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Transcript
MICROBIOTIX
A product-focused,
small molecule,
anti-infective drug
discovery company
September 10, 2007
CONFIDENTIAL
The development of novel broadspectrum 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
September 10, 2007
5 Steps overall
Synthesis of MBX 1090
September 10, 2007
7 Steps overall
Synthesis of MBX 1113
September 10, 2007
Synthesis of MBX 1113
September 10, 2007
8 Steps overall
Synthesis of MBX 1128
September 10, 2007
Synthesis of MBX 1128
September 10, 2007
Synthesis of MBX 1128
September 10, 2007
13 Steps overall
Analogs of MBX 1066
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
MBX BLS1/2 MIC Data
Average MIC (µg/mL)
MBX 1090
MBX 1113
(NSC-317880)
(NSC-330687)
Bacterial Strain
MBX 1066
(NSC-317881)
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
September 10, 2007
MBX 1128
(NSC-369718)
MBX BLS1/2 MIC Data (continued)
Average MIC (µg/mL)
MBX 1090
MBX 1113
(NSC-317880)
(NSC-330687)
Bacterial Strain
MBX 1066
(NSC-317881)
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)
September 10, 2007
MBX 1066
MBX 1090
MBX 1113
MBX 1128
40-80
80-160
20-40
20-40
U. Calgary and USAMRIID BSL3 Data for MBX Compounds
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
*Value determined only once.
September 10, 2007
BLS1/2 MIC Data for MBX 1066 Analogs
Average MIC (µg/mL)
Bacillus subtilis BD54
MBX 1066
(NSC-317,881)
0.117
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
Bacterial Strain
September 10, 2007
MBX 1142
MBX 1143
MBX 1162
0.068
0.034
0.063
BLS1/2 MIC Data for MBX 1066 Analogs (continued)
Average MIC (µg/mL)
Escherichia coli J53, lab strain
MBX 1066
(NSC-317881)
0.391
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
Stenotrophomonas maltophilia ATCC
13637
6.25
22.5
0.352
0.352
0.176
0.156
0.078
0.156
Bacterial Strain
MBX 1142
MBX 1143
MBX 1162
0.43
0.195
0.274
Estimated Maximum Solubility in PBS (µg/mL)
September 10, 2007
MBX 1066
MBX 1142
MBX 1143
MBX 1162
40-80
≥160
ND
40-80
U. Calgary and USAMRIID BSL3 Data for MBX 1066 Analogs
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
September 10, 2007
CC50 of MBX 1066 and its analogs on HeLa cells
Compounds
CC50
(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
MECHANISM
Donald 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
Rapid Bactericidal Activity of MBX 1066, 1090, 1142 and 1162
MBX Compounds vs. S. aureus in a Time Kill
Assay at 4x MIC
10
8
MBX Compounds vs. S. aureus in a Time Kill
Assay at 4x MIC
Control
6
MBX 1066
12
4
MBX 1090
10
2
MBX 1142
0
MBX 1162
0
10
20
30
Log CFU/mL
Log CFU/mL
12
Time (hours)
8
Control
6
MBX 1066
4
MBX 1090
2
MBX 1142
0
MBX 1162
0
1
2
3
4
Time (hours)
Compound
Time to reach cidal
effect (hours)
September 10, 2007
MBX 1066
MBX 1090
MBX 1142
MBX 1162
2
4
1.5
1
Macromolecular Synthesis Assays in S. aureus — 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
DNA synthesis is the most sensitive macromolecular pathway to
MBX 1066 treatment – effects are observed at >10 μg/ml
September 10, 2007
Fluorescence Enhancement of MBX 1066 in the Presence of DNA –
Concentration Dependence
Conclusion: Half-maximal DNA interaction by MBX 1066 occurs at
about 0.4 μM (~0.3 μg/ml)
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
Helicase Inhibition by MBX 1066 & 1090 as Measured by 32P-Based
of Helicase Activity vs. Concentration
Unwinding Assay%–Inhibition
Comparison
to Other Helicase Inhibitors
P-based Asssay
32
120.00
100.00
M02
% Inhibition of Helicase Activity vs. Concentration
E10
80.00
P-based Asssay
% Inhibition
32
N18
F21
MBX1066
MBX1090
60.00
Log. (E10)
Log. (M02)
Log. (N18)
Log. (F21)
40.00
Log. (MBX1066)
M02
Log. (MBX1090)
E10
N18
20.00
F21
MBX1066
MBX1090
0.00
Log. (E10)
0
10
20
30
40
Concn (uM)
50
60
70 (M02)
80
Log.
Log. (N18)
Log. (F21)
Log. (MBX1066)
Conclusion: MBX 1066 & 1090 are very potent B. anthracis
helicase inhibitors with
Log. (MBX1090)
IC50’s of <1 μM (<0.6 μg/ml)
September 10, 2007
DNA Interaction with MBX 1066 & Hoechst 33258 in the Presence
of Increasing Concentrations of Calf Thymus or B. anthracis
Genomic DNA
Hoechst fluorescent enhancement from B.anthracis or Calf thymus
MBX-1066 fluorescent enhancement from B. anthracis or calf thymus genomic
0.8
1
1066+B.anthracis DNA
1066+Calf Thym us DNA
33258+B.anthracis DNA
33258+Calf thym us DNA
0.6
0.4
0.2
0.001
0
0.01
-0.2
0.1
1
-0.4
10
100
ratio of 1-(freedrug/drug+DNA)
ratio of 1-(freedrug/drug+DNA)
1
0.001
-0.6
[uM ] nt bp's
0.8
0.6
0.4
0.2
0
0.01
-0.2
0.1
1
10
[uM ] nt bp's
Average A+T content: 64% for B. anthracis DNA vs. 58% for calf thymus DNAc
Conclusion: Affinity of both MBX 1066 and Hoechst 33258 for
AT-rich B. anthracis DNA is ~2-fold stronger than for calf
thymus DNA
September 10, 2007
100
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
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
September 10, 2007
10X zoom
Contrast enhanced
MBX MIC Data for MBX 1066 & Analogs
Isogenic P. aeruginosa Strains +/- a Major Efflux Pump
Average MIC (µg/mL)
Bacterial Strain
MBX 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
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
Highest Sublethal Concentration
(Fold MIC)
S. aureus NCTC 8325
0.125
0.25
0.5
1
2
4
8
16
32
64
128
A
MBX 1066
1
5
10
Time (days)
B
C
20
E
F
G
H
MBX 1090
0.125
0.25
0.5
1
2
4
8
16
32
64
128
15
D
1
5
10
15
20
0.125
0.25
0.5
1
2
4
8
16
32
64
128
MBX 1113
1
Time (days)
Resistant mutants-16X MIC
September 10, 2007
5
10
Time (days)
15
20
MBX 1090 Resistant Mutants are not CrossResistant to MBX 1066
MICs vs MBX 1090, MBX 1066, and MBX 1113
Clone
A1
A2
A3
C1
C2
C3
C4
G1
WT
RESISTANCE (FOLD MIC)
MBX-1066 MBX-1090 MBX-1113
1
32
4
1
64
4
1
32
4
2
32
4
2
32
4
2
32
4
2
32
4
1
32
4
2
2
2
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)
Ex-622
Em-670
2H+
DiSC3(5)
No membrane potential
perturbation by compound
e- transport
QUENCH
2H+
Membrane potential
perturbation by compound
Ex-622
Em-670
September 10, 2007
Membrane
disrupter
Membrane
potential
perturbation
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
DiSC3(5) Membrane Perturbation Assay of
MBX 1066 & Analog MBX 1162
800
700
RFU
600
500
400
300
200
100
1066-32X
1066-4X
1066-1X
1066-0.25X
1162-32X
1162-4X
1162-1X
1162-0.25X
Van-32X
CCCP
No cmpd
0
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
20
RFU x103
15
10
5
Total Lysis
1X MIC
64X MIC
16X MIC
VAN
No antibiotic
MBX-1066
1X MIC
16X MIC
64X MIC
0
• 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 overexpression 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 STUDIES
Terry Bowlin, Ph.D.
September 10, 2007
In Vivo Testing of Lead Antimicrobial Compounds in B. anthracis
Testing of antimicrobials in Ames challenge model
control
100
PW 317881
MBX 1090
% mice survival
80
MBX 1113
60
40
20
0
0
50
100
150
Time (Hrs)
September 10, 2007
200
250
Note: MBX 1066
protected 5/5 mice
for 14 days in a
previous Ames
challenge
experiment
In Vivo Testing Against F. Tularensis
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
September 10, 2007
150
In Vivo Testing in Y. Pestis Murine Model
Y.Pestis survival study
MBX 1066
% mice survival
100
MBX 1142
MBX 1162
80
60
40
20
0
0
5
10
15
20
Days post-infection
September 10, 2007
control
25
30
Efficacy of MBX 1162 in a murine IP/IP
B. pseudomallei infection model
Group
n
Treatment
Dose, mg/kg
1
2
3
5
5
5
D/po
Tetracycline
MBX 1162
10
10
#
%
surviving
survival
at 48 hr
0
0
5
100
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
1
2
3
10
10
10
D/po
Dapto
MBX 1066
10
1
7
9
10
1
9
10
1
9
10
1
9
10
%
surviv
al
10
90
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
Efficacies of MBX compounds in a murine IP/IV S. aureus
infection model
survivors
24 hr
48 hr
%
survival
2
2
2
20
10
10
10
10
100
10
9
8
8
8
80
MBX 1090
10
10
9
9
9
90
2
MBX 1113
10
5’
8
MBX 1113
1
6
1
MBX 1128
10
6'
9
MBX 1128
1
Group
n
treatment
Dose, mg/kg
1
10
DMA/D5W,
pH 3.52
-
2
2
10
Dapto
10
3
10
MBX 1066
4
10
5
September 10, 2007
8 hr 18 hr
2 mice died immediately after injection
2
1
1
1
12.5
1 mouse died immediately after injection
5
2
0
0
0
Toxicity Determination in Mice
NCI ID
NSC 317880
(MBX 1090)
NSC 317881
(MBX 1066)
NSC 330687
(MBX 1113)
NSC 369718
(MBX 1128)
Dose (mg/kg/
injection)
100
200
400
100
200
400
25
50
100
200
400
50
100
200
September 10, 2007
Sched.
Q04DX003
Q04DX003
Q04DX003
Q04DX003
Q04DX003
Q04DX003
Q04DX003
Q04DX003
Q04DX003
Q04DX003
Q04DX003
Q01DX005
Q01DX005
Q01DX005
Route
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
i.p.
#Mice
6
6
6
6
6
6
6
6
6
6
6
6
6
6
#Surviving
Day 5
6
6
6
6
6
6
6
6
5
5
2
6
6
6
on
MBX 1066 Product Development
Jennifer Brooks
Regulatory Affairs Manager
Microbiotix, Inc.
September 10, 2007
MBX 1066 Development
Goal
Develop a new chemical class of therapeutic agents for use
against intracellular bacterial biowarfare threats.
Indication
Treatment 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 Plan
Chemical 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
Task
4.1 Efficacy
4.1.1 Small Scale Synthesis
4.1.2 Large Scale Process
4.1.3 In vitro potency
4.1.4 In vivo potency
4.1.4.1 F. tularensis
4.1.4.2 Y. pestis
4.1.4.3 C. burnetii
4.1.4.4 B. pseudomallei
4.1.4.5 B. mallei
4.1.5 Murine toxicity
Milestone: Identify a Lead Compound
4.2 Mechanism of Action
4.2.1 Macromolecular Synthesis
4.2.2 Membrane Perturbation
4.2.3 Map Loci
4.2.4 Genetic Expression Profile
4.2.5 Hypersensitivity and Resistance
Milestone: Defined MOA
September 10, 2007
1Q
Year 1
2Q
3Q
4Q


1Q
Year 2
2Q
3Q
4Q
TIMELINE (continued)
Task
4.3 SAR
4.3.1 Molecular Modeling
4.3.2 Synthesis of Analogs
4.3.2.1 NSC 317,880
4.3.2.2 NSC 317,881
4.3.2.3 NSC 330,687
4.3.3 Biological Evaluation
4.3.3.1 Potency
4.3.3.2 Cytotoxicity
4.3.3.3 Kinetics
4.3.3.4 Frequency of Resistance
4.3.3.5 Bioavailability (in vitro)
4.3.3.6 Drug Interactions
4.3.3.7 Metabolic Stability
4.3.3.8 QT Interval
Milestone: Backup Compounds Identified
September 10, 2007
1Q
Year 1
2Q
3Q
4Q
1Q
Year 2
2Q
3Q
4Q

TIMELINE (continued)
Task
4.4 IND-Enabling Studies
4.4.1 Compound Scaleup
4.4.2 PK and Bioavailablity
4.4.3 Toxicology Studies
4.4.3.1 Rat
4.4.3.2 Primate
4.4.3.3 Genetic
4.4.4 Safety Pharmacology
Milestone: 2 spec. Tox & Safety Profile
4.5 Prepare and File IND
4.5.1 Hire Regulatory Manager
4.5.2 Project Reports
4.5.3 Clinical Protocols
4.5.4 FDA Pre-meeting
4.5.5 Compile IND
Milestone: IND Filing
September 10, 2007
1Q
Year 1
2Q
3Q
4Q
1Q
Year 2
2Q
3Q
4Q

