1. C-1 Microbiology Jeff Alder, Ph.D. Vice President, Drug Discovery and Evaluation Cubist Pharmaceuticals

2. C-2 Analysis of MIC Shifts Unprecedented microbiology database Unprecedented microbiology database – 1,215 S. aureus isolates were tested for MICs – First study to examine serial isolate susceptibility in SAB patients MIC shifts to  2 noted MIC shifts to  2 noted – Daptomycin- and vancomycin-treated patients – Different susceptibility criteria Scientific investigations with these isolates Scientific investigations with these isolates – Bacteria, drug, patient

3. C-3 Wild-type Distribution of S. aureus Isolates Includes Daptomycin MICs of 2 µg/mL No. of Isolates MIC 90 (µg/mL) Range (µg/mL) MIC of 2 µg/mL n (%) Regional Surveys 1998-20035,2480.25 - 1  0.12 - 2 8 (0.15) Global Surveillance Studies 1999-20012,7870.25 - 0.5  0.12 - 2 1 (0.04) 20022,6230.5  0.12 - 2 2 (0.08) 20034,3620.5  0.12 - 2 1 (0.02) 20045,2600.5  0.12 - 2 2 (0.04) 20056,3740.5  0.12 - 2 3 (0.05) Total26,654NA  0.12 - 2 17 (0.06) MICs of 2 µg/mL were observed prior to approval (Sept ’03)MICs of 2 µg/mL were observed prior to approval (Sept ’03)

4. C-4 Daptomycin-treated Patients with Post-baseline Daptomycin MICs of 2 and 4 MIC 4 µg/mL (N = 1) MIC 4 µg/mL (N = 1) – 1 failure (cRIE; large septic pulmonary emboli, tunnel infection) MIC 2 µg/mL (N = 6) MIC 2 µg/mL (N = 6) – 1 success (cBAC; vertebral osteomyelitis, debrided x2) – 3 failures (cBAC; IV port infection, septic arthritis, retroperitoneal abscess) – 2 failures (LIE; no valve replacement surgery) Failed patients did not or could not receive adjunctive therapy (e.g., surgery, drainage) Failed patients did not or could not receive adjunctive therapy (e.g., surgery, drainage)

5. C-5 Vancomycin-treated Patients with Post-baseline Vancomycin MICs of  2 2 patients by Central Lab testing 2 patients by Central Lab testing – 1 success (cRIE) – 1 failure (cBAC; septic thrombophlebitis) 5 additional patients by Local Lab testing 5 additional patients by Local Lab testing – 1 failure (LIE; no valve replacement surgery) – 3 failures (cBAC; sternal osteomyelitis, abscesses, ulcers) – 1 failure (uBAC; abdominal wound with mesh) Failed patients did not or could not receive adjunctive therapy (e.g., surgery, drainage) Failed patients did not or could not receive adjunctive therapy (e.g., surgery, drainage)

6. C-6 Serial Passage Experiments: Magnitude of MIC Shifts Daptomycin Ciprofloxacin20 40 60 80 100 120 140 12345678910111213141516 Day Fold Change [(Strain MIC/Parent MIC)-1] 0Vancomycin

7. C-7 Genes Involved in S. aureus Susceptibility Genetic Change * Isolate Source Wild-type (Non-exposed) SAB/SAI EStudy Clinical Use Serial Passage (MIC  1) (MIC 2) (MIC 2- 4) (MIC 2- 8) (MIC 2-16) mprF- yycG--- rpoB---- rpoC---- *Genetic loci identified using whole genome comparisons between CA-MRSA MW2 and lab-derived MW2 strains with increasing daptomycin MICs mprF mutations: Part of the wild-type distribution mprF mutations: Part of the wild-type distribution yycG mutations: First unique change seen in MIC  4 clinical isolates, but not seen in wild-type isolates yycG mutations: First unique change seen in MIC  4 clinical isolates, but not seen in wild-type isolates

8. C-8 Genes Involved in S. aureus Susceptibility *Genetic loci identified using whole genome comparisons between CA-MRSA MW2 and lab-derived MW2 strains with increasing daptomycin MICs Genetic Change * Isolate Source Wild-type (Non-exposed) SAB/SAI E Study Clinical Use Serial Passage (MIC  1) (MIC 2) (MIC 2- 4) (MIC 2- 8) (MIC 2-16) mprF- yycG--- rpoB---- rpoC---- mprF mutations: Part of the wild-type distribution mprF mutations: Part of the wild-type distribution yycG mutations: First unique change seen in MIC  4 clinical isolates, but not seen in wild-type isolates yycG mutations: First unique change seen in MIC  4 clinical isolates, but not seen in wild-type isolates

9. C-9 1766 1031 453 250 275 Pharmacodynamics of S. aureus in Mice Lab-derived Isolates of CA-MRSA MW2 Median AUC = 543 µg·hr/mL with the human 6 mg/kg dose MIC Value (µg/mL) Total AUC for 3 log 10 Reduction (µg·hr/mL) 0 200 400 600 800 1000 1200 1400 1600 1800 2000 24816110100 MICs  2 respond to similar drug exposure (AUC) MICs  2 respond to similar drug exposure (AUC) MICs  4 require increasing levels of drug exposure MICs  4 require increasing levels of drug exposure

10. C-10 Efficacy in Mice Against Baseline and Post-baseline Isolates from the SAB/SAIE Study 152212105037183136172 Patient Number (Daptomycin-treated) Total Plasma AUC for 3 log 10 Reduction (µg·hr/mL) 0 250 500 750 1000 1250 1500 Baseline Post-baseline 1100 537 442 651 413 1420 713 Patient-specific AUC

11. C-11 Daptomycin Penetration into Simulated Endocardial Vegetations Daptomycin penetrates into rat fibrin clots; exerts bactericidal activity Daptomycin penetrates into rat fibrin clots; exerts bactericidal activity Mortin et al. ASM 2003; Tsuji and Rybak. AAC 2005; Caron et al. AAC 1992. T = 72h; 2 doses T = 72h; No treatment

12. C-12 Daptomycin Penetration into Simulated Endocardial Vegetations Daptomycin penetrates into rat fibrin clots; exerts bactericidal activity Daptomycin penetrates into rat fibrin clots; exerts bactericidal activity Daptomycin achieved efficacy at simulated 6 mg/kg dose Daptomycin achieved efficacy at simulated 6 mg/kg dose Mortin et al. ASM 2003; Tsuji and Rybak. AAC 2005; Caron et al. AAC 1992. T = 72h; 2 doses T = 72h; No treatment

13. C-13 Daptomycin Penetration into Simulated Endocardial Vegetations Daptomycin penetrates into rat fibrin clots; exerts bactericidal activity Daptomycin penetrates into rat fibrin clots; exerts bactericidal activity Daptomycin achieved efficacy at simulated 6 mg/kg dose Daptomycin achieved efficacy at simulated 6 mg/kg dose 14 C-daptomycin achieved homogenous penetration into rabbit vegetations 14 C-daptomycin achieved homogenous penetration into rabbit vegetations Mortin et al. ASM 2003; Tsuji and Rybak. AAC 2005; Caron et al. AAC 1992. T = 72h; 2 doses T = 72h; No treatment

14. C-14 Global Surveillance Data (MRSA) Study Year % Incidence 0 10 20 30 40 50 60 70 2000-20012002200320042005  0.12 0.25 0.5 1 2 MIC Jones et al. Annual Surveillance Reports. 2002-2005.

15. C-15 Microbiology Summary Additional investigations due to failures at MIC  2 Additional investigations due to failures at MIC  2 – Bacteria, drug, patient No decisive bacterial or daptomycin factors No decisive bacterial or daptomycin factors – No trends in surveillance – Difficult to select for large MIC increases – Incremental genetic changes – Modeling shows adequate drug exposure and penetration Patient-specific factors likely play a role Patient-specific factors likely play a role – Complicated infections and outcomes – Adjunctive care important – Similar observations with vancomycin