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Antifungal Agent from Merck Research Laboratories

Caspofungin. Discovery and Development of a Novel, Potent. Antifungal Agent from Merck Research Laboratories. Mortality Rates of Candida and Aspergillus Infections. Mortality. Candida 40%. Aspergillus up to 90% *. *In transplant recipients.

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Antifungal Agent from Merck Research Laboratories

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  1. Caspofungin Discovery and Development of a Novel, Potent Antifungal Agent from Merck Research Laboratories

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

  3. Current Options in Antifungal Therapy Drug Advantages Disadvantages Amphotericin B Fungizone™ Active against Candida, Aspergillus Nephrotoxicity Potential for toxicity Lipid formulation Active against Candida, Aspergillus, Cryptococcus Nephrotoxicity is lower than 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 Azoles Fluconazole Active against Candida, Cryptococcus 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 Nucleoside analog Flucytosine Selective toxicity Weak activity against Candida, Cryptococcus Rapid resistance development 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) InjectionPrescribing Information; Fluconazole Prescribing Information.

  4. Isolated in 1974, from Aspergillus species Cyclic hexapeptides, N-acylated with afatty acid side chain Identified by severalpharmaceutical companies Potent in vitro and in vivo activityagainst Candida with low toxicity Limited antifungal spectrum, low water solubility, poor oral bioavailability, hemolytic Antifungal Screening: History of the Echinocandins 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. 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) Medicinal Chemistry to Optimize Compound Characteristics In vivoTesting Additionalin vitro Testing Produce Quantities for Clinical Trials

  6. Discovery of Antifungal Activity In vitro activity against Candida was observedin a fermentation extract of Glarea lozoyensis Bartizal K. Presented at Antibacterial & Antifungal Drug Discovery & Development Summit. March 2001.

  7. A Novel Glucan Synthesis Inhibitor(L-688786) HO OH O O HO H N H N O O N OH HN O H2N O HO CH3 NH N O H N HO OH O OH HO Groll AH Adv Pharmacol 1998;44:343–500.

  8. L-688786 – Stepping Up Production

  9. Chemical Modifications Synthesizing Caspofungin (L-743872) from L-688786 • Water soluble • Improved potency • Expanded spectrum • Favorablepharmacokinetics H3N NH hemiaminal HO OH O O HO H N H N O N OH HN H2NOC O O CH3 HO NH glutamine N O H N HO OH H3N O HO • 2 CH3COOH HO 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. Compound Nomenclature Inhibitor Class: glucan synthesis inhibitor Generic Class: lipopeptide Structural Class: echinocandin Semisynthetic derivative of: L-688786 (Pneumocandin BO) 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.  (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%) 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. Fungal Cell Wall  (1,6)-glucan Fungal cell wall  (1,3)-glucan Phospholipid bilayer of the fungal cell membrane Ergosterol  (1,3)-glucan synthase Kartsonis NA. Presented at the 12th European Congress of Clinical Microbiology and Infectious Diseases. April 24–27, 2002.

  13. Glucan Synthesis and Its Inhibition

  14. Caspofungin: Preclinical In Vitro Data

  15. Caspofungin In Vitro Spectrum of Activity Minimum Inhibitory Concentrations* (g/mL) Potent (0.03–2.0 g/mL) Intermediate (2.0–16.0 g/mL) Weak (16.0–>64.0 g/mL) Candida species Histoplasma capsulatum Cryptococcus neoformans Aspergillus species Coccidiodes imitis Fusarium species Saccharomyces cerevisiae Paracoccidiodes species Trichosporon species Alternaria species Blastomyces dermatitidis Rhizopus species Curvularia species Sporothrix schenckii Trichophyton species Fonseca pedrosoi Phiolophora species Scedosporium species Pneumocystis carinii** Epidermophyton species Pseudallescheria boydii *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.

  16. Glucan Synthesis Inhibition vs. MIC Value Glucan Synthesis IC50 (nM) Mean MIC µg/mL ± SE Candida albicans (MY1028) 0.6 0.125 Aspergillus fumigatus (MF4839) 9.6 0.125 Data on file, MSD.

  17. In Vitro Susceptibility Minimum Inhibitory Concentrations MIC90 (g/mL) Caspofungin AmB Fluconazole C. albicans 0.12 0.25 0.50 Candida spp. (non-albicans) 0.50 0.50 16.0 Aspergillus spp. 0.38 0.43 >64.0 Cryptococcus neoformans 16.0 0.50 0.50 AmB = Amphotericin B Bartizal K. Presented at Antibacterial & Antifungal Drug Discovery & Development Summit. March 2001.

  18. Fluconazole-Susceptible and -Resistant Candida albicans MIC (g/mL) Organism(no. of isolates) Antifungal Agent Range MIC90 C. albicans (40) Susceptible C. albicans (10) Resistant Caspofungin Amphotericin B Flucytosine Fluconazole Caspofungin Amphotericin B Flucytosine Fluconazole 0.05–0.8 0.40 0.01–0.8 0.20 0.04–>40 0.64 0.08–10 2.5 0.1–0.8 0.40 0.02–0.4 0.20 0.08–1.25 0.64 20–80 80 Vazquez JA Antimicrob Agents Chemother 1997;41(7):1612–1614.

  19. Drug Combination Studies FIC* (μg/ml)MK-0991 PlusAmphotericin B Fungus (n=2) Candida albicans Cryptococcus neoformans Aspergillus fumigatus 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.

  20. Caspofungin: Genetics Show Target Is Essential in Candida Step 1 URA3 FKS1 FKS1 FKS1 URA3 Or URA3 Select for FOA resistance

  21. Genetics Prove Caspofungin Target Is Essential in Candida Step 2 URA3 FKS1 FKS1 FKS1 If CaFKS1is not essential: No functional FKS1 remains URA3 URA3 If CaFKS1is essential: Always retain a functional copy of FKS1 Result: Candida  (1,3)-D-glucan synthase is essential

  22. Colony Forming Unit Quantitation Candida spp. and Other Yeasts Aspergillus spp. 10 Colony Forming Units 1 Colony Forming Unit 4 Colony Forming Units 1 Colony Forming Unit

  23. Evaluation of Caspofungin Effect Against Aspergillus spp. In Vitro Using Vital Dyes Viable Stain(CFDA) Non-viable Stain (DiBAC4[3]) Status of Cell Alive Fluorescent Non-fluorescent Dead Non-fluorescent Fluorescent Data on file, MSD.

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

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

  26. Effects of Caspofungin on Aspergillus fumigatus Bowman JC Antimicrob Agents Chemother 2002;46(9):3001–3012.

  27. Caspofungin: Preclinical In Vivo Data

  28. In Vivo Efficacy of Caspofungin & Amphotericin B Against Candida Species in Mice ED (Effective Dose)90 (mg/kg/dose) Caspofungin Amphotericin B Isolate Candida albicans 0.02 0.03 Candida glabrata 0.06 0.17 Candida lusitaniae 0.16 0.11 Candida tropicalis 0.05 0.24 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.

  29. 8 7 6 5 4 3 18 2 4 6 8 10 12 14 16 Caspofungin, AmB* or FCZ** in DBA/2 Mice vs. Disseminated Renal Candidiasis after Delayed Treatment Fluconazole @ 10 mg/kg0% Sterilization Vehicle Control AmB* @ 0.5 mg/kg40% Sterilization Caspofungin @ 0.38mg/kg60% Sterilization Log CFU C. albicans/g kidneys Dose I.V. q.d. x 4 2 0 Days After Challenge Challenge I.V. *AmB = Amphotericin B **FCZ = Fluconazole Bartizal K. Presented at Antibacterial & Antifungal Drug Discovery & Development Summit. March 2001.

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

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

  32. Disseminated Candidiasis in Chronically Pancytopenic Mice: Tissue Burden and Sterilization Kidney Burden Reduction from Control at Day 28* Log10 CFU Reduction % Kidney Tissue Sterilization Caspofungin 1 mg/kg -4.84 100 0.25 mg/kg -3.13 40 Amphotericin B 1 mg/kg -3.52 80 0.25 mg/kg -2.49 50 Fluconazole 80 mg/kg -0.92 205 20 mg/kg +0.75 01 * Mice were challenged IV with C. albicans MY1055 at 5.6  104 CFU/mouse and 1.22  105 CFU/mouse. Kidneys aseptically collected at Days 14 and 28 after challenge. Mean log10 CFU/g at time points after challenge for paired kidneys. Ten mice per group unless indicated by superscript number. Data on file, MSD.

  33. 7 Control 6 Log10 Mean Number of Cysts/Lung TMP-SMZ* 5 90% Reduction 4 99% Reduction MK-0991 3 0 2 4 6 8 10 12 14 16 Days of Treatment P. carinii Cyst Clearance in Mice with Caspofungin *TMP-SMZ = Trimethoprim-sulfamethoxazole. Powles MA Antimicrob Agents Chemother 1998;42(8):1985–1989.

  34. 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 againstAspergillus 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. Additional In Vivo Studies • Reduction of infections in CD4+ deficient micewith 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. Comparative Pharmacokinetics:Caspofungin Single IV Dose 100 Species T1/2 hrs Mouse 7.6 Rat 6.1 Rhesus 5.0 Chimpanzee 5.2 Human 10.0 10 1 mg/kg Mean Plasma Concentration (g/ml) 0.5 mg/kg 0.75 mg/kg 1 Candida spp., A. fumigatus MIC90 C. albicans MIC90 0.1 0 4 8 12 16 20 24 Time (Hours) Bartizal K. Presented at Antibacterial & Antifungal Drug Discovery & Development Summit. March 2001.

  37. H2N NH OH O O HO H N N H O H2N N H3C HN O OH CH3 CH3 O CH3 HO NH N ·2CH3CO2H O H N HO OH O OH HO 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 *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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