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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