1. Download from www.cancidas.aewww.cancidas.ae Antifungal Agent from Merck Research Laboratories Caspofungin Discovery and Development of a Novel, Potent

2. Download from www.cancidas.aewww.cancidas.ae Mortality Rates of Candida and Aspergillus Infections *In transplant recipients. Edmond MB Clin Infect Dis 1999;29:239–244; Paterson DL Medicine 1999;78(2):123–138. Aspergillus up to 90% * Candida 40% Mortality

3. Download from www.cancidas.aewww.cancidas.ae Current Options in Antifungal Therapy DisadvantagesDrugAdvantages Fungizone ™ Active against Candida, Aspergillus Nephrotoxicity Potential for toxicity Nephrotoxicity Potential for toxicity Lipid formulation Active against Candida, Aspergillus, Cryptococcus Nephrotoxicity is lower than with conventional amphotericin B Active against Candida, Aspergillus, Cryptococcus Nephrotoxicity is lower than with conventional amphotericin B Fluconazole Active against Candida, Cryptococcus Ineffective against Aspergillus Potential resistance development Ineffective against Aspergillus Potential resistance development Itraconazole Active against Candida, Aspergillus, Cryptococcus Potent inhibitor of cytochrome P450 3A4 system – may cause serious cardiovascular events in combination with certain drugs, contraindicated in severe renal dysfunction. Idiosyncratic hepatitis and hepatotoxicity, resistance observed Flucytosine Selective toxicity Weak activity against Candida, Cryptococcus Rapid resistance development Weak activity against Candida, Cryptococcus Rapid resistance development Amphotericin B Azoles Nucleoside analog Andriole VT J Antimicrob Chemother 1999;44:151–162; Groll AH Adv Pharmacol 1998;44:343-500; Onishi J Antimicrob Agents Chemother 2000;44:368–377; Stone EA Clin Ther 2002;24(3):351-377; Sporanox ™ (Itraconazole) Injection Prescribing Information; Fluconazole Prescribing Information. Nephrotoxicity Potential for toxicity Rates of acute infusion-related reactions do not differ substantially from those observed with conventional amphotericin B Nephrotoxicity Potential for toxicity Rates of acute infusion-related reactions do not differ substantially from those observed with conventional amphotericin B

4. Download from www.cancidas.aewww.cancidas.ae Antifungal Screening: History of the Echinocandins Limited antifungal spectrum, low water solubility, poor oral bioavailability, hemolytic Potent in vitro and in vivo activity against Candida with low toxicity Identified by several pharmaceutical companies Cyclic hexapeptides, N-acylated with a fatty acid side chain Isolated in 1974, from Aspergillus species Groll AH Adv Pharmacol 1998;44:343–500; Bartizal K. Presented at Antibacterial & Antifungal Drug Discovery & Development Summit. March 2001; Onishi J Antimicrob Agents Chemother 2000;44(2):368–377; Tkacz JS Emerging Targets in Antibacterial and Antifungal Chemotherapy 1992:495–523; Debono M Annu Rev Microbiol 1994;48:471–497; Bartizal K Antimicrob Agents Chemother 1997;41(11):2326–2332.

5. Download from www.cancidas.aewww.cancidas.ae Screening for Natural Products Identify a Therapeutic Target Develop Assays that Look for Biological Activity Against Target Test Fungal, Bacterial, Plant, or Other Extracts for Desired Biological Activity Purify and Identify Lead Compound(s) Produce Quantities for Clinical Trials Medicinal Chemistry to Optimize Compound Characteristics Additional in vitro Testing In vivo Testing

6. Download from www.cancidas.aewww.cancidas.ae Discovery of Antifungal Activity In vitro activity against Candida was observed in a fermentation extract of Glarea lozoyensis Bartizal K. Presented at Antibacterial & Antifungal Drug Discovery & Development Summit. March 2001.

7. Download from www.cancidas.aewww.cancidas.ae A Novel Glucan Synthesis Inhibitor (L-688786) OH N N HNHN HNHN HN O O HO CH 3 OH O O NH HO N N H2NH2N H2NH2N O O OH O O O O O O O O HO O O HNHN HNHN HNHN HNHN Groll AH Adv Pharmacol 1998;44:343–500.

8. Download from www.cancidas.aewww.cancidas.ae L-688786 – Stepping Up Production

9. Download from www.cancidas.aewww.cancidas.ae Chemical Modifications Water soluble Improved potency Expanded spectrum Favorable pharmacokinetics Water soluble Improved potency Expanded spectrum Favorable pharmacokinetics Synthesizing Caspofungin (L-743872) from L-688786 HO H3NH3N H3NH3N glutamine HO O O HNHN HNHN HNHN HNHN NH O O O O O O O O N N HNHN HNHN HO O O OH O O CH 3 OH NH H3NH3N H3NH3N HN hemiaminal H 2 NOC N N 2 CH 3 COOH Bartizal K Antimicrob Agents Chemother 1997;41(11):2326–2332; Groll AH Adv Pharmacol 1998;44:343–500; Powles MA Antimicrob Agents Chemother 1998;42(8):1985–1989; Debono M Annu Rev Microbiol 1994;48:471–497; Stone EA Clin Ther 2002;24(3):351–377.

10. Download from www.cancidas.aewww.cancidas.ae Compound Nomenclature Inhibitor Class: glucan synthesis inhibitor Generic Class:lipopeptide Structural Class:echinocandin Semisynthetic derivative of:L-688786 (Pneumocandin B O ) Produced by:Glarea lozoyensis Merck L#:L-743872 Merck MK#:MK-0991 Generic Name:caspofungin acetate Trade Name:CANCIDAS ™ Inhibitor Class: glucan synthesis inhibitor Generic Class:lipopeptide Structural Class:echinocandin Semisynthetic derivative of:L-688786 (Pneumocandin B O ) Produced by:Glarea lozoyensis Merck L#:L-743872 Merck MK#:MK-0991 Generic Name:caspofungin acetate Trade Name:CANCIDAS ™ Georgopapadakou NH Exp Opin Invest Drugs 2001;10(2):269–280; Bartizal K Antimicrob Agents Chemother 1997;41(11):2326–2332; Data on file, MSD. CANCIDAS (caspofungin acetate) is a trademark of Merck & Co., Inc., Whitehouse Station, NJ, USA.

11. Download from www.cancidas.aewww.cancidas.ae  (1,3)-D-glucan: Integral Part of Fungal Cell Wall Fungal specific, not found in mammalian cells Important cell wall component of many fungal species: –Candida species mostly  -1,3 &  -1,6 (~40–60%) –Aspergillus species (hyphae mostly  -1,3) –Pneumocystis carinii (cyst form) C. neoformans mostly  -1,3 and  -1,3 (~30–50%) Fungal specific, not found in mammalian cells Important cell wall component of many fungal species: –Candida species mostly  -1,3 &  -1,6 (~40–60%) –Aspergillus species (hyphae mostly  -1,3) –Pneumocystis carinii (cyst form) C. neoformans mostly  -1,3 and  -1,3 (~30–50%) Data on file, MSD; Debono M Annu Rev Microbiol 1994;48:471–497; Tkacz JS Emerging Targets in Antibacterial and Antifungal Chemotherapy 1992:495–523; Maertens J Current Medicinal Chemistry–Anti-Infective Agents 2002;1(1); Bartizal K. Presented at Antibacterial & Antifungal Drug Discovery & Development Summit. March 2001.

12. Download from www.cancidas.aewww.cancidas.ae Phospholipid bilayer of the fungal cell membrane Fungal cell wall  (1,3)-glucan  (1,6)-glucan  (1,3)-glucan synthase Ergosterol Fungal Cell Wall Kartsonis NA. Presented at the 12th European Congress of Clinical Microbiology and Infectious Diseases. April 24–27, 2002.

13. Download from www.cancidas.aewww.cancidas.ae Glucan Synthesis and Its Inhibition

14. Download from www.cancidas.aewww.cancidas.ae Caspofungin: Preclinical In Vitro Data

15. Download from www.cancidas.aewww.cancidas.ae Caspofungin In Vitro Spectrum of Activity Candida speciesHistoplasma capsulatumCryptococcus neoformans Aspergillus species Coccidiodes imitisFusarium species Saccharomyces cerevisiaeParacoccidiodes species Trichosporon species Alternaria species Blastomyces dermatitidisRhizopus species Curvularia species Sporothrix schenckiiTrichophyton species Fonseca pedrosoiPhiolophora species Scedosporium species Pneumocystis carinii**Epidermophyton species Pseudallescheria boydii Candida speciesHistoplasma capsulatumCryptococcus neoformans Aspergillus species Coccidiodes imitisFusarium species Saccharomyces cerevisiaeParacoccidiodes species Trichosporon species Alternaria species Blastomyces dermatitidisRhizopus species Curvularia species Sporothrix schenckiiTrichophyton species Fonseca pedrosoiPhiolophora species Scedosporium species Pneumocystis carinii**Epidermophyton species Pseudallescheria boydii Potent (0.03–2.0  g/mL) Potent (0.03–2.0  g/mL) Minimum Inhibitory Concentrations* (  g/mL) *Data derived from MRL, National Fungus Testing Laboratory and National Mycology Reference Laboratory **Based on in vivo data Bartizal K. Presented at Antibacterial & Antifungal Drug Discovery & Development Summit. March 2001. Intermediate ( 2.0–16.0  g/mL) Intermediate ( 2.0–16.0  g/mL) Weak (16.0–>64.0  g/mL) Weak (16.0–>64.0  g/mL)

16. Download from www.cancidas.aewww.cancidas.ae Glucan Synthesis Inhibition vs. MIC Value Candida albicans (MY1028)0.60.125 Aspergillus fumigatus (MF4839)9.60.125 Candida albicans (MY1028)0.60.125 Aspergillus fumigatus (MF4839)9.60.125 Glucan Synthesis IC 50 (nM) Glucan Synthesis IC 50 (nM) Mean MIC µg/mL ± SE Mean MIC µg/mL ± SE Data on file, MSD.

17. Download from www.cancidas.aewww.cancidas.ae In Vitro Susceptibility C. albicans0.120.250.50 Candida spp. (non-albicans)0.500.5016.0 Aspergillus spp.0.380.43>64.0 Cryptococcus neoformans16.00.500.50 C. albicans0.120.250.50 Candida spp. (non-albicans)0.500.5016.0 Aspergillus spp.0.380.43>64.0 Cryptococcus neoformans16.00.500.50 Caspofungin AmB Fluconazole MIC 90 (  g/mL) Minimum Inhibitory Concentrations AmB = Amphotericin B Bartizal K. Presented at Antibacterial & Antifungal Drug Discovery & Development Summit. March 2001.

18. Download from www.cancidas.aewww.cancidas.ae Fluconazole-Susceptible and -Resistant Candida albicans 0.05–0.80.40 0.01–0.80.20 0.04–>400.64 0.08–102.5 0.1–0.80.40 0.02–0.40.20 0.08–1.250.64 20–8080 0.05–0.80.40 0.01–0.80.20 0.04–>400.64 0.08–102.5 0.1–0.80.40 0.02–0.40.20 0.08–1.250.64 20–8080 Range MIC 90 MIC (  g/mL) Caspofungin Amphotericin B Flucytosine Fluconazole Caspofungin Amphotericin B Flucytosine Fluconazole Caspofungin Amphotericin B Flucytosine Fluconazole Caspofungin Amphotericin B Flucytosine Fluconazole C. albicans (40) Susceptible C. albicans (10) Resistant C. albicans (40) Susceptible C. albicans (10) Resistant Vazquez JA Antimicrob Agents Chemother 1997;41(7):1612–1614. Antifungal Agent Organism (no. of isolates)

19. Download from www.cancidas.aewww.cancidas.ae Drug Combination Studies Candida albicans Cryptococcus neoformans Aspergillus fumigatus Candida albicans Cryptococcus neoformans Aspergillus fumigatus FIC* (μg/ml) MK-0991 Plus Amphotericin B 0.82 0.50 0.45 0.82 0.50 0.45 *≤0.5 = Synergistic ≤4.0 = Additive-to-indifferent ≥4.0 = Antagonistic Bartizal K. Presented at Antibacterial & Antifungal Drug Discovery & Development Summit. March 2001. Fungus (n=2) Fungus (n=2)

20. Download from www.cancidas.aewww.cancidas.ae Caspofungin: Genetics Show Target Is Essential in Candida URA3 Step 1 Or Select for FOA resistance URA3 FKS1

21. Download from www.cancidas.aewww.cancidas.ae Genetics Prove Caspofungin Target Is Essential in Candida URA3 If CaFKS1 is not essential: If CaFKS1 is essential: No functional FKS1 remains Always retain a functional copy of FKS1 Result: Candida  (1,3)-D-glucan synthase is essential Step 2 URA3 FKS1

22. Download from www.cancidas.aewww.cancidas.ae Colony Forming Unit Quantitation 10 Colony Forming Units 4 Colony Forming Units 1 Colony Forming Unit Candida spp. and Other YeastsAspergillus spp.

23. Download from www.cancidas.aewww.cancidas.ae Evaluation of Caspofungin Effect Against Aspergillus spp. In Vitro Using Vital Dyes AliveFluorescentNon-fluorescent Dead Non-fluorescentFluorescent AliveFluorescentNon-fluorescent Dead Non-fluorescentFluorescent Status of Cell Viable Stain (CFDA) Non-viable Stain (DiBAC 4 [3]) Non-viable Stain (DiBAC 4 [3]) Data on file, MSD.

24. Download from www.cancidas.aewww.cancidas.ae Viable Stain — Caspofungin Halts Growth of Aspergillus fumigatus Amphotericin B 0.15 µg/mL Caspofungin 0.30 µg/mL Itraconazole 2.5 µg/mL Control Data on file, MSD; Bowman JC Antimicrob Agents Chemother 2002;46(9):3001–3012.

25. Download from www.cancidas.aewww.cancidas.ae Non-viable Stain — Cellular Lysis in Aspergillus fumigatus Confirmed Amphotericin B 0.15 µg/mL Caspofungin 0.30 µg/mL Itraconazole 2.5 µg/mL Control Data on file, MSD; Bowman JC Antimicrob Agents Chemother 2002;46(9):3001–3012.

26. Download from www.cancidas.aewww.cancidas.ae Effects of Caspofungin on Aspergillus fumigatus Bowman JC Antimicrob Agents Chemother 2002;46(9):3001–3012.

27. Download from www.cancidas.aewww.cancidas.ae Caspofungin: Preclinical In Vivo Data

28. Download from www.cancidas.aewww.cancidas.ae In Vivo Efficacy of Caspofungin & Amphotericin B Against Candida Species in Mice Candida albicans0.020.03 Candida glabrata0.060.17 Candida lusitaniae0.160.11 Candida tropicalis0.050.24 Candida albicans0.020.03 Candida glabrata0.060.17 Candida lusitaniae0.160.11 Candida tropicalis0.050.24 Caspofungin ED (Effective Dose) 90 (mg/kg/dose) Isolate Data on file, MSD; Bartizal K. Presented at Antibacterial & Antifungal Drug Discovery & Development Summit. March 2001; Abruzzo GK Antimicrob Agents Chemother 1997;41(11):2333–2338. Amphotericin B

29. Download from www.cancidas.aewww.cancidas.ae Caspofungin, AmB* or FCZ** in DBA/2 Mice vs. Disseminated Renal Candidiasis after Delayed Treatment Days After Challenge Challenge I.V. 2 0 3 3 4 4 5 5 6 6 7 7 8 8 2 2 4 4 6 6 8 8 10 12 14 16 18 Log CFU C. albicans/g kidneys Dose I.V. q.d. x 4 Fluconazole @ 10 mg/kg 0% Sterilization Vehicle Control AmB * @ 0.5 mg/kg 40% Sterilization Caspofungin @ 0.38mg/kg 60% Sterilization *AmB = Amphotericin B **FCZ = Fluconazole Bartizal K. Presented at Antibacterial & Antifungal Drug Discovery & Development Summit. March 2001.

30. Download from www.cancidas.aewww.cancidas.ae Caspofungin vs. AmB in Chronic Pancytopenic Mouse Model of Disseminated Aspergillosis Dosing Percent Survival 20 40 60 80 100 0 0 5 5 10 15 20 25 Days Postinfection Day +28 Day –3 Immunosuppression with Cyclophosphamide 1 mg/kg 0.25 mg/kg 1 mg/kg 0.25 mg/kg Sham-Rx Caspofungin AmB Data on file, MSD.

31. Download from www.cancidas.aewww.cancidas.ae Disseminated Candidiasis in Chronically Pancytopenic Mice: Survival Dosing Percent Survival 0 0 20 30 40 50 60 70 80 90 100 0 0 4 4 8 8 12 16 20 24 1 mg/kg 0.25 mg/kg Days After Challenge 1 mg/kg 0.25 mg/kg Sham-Rx Day +28 Day –3 Immunosuppression with Cyclophosphamide Caspofungin AmB 10 28 80 mg/kg 20 mg/kg Fluconazole Data on file, MSD.

32. Download from www.cancidas.aewww.cancidas.ae Disseminated Candidiasis in Chronically Pancytopenic Mice: Tissue Burden and Sterilization Caspofungin 1 mg/kg  4.84100 0.25 mg/kg  3.1340 Amphotericin B 1 mg/kg  3.5280 0.25 mg/kg  2.4950 Fluconazole 80 mg/kg-0.9220 5 20 mg/kg+0.750 1 Caspofungin 1 mg/kg  4.84100 0.25 mg/kg  3.1340 Amphotericin B 1 mg/kg  3.5280 0.25 mg/kg  2.4950 Fluconazole 80 mg/kg-0.9220 5 20 mg/kg+0.750 1 Log 10 CFU Reduction % Kidney Tissue Sterilization Kidney Burden Reduction from Control at Day 28* Kidney Burden Reduction from Control at Day 28* *Mice were challenged IV with C. albicans MY1055 at 5.6  10 4 CFU/mouse and 1.22  10 5 CFU/mouse. Kidneys aseptically collected at Days 14 and 28 after challenge. Mean log 10 CFU/g at time points after challenge for paired kidneys. Ten mice per group unless indicated by superscript number. Data on file, MSD.

33. Download from www.cancidas.aewww.cancidas.ae P. carinii Cyst Clearance in Mice with Caspofungin Control TMP-SMZ* MK-0991 90% Reduction 99% Reduction 7 7 6 6 5 5 4 4 3 3 0 0 2 2 4 4 6 6 8 8 10 12 14 16 Log 10 Mean Number of Cysts/Lung Days of Treatment *TMP-SMZ = Trimethoprim-sulfamethoxazole. Powles MA Antimicrob Agents Chemother 1998;42(8):1985–1989.

34. Download from www.cancidas.aewww.cancidas.ae Preclinical Microbiology Summary Spectrum of activity includes Candida albicans, non-albicans Candida spp., and Aspergillus spp. Caspofungin is fungicidal for Candida spp. based on in vitro and in vivo studies Caspofungin demonstrates clear activity against Aspergillus spp. –In vitro Kills cells with active cell wall synthesis Effects are consistent with the mechanism of action –In vivo, there is a sustained activity in severely immunosuppressed mice with disseminated aspergillosis Spectrum of activity includes Candida albicans, non-albicans Candida spp., and Aspergillus spp. Caspofungin is fungicidal for Candida spp. based on in vitro and in vivo studies Caspofungin demonstrates clear activity against Aspergillus spp. –In vitro Kills cells with active cell wall synthesis Effects are consistent with the mechanism of action –In vivo, there is a sustained activity in severely immunosuppressed mice with disseminated aspergillosis Data on file, MSD; Maertens J Current Medicinal Chemistry–Anti-Infective Agents 2002;1(1); Bowman JC Antimicrob Agents Chemother 2002;46(9):3001–3012.

35. Download from www.cancidas.aewww.cancidas.ae Additional In Vivo Studies Reduction of infections in CD4 + deficient mice with chronic oropharyngeal and gastrointestinal mucosal candidiasis Effective prophylactic and therapeutic treatment of immunocompromised rats with pulmonary aspergillosis Limited role in the treatment of Histoplasma capsulatum No activity against Cryptococcus neoformans Reduction of infections in CD4 + deficient mice with chronic oropharyngeal and gastrointestinal mucosal candidiasis Effective prophylactic and therapeutic treatment of immunocompromised rats with pulmonary aspergillosis Limited role in the treatment of Histoplasma capsulatum No activity against Cryptococcus neoformans Flattery AM Antimicrob Agents Chemother 1996:40(7):1604–1609; Data on file, MSD; Stone EA Clin Ther 2002; 24(3):351–377.

36. Download from www.cancidas.aewww.cancidas.ae Comparative Pharmacokinetics: Caspofungin Single IV Dose 100 10 1 1 0.1 0 0 4 4 8 8 12 16 20 24 Mean Plasma Concentration (  g/ml) Time (Hours) C. albicans MIC 90 Candida spp., A. fumigatus MIC 90 0.5 mg/kg 0.75 mg/kg 1 mg/kg Mouse7.6 Rat6.1 Rhesus5.0 Chimpanzee5.2 Human10.0 Species T 1/2 hrs Bartizal K. Presented at Antibacterial & Antifungal Drug Discovery & Development Summit. March 2001.

37. Download from www.cancidas.aewww.cancidas.ae Merck Antifungal Caspofungin (MK-0991) Preclinical Summary Water soluble  (1,3)-D-glucan synthesis inhibitor: –0.6 nM–Candida –9.6 nM–Aspergillus Relevant spectrum: –Candida & Aspergillus Fungicidal against C. albicans Low resistance induction potential Effective vs. FCZ*, AmB**, 5FC*** resistors No antagonism –In combination with AmB** or FCZ* Excellent animal efficacy: –Candida, Aspergillus Pharmacokinetics supports once-daily dosing Water soluble  (1,3)-D-glucan synthesis inhibitor: –0.6 nM–Candida –9.6 nM–Aspergillus Relevant spectrum: –Candida & Aspergillus Fungicidal against C. albicans Low resistance induction potential Effective vs. FCZ*, AmB**, 5FC*** resistors No antagonism –In combination with AmB** or FCZ* Excellent animal efficacy: –Candida, Aspergillus Pharmacokinetics supports once-daily dosing OH N N NHNH NHNH HNHN HNHN HN O O NH CH 3 OH O O HNHN HNHN NH HO N N O O OH O O O O O O O O H2NH2N H2NH2N H2NH2N H2NH2N ·2CH 3 CO 2 H CH 3 H3CH3C H3CH3C *FCZ = fluconazole **AmB = amphotericin B ***5FC = flucytosine Data on file, MSD; Franzot SP Antimicrob Agents Chemother 1997;41(2):331–336; Bartizal K Antimicrob Agents Chemother 1997;41(11):2326–2332.

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