infection
15
18
Infection: Y. pestis (100 cfu, CO92
strain) given i.p
Treatment: 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 postinfection
MBX compounds are efficacious in a murine
Burkholderia pseudomallei infection model (i.p./i.p.)
B. pseudomallei i.p. Treatment
120
% Survival
100
80
Vehicle control
60
Tetracycline
40
MBX 1090
MBX 1066
20
MBX 1162
0
0
1
2
3
Days Post-infection
November 7, 2008
4
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
MBX 1090 and 1162 are somewhat efficacious in a Burkholderia
mallei murine infection model (intranasal/i.v.)
Burkholderia mallei IV Treatment
120
% Survival
100
80
Control
60
Tetracycline
40
MBX 1090
20
MBX 1162
0
0
1
2
3
4
5
Days Post-challenge
*MBX 1066 was not tested due to the deaths observed in the B. pseudomallei model.
November 7, 2008
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 compounds were not effective in a multiple-dose
F. tularensis infection model (i.p./i.p.)
F.Tularensis model
100
control
% mice survival
80
MBX 1090
MBX 1113
60
MBX 1142
MBX 1162
40
20
0
0
25
50
75
100
125
hours post challange
November 7, 2008
150
Infection: F. tularensis
(Schu4 strain) given i.p
Treatment: 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
Demonstrated efficacy with MBX-1066 in a single-dose
Bacillus anthracis infection model (i.p./i.p.)
Efficacy of MBX 1066 Given at Varying
Times Post-infection
120
% Survival
100
80
Control
6 hrs
60
12 hrs
40
18 hrs
20
24 hrs
0
0
5
10
15
20
Days Post-Infection
November 7, 2008
25
30
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
Efficacy in a single-dose Bacillus anthracis infection
model (i.p./i.v.)
B. anthracis Single IV treatment
100
control
75
MBX 1066
% survival
MBX 1090
MBX 1162
50
25
0
0
5
10
Days post challenge
November 7, 2008
15
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
Efficacy in a multiple-dose Bacillus anthracis murine
infection model (i.p./i.v.)
B. anthracis Triple IV treatment
100
control
MBX 1066
75
% survival
MBX 1090
MBX 1162
50
25
0
0
5
10
15
Days post challenge
November 7, 2008
20
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
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, singledose 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
S. aureus
Controls
MBX-1066
Rifampicin (RNA), Chloramphenicol (protein), Ciprofloxacin
(DNA), Vancomycin (cell wall) and Irgasan (lipid)
MBX-1066 (5x, 10x, 20x, 40x MIC)
120
140
120
% of Control
MBX-1066-5x
MBX-1066-10x
MBX-1066-20x
MBX-1066-40x
0
DNA
RNA
Protein
Macromolecule
Cell
wall
40
Irgasan-2x
Vancomycin-10x
Chloram-10x
Rifampicin-10x
Ciprofloxacin
20
0
Lipid
Macromolecule
Lipid
20
60
Cell wall
40
80
Protein
60
100
RNA
80
DNA
% of Control
100
•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)
November 7, 2008
Membrane perturbation assays
Bacterial membrane perturbation
DiOC(2)/FACS
Mammalian membrane lysis
LDH release assay
20
6
15
5
4
3
2
RFU x103
Flourescence ratio (red/green)
7
10
5
1
MBX-1066 does not perturb bacterial or mammalian cellular
membranes at bactericidal concentrations
November 7, 2008
Total Lysis
1X MIC
64X MIC
16X MIC
VAN
No antibiotic
MBX-1066
1X MIC
16X MIC
64X MIC
0
0
Map loci responsible for bis(imidazolinylindole) resistance
Serial passage of S. aureus NCTC-8325 in subinhibitory compound
concentrations to select resistance mutants
Highest Sublethal Concentration
(Fold MIC)
A
B
C
D
E
F
G
H
64
32
MBX-1066
MBX-1090
MBX-1162
16
8
4
2
1
0.5
0.25
0.125
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Passage Number
MBX-1066 resistance
is not detectable
November 7, 2008
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Passage Number
Resistant mutants16X MIC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Passage Number
MBX-1162 resistance
is not detectable
MBX-1090 resistance maps to mepR, regulator
of a drug-sodium antiporter
No cross resistance vs. other bis(imidazolinylindole) compounds
WT
OFF
SAOUHSC_00314
mepR
MarR-like repressor
SAOUHSC_00315
mepA
Multi Antimicrobial
Extrusion (MATE)
drug/sodium antiporter
SAOUHSC_00316
mepB
Unknown function
1090R mutants
(A1, B1, and C1)
X
ON
mepA
mepR
328800
329600
mepB
330400
331200
Model confirmed by extensive
genetic and transcription
profiling analyses
November 7, 2008
Compound
MBX-1066
MBX-1090
MBX-1113
MBX-1128
MBX-1162
MBX-1195
MBX-1196
MBX-1335
distamycin
MIC (µg/ml)
NCTC 8325 (WT) 1090R d20A1
0.5
0.5
1
8
1
2
1
1
0.5
0.5
16
32
1
1
0.0625
0.0625
50
200
•MBX-1090 is a MepA substrate
•Other bis-(imidazolinylindole)
compounds are NOT MepA
substrates
Compound MBX-1162 is not susceptible to
bacterial efflux
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
November 7, 2008
Analysis of DNA binding activity of bis(imidazolinylindole) compounds
Fluorescence Enhancement of MBX1066 in the Presence of DNA –
Concentration Dependence
DNA Interaction with MBX-1066 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
ratio of 1-(freedrug/drug+DNA)
1
0.8
1066+B.anthracis DNA
1066+Calf Thymus DNA
0.6
0.4
0.2
0.001
0
0.01
-0.2
0.1
1
10
100
-0.4
-0.6
Half-maximal DNA interaction by MBX1066 occurs at about 0.4 μM (~0.3 μg/ml)
November 7, 2008
[uM] nt bp's
Affinity of MBX 1066 for AT-rich B.
anthracis DNA is ~2-fold stronger than for
calf thymus DNA
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
DIC
DAPI
4 X MBX-1113
DIC
Intracellular fluorescence readily detected at 1X MIC
Consistent with DNA-dependent fluorescence enhancement
1 X MBX-1066
cytoplasmic
localization
DAPI
November 7, 2008
10X zoom
Contrast enhanced
DNA sequence preference for MBX-1162 binding and
affinity constant
Fluorescent displacement assay
MBX-1162
Relative affinity for AATT
Scatchard plot (Kapp)
A A
5’-CGXXXXC
A
3’-GCXXXXG A
A
136 possible sequences
Slope = Kapp
Dr. Eric Long (IUPUI)
November 7, 2008
Preference for A/T rich sequences
Highest affinity for AATT
Absence of correlation between DNA binding
and biological activity
Compound
MBX-1066
MBX-1090
MBX-1195
MBX-1196
MBX-1162
MIC (μg/ml)
0 μg reserpine/ml
20 μg reserpine/ml
0.25
1
16
1
0.5
CC50 (μg/ml)
Kapp x 106 (M-1)
32.5
10
4
15
4
0.5
0.25
ND (too weak)
1
31
0.125
0.5
2
0.25
0.125
cytotoxicity
antibacterial
100
100
Kapp x 106 (M-1)
Kapp x 106 (M-1)
1162
10
1
1196
10
1
y = 0.0632x -1.988
R² = 0.38
1066
y = 130.74x-1.836
R² = 0.5601
1090
0.1
0.1
0.1
1
MIC (µg/ml) in 20 µg reserpine/ml
1
10
100
CC50 (µg/ml)
MIC and cytotoxicity correlate poorly with DNA binding
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 12X 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.
November 7, 2008
Samples prepared for profiling
1
2
3
4
5
6
7
8
9
10
11
12
antibiotic
control
1090Rd20A1
MBX 1066
MBX 1090
Distamycin
Novobiocin
Trimethoprim
Nalidixic Acid
Ciprofloxacin
actinomycin D
Rifampicin
Phleomycin
concentration
NA
NA
1X MIC
1X MIC
2X MIC
2X MIC
1X MIC
1X MIC
2X MIC
1X MIC
1X MIC
2X MIC
Genes affected by MBX-1066 and -1090
MBX-1090 selected genes
Numbers of genes Up- and Downregulated (4X, 90% confidence)
MBX-1066
691
74
peptide ABC transporter, ATP-binding protein, putative
superoxide dismutase, putative
chaperonin, 10 kDa, GroES, putative
prophage genes
phosphoribosylformylglycinamidine synthase, PurS protein
phosphoribosylformylglycinamidine synthase II
5.511 up
5.103 up
9.694 up
5-23 up
4.687 up
4.924 up
capsular polysaccharide synthesis enzyme Cap5B
acetyl-CoA acetyltransferase, putative
iron (chelated) ABC transporter, permease protein, putative
clumping factor
9.603 down
5.995 down
9.980 down
4.173 down
MBX-1090
131
Up=44
Down=87
conserved hypothetical protein = 65
Up=333
Down=358
conserved hypothetical protein = 325
November 7, 2008
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.
November 7, 2008
3. Use statistical clustering program to group
antibiotics based on SAVs. Antibiotics with like
MOAs cluster together.
.
79
130
Clarithromycin
Macrolide
Ribosome, 50S, 23S RNA,.
.
135
293
Oleandomycin
Macrolide, 14 Ring Ribosome 50S, 23S RNA,.
.
230
511
OlePear
Macrolide, 14 Ring Ribosome 50S, 23S RNA,.
.
94
384
Spiramycin
Macrolide, 16 ring Ribosome 50S, 23S RNA,.
.
130
457
Tylosin
Ketolide
.
109
179
Erythromycin
Macrolide, 14 ring Ribosome, 50S, 23S RNA,.
.
125
179
Erythromycin
Macrolide, 14 ring Ribosome, 50S, 23S RNA,.
.
110
179
Erythromycin
Macrolide, 14 ring Ribosome, 50S, 23S RNA,.
.
145
226
Josamycin
Macrolide, 16 ring Ribosome, 50S, 23S RNA,.
.
57
163
Dirithromycin
Macrolide, 14 ring . Ribosome, 50S, 23S RNA,.
.
65
163
Dirithromycin
Macrolide, 14 ring . Ribosome, 50S, 23S RNA,.
.
202
348
Roxithromycin
Macrolide
Ribosome 50S, 23S RNA,.
.
67
132
Clindamycin
Lincosamine
Ribosome, 50S, Peptidyltr.
.
55
240
Lincomycin
Lincosamine
Ribosome, 50S, Peptidyltr.
.
212
74
Cadmium Chloride
toxic cation
Cation toxicity
.
206
458
Tyrothricin
.
196
270
Monensin
Polyether
Ionophore, K?
.
8
52
Antimony (III) Chloride
toxic cation
Cation toxicity
.
231
139
Coumarin
AminoCoumerins
DNA Topoisimerase II (D.
.
282
515
MBX-1066
.
280
516
MBX-1090
.
244
10
2,4-Dintrophenol
Uncoupler, Respiration
.
245
81
CCCP
Uncoupler, Respiration
.
217
313
Pentachlorophenol
.
152
351
Salicylanilide
membrane permeability, .
.
224
361
Sodium Azide
respiration, uncoupler
.
2
4
1,10-Phenanthroline
.
101
211
Hexachlorophene
.
247
62
b-Chloro-L-Alanine
Phenotypic Microarray-Clustering results
Ribosome 50S, 23S RNA,.
membrane, cyclic peptide
phenol
Phenanthroline
membrane permeability, .
Chelator, Fe (Zinc?)
membrane permeability, .
aa analog, alanine aminotransferase inhibitor
MBX-1066 and MBX-109070
with toxic cations—suggests
non. cluster
157 Diamide
Non-Native Sulfhydryl
Bo.
. specific
143
369 Sodium MOA
m-Arsenite
toxic anion
Anion toxicity
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 2008
Results 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)



CHO Study (MBX-1143)



Completed in August 2008
Did not induce chromosomal aberrations
Rat Micronucleus Study (MBX-1143)



Completed in March 2008
Neither compound induced mutations
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 Parameters
Compound
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
Single Dose Rat MBX-1143 (free base
of MBX-1162) Acute Toxicity – Bolus
Findings
Unscheduled Deaths*
Clinical Signs
0
Dose (mg/kg; n=6/sex/group)
5
10
15
20
M
F
M
F
M
F
M
F
M
F
30
M
F
0 (1)
0 (1)
0
0
0
0 (2)
3
1 (3)
6
1 (2)
6
6
+
+
+
+
+
+
++
++
++
++
+++
+++
↑↑
↑
↑↑
↑
↑↑
↓
↑
↓
↓
↓↓
↓↓
-
N/A
N/A
↓
↓
↓
↓
↓↓
↓
↓↓
↓
Body Weight (thru Day
↑↑
11)
Food Consumption (Days
-1 to4 compared to
control)
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).
November 7, 2008
Single Dose Rat MBX-1143 (free base
of MBX-1162) Acute Toxicity – Bolus

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
Single Dose Rat MBX-1143 (free base
of MBX-1162) Acute Toxicity – Bolus

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

Single Dose Rat Acute Toxicity
MBX-1143 (free base of MBX-1162)
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
Single Dose Rat MBX-1143 (free base of
MBX-1162) Acute Toxicity - Infusion
Findings
M
Dose (mg/kg; n=6/sex/group, except control n=2/sex)
0
10
30
50
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
+
+
+
+
↑↑
↑↑
↑
↑
↓↓
↓↓
↓↓
↓↓
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
Body Weight (thru Day 11)
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
Single Dose Rat Acute Toxicity
MBX-1143 (free base of MBX-1162)
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
Dose Escalation MBX-1143 (free
base of MBX-1162) Dog Study

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
Dose Escalation MBX-1143 (free
base of MBX-1162) Dog Study

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
Dose Escalation MBX-1143 (free
base of MBX-1162) Dog Study

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
Summary of MBX-1143 Toxicity
Studies

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





November 7, 2008
Future Alternatives



Further development of backups – e.g., MBX-1090
Topical treatment for wounds – e.g., MDR A.
baumannii
Alternative indications – device-related infections
November 7, 2008
Future SAR Plans: Analogs of MBX
1090
November 7, 2008