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

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

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 ,  (0.15) Global Surveillance Studies ,  (0.04) 20022,  (0.08) 20034,  (0.02) 20045,  (0.04) 20056,  (0.05) Total26,654NA  (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)

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)

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)

C-6 Serial Passage Experiments: Magnitude of MIC Shifts Daptomycin Ciprofloxacin Day Fold Change [(Strain MIC/Parent MIC)-1] 0Vancomycin

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

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

C 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) 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

C-10 Efficacy in Mice Against Baseline and Post-baseline Isolates from the SAB/SAIE Study Patient Number (Daptomycin-treated) Total Plasma AUC for 3 log 10 Reduction (µg·hr/mL) Baseline Post-baseline Patient-specific AUC

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 T = 72h; 2 doses T = 72h; No treatment

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 T = 72h; 2 doses T = 72h; No treatment

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 T = 72h; 2 doses T = 72h; No treatment

C-14 Global Surveillance Data (MRSA) Study Year % Incidence  MIC Jones et al. Annual Surveillance Reports

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