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MICROBIOTIX. A product-focused, small molecule, anti-infective drug discovery company. CONFIDENTIAL. The development of novel broad-spectrum anti-bacterials for intracellular BW threats. AGENDA. Terry Bowlin, Ph.D. – Introduction/Welcome John Williams, Ph.D. – Chemistry

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slide1
MICROBIOTIX

A product-focused,

small molecule,

anti-infective drug discovery company

CONFIDENTIAL

agenda
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
slide4
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 safetyevaluation.
slide5
CHEMISTRY

John Williams, Ph.D.

synthesis of mbx 1066
Synthesis of MBX 1066

5 Steps overall

synthesis of mbx 1090
Synthesis of MBX 1090

7 Steps overall

synthesis of mbx 11131
Synthesis of MBX 1113

8 Steps overall

synthesis of mbx 11282
Synthesis of MBX 1128

13 Steps overall

aim 1 2 microbiology studies
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
slide23
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
aim 2
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

slide26
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) compoundsinteract with DNA
    • Fluorescence enhancement in the presence of DNA (Max1/2~0.4 μM)
    • Inhibition of ReplixTM (IC50 ~2 μM) & replicativehelicase (IC50~1 μM)
    • ~2x preference for AT-rich B. anthracisDNA 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
macromolecular synthesis assays in s aureus mbx 1066
Macromolecular Synthesis Assays in S. aureus — MBX 1066

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

slide29
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)

slide31
Helicase Inhibition by MBX 1066 & 1090 as Measured by 32P-Based Unwinding Assay – Comparison to Other Helicase Inhibitors

Conclusion: MBX 1066 & 1090 are very potent B. anthracis helicase inhibitors with IC50’s of <1 μM (<0.6 μg/ml)

slide32
DNA Interaction with MBX 1066 & Hoechst 33258 in the Presence of Increasing Concentrations of Calf Thymus or B. anthracisGenomic DNA

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. anthracisDNA is ~2-fold stronger than for calf thymus DNA

in situ fluorescence of mbx 1066 in s aureus cells is consistent with cell penetration dna binding
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

Intracellular fluorescence readily detected at 1X MIC

Consistent with DNA-dependent fluorescence enhancement

DIC

1 X MBX 1066

cytoplasmic

localization

DAPI

Contrast enhanced

10X zoom

slide34
MBX MIC Data for MBX 1066 & Analogs

IsogenicP. aeruginosaStrains +/- a Major Efflux Pump

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

mutation to resistance to mbx 1066 is rare in s aureu s nctc 8325 serial passage
Mutation to Resistance to MBX 1066 is Rare in S. aureus NCTC-8325 Serial Passage

S. aureus NCTC 8325

E

D

B

F

G

H

A

C

MBX 1066

MBX 1113

MBX 1090

0.125

0.125

0.125

0.25

0.25

0.25

0.5

0.5

0.5

1

1

1

2

2

2

4

4

4

8

8

8

Highest Sublethal Concentration (Fold MIC)

16

16

16

32

32

32

64

64

64

128

128

128

1

5

10

15

20

1

5

10

15

20

1

5

10

15

20

Time (days)

Time (days)

Time (days)

Resistant mutants-16X MIC

mbx 1090 resistant mutants are not cross resistant to mbx 1066
MBX 1090 Resistant Mutants are not Cross-Resistant to MBX 1066

MICs vs MBX 1090, MBX 1066, and MBX 1113

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

bacterial membrane perturbation assay using disc 3 5
Bacterial membrane perturbation assay using DiSC3(5)

Ex-622

Em-670

2H+

DiSC3(5)

No membrane potential perturbation by compound

e-transport

QUENCH

Membrane potential perturbation by compound

2H+

Ex-622

Em-670

Membrane potential perturbation

Membrane disrupter

slide38
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

slide39
DiSC3(5) Membrane Perturbation Assay of

MBX 1066 & Analog MBX 1162

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

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

mbx 1066 1090 do not disrupt hela cell membranes
MBX 1066 & 1090 do not disrupt HeLa cell membranes
  • 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
slide41
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
future mechanism studies
Future Mechanism Studies
  • Perform genetic expression profile analysis. Expression profiling in the presence of various concentrations of bis(imidazolinylindole) compounds to identify genes up- and down-regulated in response to compound treatment
  • Perform target under-expression hypersensitivity and over-expression resistance assays. For implicated single gene targets, construct and test strains over- and under-expressing those putative targets to confirm MOA in the cell
  • Map loci responsible for resistance. Select resistant strains and map resulting mutations to identify genes which can confer resistance
slide44
In Vivo Testing of Lead Antimicrobial Compounds in B. anthracis

Note: MBX 1066 protected 5/5 mice for 14 days in a previous Ames challenge experiment

slide47
Efficacy of MBX 1162 in a murine IP/IP

B. pseudomalleiinfection model

Three groups of 5 Balb/C mice (female, 20-22g) were inoculated intraperitoneally with 106 cells of Burkholderiapseudomalleistrain 1026b. Mice were treated intraperitoneally ten minutes post infection with tetracycline (10 mg/kg), MBX 1162, or vehicle alone

mbx 1066 product development

MBX 1066 Product Development

Jennifer Brooks

Regulatory Affairs Manager

Microbiotix, Inc.

efficacy study design
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
clinical studies
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)
regulatory
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
mbx 1066 summary
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