Novel Antifungal Agents Dr. Khaled H. Abu-Elteen Dr. Mawieh A. Hamad

1. Novel Antifungal Agents Dr. Khaled H. Abu-Elteen Dr. Mawieh A. Hamad Department of Biological Sciences Faculty of Science- Hashemite University

2. Most Important Antifungal Agents Used in Treatment of Fungal Infections Griseofulvina 1947 Amphotericin B 1957 Fluocytosine 1971 Miconazole 1978 Ketoconazole 1981 Fluconazole 1989 Itraconazole 1990 2003 Voriconazole 2003 Caspofungin

3. Polyene Macrolide Antibiotics The discovery of nystatin (fungicidin) by Rachel Brown and Elizabeth Hazen in the early 1950s had led to the isolation and characterization of numerous antibiotics. Amphotericin B (fungizone), first isolated in the 1957 from Streptomyces nodosus, an actinomycete cultured from the soil of the Orinoco Valley in Venezuela, was the first commercially available systemic antifungal drug; so far, about 200 antifungal agents of this class exist. However, problems associated with the stability, solubility, toxicity and absorption of most such compounds, cut down the number of polyenes approved for therapeutic use to only a few.

4. Polyenes are characterized by a large macrolide ring of carbon atoms closed by the formation of an internal ester of lactone (Figure 1). The macrolide ring contains 12- 37 carbon atoms, the conjugated double bond structure is contained exclusively within the cyclic lactone. A number of hydroxyl groups (6-14) are distributed along the macrolide ring on alternate carbon atoms. Amphotericin B has a free carboxyl group and a primary amine group that confer amphoteric properties on the compound, hence the drug’s name. Being amphoteric, amphotericin B tends to form channels through the cell membrane causing cell leakage.

6. Although amphotericin B remains the preferred compound for treating systemic mycoses, problems associated with solubility in water, toxicity and ineffectiveness against mold diseases in immunocompromised patients limit its therapeutic potential. Three lipid formulations of amphotericin B (amphotericin B lipid complex, amphotericin B cholesteryl sulfate and liposomal amphotericin B) have been developed and approved for use in the US. These drug delivery systems offer several advantages over conventional amphotericin B. The parent drug can be introduced in much higher doses (up to 10-fold) compared with conventional amphotericin B.

7. Mechanism of Action of Polyenes Polyene antibiotics increase cell membrane permeability, which causes leakage of cellular constituents (amino acids, sugars and other metabolites), cell lysis and death. Inhibition of aerobic and anaerobic respiration observed in cells treated with polyenes is though to be a consequence of leakage of cellular constituents. Polyenes could also cause oxidative damage to the fungal plasmalemma, which may contribute to the fungicidal activity of the drug. Inhibition of fungal growth by polyenes depends, to a large extent, on the binding of the drug to the cell; only cells that bind appreciable amounts of the drug are sensitive. Bacterial cells and protoplasts do not take up polyenes; therefore, they are resistant to the drug. Polyene antifungals selectively bind to membrane sterols; ergosterol in fungal cells and cholesterol in mammalian cells.

8. The interaction of larger polyenes like amphotericin B with fungal membrane sterols results in the production of aqueous pores consisting of an annulus of eight amphotericin B molecules linked hydrophobically to membrane sterols (Figure 6.2). This leads to the formation of pores in which the hydroxyl residues of the polyene face inwards to give an effective pore diameter of 0.4 to 1.0 nm. Leakage of vital cytoplasmic components and death of the cell follows. The selective mode of action of polyenes is also related to the differential affinity of different polyenes to membrane sterols on target cells. Amphotericin B binds with high affinity to ergosterol in fungal cell membrane.

10. PHARMACOKINETICS PHARMACODYNAMICS dose absorption high serum or tissue levels MIC KILLING SERUM TISSUE distribution LEVELS LEVELS low or absent serum or tissue levels metabolism PAFE PAFSE elimination PK PD CORRELATION (T>MIC AUC/MIC Cmax/MIC) dose optimisation TOXICITY EFFICACY RESISTANCE (?)

11. Sites of action of antifungal agents (A) Mechanism of azole action (B) Mechanism of polyene Endocellular space Ergosterolo Lanosterolo Zymosterolo Cytoplasm Cytoplasm Pore/channel formed by AmB results in cell death FLU inhibits ergosterol biosynthesis , resulting In depletion of this sterol in the cell membrane Ergosterolo Cell membrane Amphotericin B Cell wall ergosterolo fluconazolo (C) Mechanism of 5-fluorocytosine action (D) Mechanism of echinocandin action Inhibition of protein synthesis -(1,3)-D-glucan -(1,6)-D-glucan Candin 5-FUMP Cell wall Cytosine permease Cell membrane 5-FU 5-FC 5-FC Candins inhibit fungal glucan synthase cytoplasm 5-FdUMP Inhibition of DNA synthesis Mukherjee PK et al., Clin Microbiol Rev, 2005 -(1,6)-D-glucan synthase Cell membrane

12. Membrane Antifungal Agents SQUALENE Squalene epoxide LANOSTEROL 14-α-demethyl lanosterol Zymosterol Fecosterol ERGOSTEROL ALLYLAMINES: Naftifine Terbinafine Squalene epoxidase AZOLES: Ketoconazole Fluconazole Itraconazole Voriconazole Lanosterol 14-α demethylase POLYENES: Amphotericin B Nystatin

13. Structural features of Amphotericin B Hydrophilic stretch Hydrophobic stretch Mycosamine ring with Carbohydrate moiety

14. AMPHOTERICIN B DEOXYCOLATE Main side effects • Nausea and vomiting • Fever, chills • Artralgia, myalgia • Headaches • Thrombophlebitis • Nephrotoxicity • Hypotension • Cardiotoxicity • Bronchospasm Main pharmacokinetic parameters (0.5-1.0 mg/kg) • Cmax 1.2-2.4 mg/ml • T1/2 initial phase: 24-48 h • T1/2 terminal phase: 15 days • Protein binding: 91 - 95 % • Elimination: biliary-renal • Fu 24 h: 3 - 5 % • CSF/serum: 2 - 4 % Modified from Como and Dismuekes, 1994; Groll et al., 1998

15. Physicochemical information on the lipid formulations of AmB in comparison to AmB deoxycholate c - AmB L - AmB ABLC Liposomal AmB AmB lipid complex Brand name Fungizone AmBisome Abelcet Lipids (molar ratio) Deoxyc holate HPC/CHOL/DSPG DMPC/DMPG (7:3) (2:1:0.8) Mol% AmB 34% 10% 35% Lipid c onfiguration Micelles Liposomes Lipid - sheets m < 0.4 0.08 1.6 - 11.0 Diameter ( m)

16. The first Liposomal formulation of AMB is made up by a mixture of two phospholipid : Dimyristoyl phosphatidylcholine [ DMPC] and dimyristoyl phosphatidylglycerol [ DMPG] in a 7:3 molar ratio with 5-10% of the mixture being AMB.

17. Mechanisms of action Amphotericin B (AMB) Amphotericin B lipid formulations (formation of transmembrane pores) Host cell Fungal cell endosome a b Free AmB lysosome AmB-lipid complex Slow release of free AmB AmB-LDL a c d d c b endosome endosome Fungal cell Fungal cell parasite LDL=low density lipoproteins cholesterol lipid peroxidation ergosterol macrophages Hartsel S and Bolard J, TiPS, 1996

18. Antifungal activity of amphotericin B

19. Physicochemical and pharmacokinetic information on the lipid formulations of AmB in comparison to AmB deoxycholate AmB, amphotericin B deoxycholate; L-AmB, liposomal AmB; ABLC, AmB lipid complex; HPC, hydrogenated phosphatidylcholine; CHOL, cholesterol; DSPG, disteaorylphosphatidylglycerol; DMPC, dimyristoyl phosphatidylcholine; DMPG, dimyristoyl phosphatidylglycerol; SUV, small unilamellar vesicles (liposomes)

20. Azole derivatives • FIRST GENERATION – Ketoconazole • SECOND GENERATION – Fluconazole – Itraconazole • THIRD GENERATION – Voriconazole ( most widly used) – Ravuconazole (BMS-207147) – Posaconazole (SCH-56592) R-120758 SYN-2869 T-8581 VR-9746 VR-9751 (D0870)

21. The inhibition of fungal growth by azole derivatives was described in the 1940s and the fungicidal properties of N-substituted imidazoles were described in the 1960s. Clotrimazole and miconazole have proven very important in combating human fungal infections. More than 40 of the β-substituted 1-phenethylimidazole derivatives are known to be potent against fungi, dermatophytes and Gram-positive bacteria. Imidazoles and triazoles are available for treatment of systemic fungal infections. Imidazoles are five –membered ring structures containing two nitrogen atoms with a complex side chain attached to one of the nitrogen atoms. The structure of triazoles is similar but they contain three nitrogen atoms in the rings (Figure). Imidazoles in clinical use are clotrimazole, miconazole, econazole and ketoconazole. Triazole compounds approved for clinical use are itraconazole, fluconazole, voriconazole, lanconazole, ravuconazole and posaconazole

23. Mechanism of Action Antifungal activity of azoles is mediated mainly through the inhibition of a cytochrome P450–dependent enzyme involved in the synthesis of ergosterol. In eukaryotic cells, these are integral components of the smooth endoplasmic reticulum and the inner mitochondrial membrane. They contain an iron protoporphyrin moiety located at the active site and play a key role in metabolic and detoxification reactions. They interact with sterols, steroids, bile acids, phenols, alkenes, epoxides, sulfones, and soluble vitamins. Azoles activity is also manifested in inhibiting cytochrome C oxidative and peroxidative enzymes, influencing cell membrane fatty acids causing leakage of proteins and amino acids, inhibiting catalase systems, decreasing fungal adherence and inhibiting germ tube and mycelia formation

24. The principle molecular target of azoles (fluconazole, itraconazole and voriconazole) is a cytochrome P450–Erg 11P or Cyp 51P according to gene–based nomenclature. Cytochrome P450–Erg 11P catalyses the oxidative removal of the 14 α-methyl group in lanosterol and/or eburicol by P450 mono–oxygenase activity. As cytochrome P450–Erg 11P contains an iron protoporphyrin moiety located at the active site, the drug binds to the iron atom via a nitrogen atom in the imidazole or triazole ring. Inhibition of 14 α–demethylase leads to depletion of ergosterol and accumulation of sterol precursors including 14 α–methylated sterol as shown in figure 6.5. With ergosterol depleted and replaced by unusual sterols, permeability and fluidity of the fungal cell membrane is altered. Miconazole and ketoconazole can inhibit the ATPase system in the cell membrane of C.albicans and other yeasts, which may account for the rapid collapse of the electrochemical gradient and the fall in intracellular ATP.

25. Additionally, at growth inhibitory concentrations, miconazoles and ketoconazoles tend to inhibit the activity of C. albicans plasma membrane glucan synthase, chitin synthase, adenylcyclase and 5-nucleotidase enzymes. Incubation of C. albicans and other yeasts at fungistatic concentrations with clotrimazole, miconazole, econazole, voriconazole, posaconazole or ketoconazole results in extensive changes in the cell envelope especially the plasma membrane; for example, the appearance of holes in the nuclear membrane. At fungicidal concentrations however, changes in the membrane are more pronounced and include the disappearance of mitochondrial internal structures and the complete loss of the nuclear membrane. Ketoconazole can affect the transformation of C. albicans from the budding form to the pseudomycelial form, the prevailing type found in infected individuals.

26. Antifungal activity of triazoles + + + very active + + average activity +/- low activity - no activity * except C.krusei and C.glabrata ** +/- for C.krusei

27. Membrane Antifungal Agents SQUALENE Squalene epoxide LANOSTEROL 14-alpha-demethyl lanosterol Zymosterol Fecosterol ERGOSTEROL ALLYLAMINES: Naftifine Terbinafine Squalene epoxidase AZOLES: Ketoconazole Fluconazole Itraconazole Voriconazole Lanosterol 14-alpha demethylase Morpholines POLYENES: Amphotericin B Nystatin

28. Dose Ketoconazole 200 mg po Itraconazole 200 mg po Fluconazole 200 mg po Voriconazole 400mg 200mg po Bioavailability (%) 75 < 70 > 90 96 Plasma Cmax (mg/l) 1.5 - 3.1 0.2 - 0.4 10.2 2 - 4 Tmax (h) 1 - 4 4 - 5 2 - 4 1 - 2 t ½ (h) 7 - 10 24 - 42 27 - 37 6 - 9 Protein binding (%) 99 > 99 11 58 CSF penetration (%) < 10 < 1 > 70 > 60 Vd (l/kg) > 1 - 2 > 1 - 2 > 3 4 .6 Urinary recovery* (%) 2 - 4 < 1 80 - Pharmacokinetic properties of oral azole agents *as active drug Dismukes W.E., CID, 2000

29. Itraconazole Combination with cyclodextrine in the oral solution Bioavailability increased of 30-60% in HIV and/or neutropenic patients IV formulation as a complex with cyclodextrine De Beule K. & Van Gestel J.V., Drugs, 2001

30. TRIAZOLESFluconazoleItraconazolePK-PD correlation = AUC/MIC • Current data suggest an exposure-dependent pharmacodynamics • Both compounds may be most effective when adequate levels are maintained at target site Groll AH et al., 1999, 2001

31. Side Effect

33. VoriconazoleMetabolism Voriconazole is metabolised in vitro via three CYP450 isozymes CYP2C19 CYP2C9 CYP3A4 In vivo the major isozyme is CYP2C19 Genetic polymorphism in CYP2C19 HL Hoffman & R Chris Rathbun, Expert Opin Investig Drugs, 2002 FDA - Briefing document for Voriconazole - Pfizer, October 2001 courtesy of Dr. N. Wood, Pfizer Central Research

34. Voriconazole metabolism • CYP2C19 genetic polymorphism results in significant inter-subject variation in plasma levels and drug exposure – Poor metabolizers (3-5% Caucasians and Blacks, 15-20% Asians) have 3-4 fold increase in systemic exposure Heterozygous poor metabolizers have about a 2-fold increase in exposure • Genotype, age and gender result in wide inter- subject variability in exposure

35. Flucytosine Flucytosine or 5–fluorocytosine (5-FC) is a synthetic fluorinated pyrimidine used as an oral antimycotic agent. It was first synthesized in the 1950s as a spin off of work in cytostatic and antineoplastic agents. 5-FC lacks such activities but it has noticeable antifungal activity. Currently, 5-FC is used as an adjunct to amphotericin B therapy because amphotericin B potentate the uptake of 5-FC through increasing fungal cell membrane permeability. The activity of 5-FC is enhanced when used in combination with fluconazole against C. neoformans and C. albicans

36. Mechanism of Action The antifungal activity of 5-FC is mediated through one of two mechanisms: (i) disruption of DNA synthesis and /or (ii) alteration of the amino acid pool. Initially, 5-FC enters susceptible cells by means of cytosine permease, which is usually responsible for the uptake of cytosine, adenine, guanine, and hypoxanthine. Once inside the cell, 5-FC is converted to 5- fluorouracil (5FU) by cytosine deaminase. Inside target cells, 5-FU is then converted by uridine monophosphate pyrophosphorylase to 5-fluorouridylic acid (FUMP), which is phosphorylated further and incorporated into RNA resulting in disruption of protein synthesis. Extensive replacement of uracil by 5-FC in fungal RNA can lead to alterations in the amino acid pool. Some 5-FU can be converted to 5-fluorodeoxyuridine monophosphate, which functions as a potent inhibitor of thymidylate synthase, one of the enzymes involved in DNA synthesis and nuclear division.

37. Inhibition of DNA synthesis in C.albicans can take place before 5-FU incorporation into RNA or inhibition of protein synthesis. Resistant strains of C. neoformans incorporate 5-FC into RNA at levels comparable with sensitive strains. This could mean that resistance inhibition of DNA synthesis is more important than the production of aberrant RNA in mediating the effects of 5-FC. The drug incorporates in large quantities into the 80S ribosomal subunits in C. albicans. The number of ribosomes synthesized in the presence of high concentrations of 5-FC is greatly reduced. Morphological and ultrastructural changes that occur in C. albicans cells include increased cell diameter if growing at sub-inhibitory concentrations of 5-FC

38. Echinocandins The fungal cell wall contains compounds, such as mannan, chitin, and α– and β-glucans, which are unique to the kingdom Fungi. A number of compounds that have the ability to affect the cell wall of fungi have been discovered and described over the past 30 years. Of the three groups of compounds (aculeacins, echinocandins, and papulacandins) that are specific inhibitors of fungal 1-3 β-glucan synthase. Echinocandins are actively pursued in clinical trials to evaluate their safety, tolerability and efficacy against candidiasis. Discovered by random screening in the 1970s, echinocandins are fungal secondary metabolites comprising a cyclic hexapeptide core with a lipid side chain responsible for antifungal activity. A modified form of echinocandin B, cilofungin, was developed to the point of phase 2 trials, but then abandoned due to increased toxicity. In the late 1990s, three echinocandin compounds (anidulafungin, caspofungin and micafungin) entered clinical development and evaluation.

39. Caspofungin: Mechanism of Action b-(1,6)-glucan Fungal cell wall b-(1,3)-glucan Phospholipid bilayer of the fungal cell membrane b-(1,3)-glucan synthase Ergosterol • Caspofungin specifically inhibits beta (1-3)-D-glucan synthesis, essential to the cell-wall integrity of many fungi, including Aspergillus and Candida spp, thereby compromising the integrity • As a result, the fungal cell wall becomes permeable, and cell lysis • Beta (1-3)-D-glucan synthesis does not occur in human cells

40. Antifungal activity of the echinocandins * Only active against cyst form, and probably only useful for prophylaxis Denning DW, Lancet, 2003

41. CASPOFUNGINPharmacokinetic parameters in healthy adults Deresinski SC & Stevens DA, CID, 2003

42. Chitin synthesis is inhibited competitively by polyoxin and nikkomycin, nucleoside–peptide antibiotics produced by soil strains of Streptomycetes. These agents specifically inhibit chitin synthase by acting as mimics or decoys of the enzyme substrate (uridine diphosphate-N-acetylglucosamine). In vitro susceptibility testing of nikkomycins X and Z against various fungi show moderate susceptibility of C. albicans and C. neoformans to these compounds. Activity against C. albicans and C. neoformans improves significantly when nikkomycin Z is used in combination with fluconazole and itraconazole

43. Allylamines and Thiocarbamates Naftifine and terbinafine are the two major allylamines in clinical use and tolnaftate is the only thiocarbamate available for use. Naftifine is used as a topical agent while terbinafine is administered orally. These are two synthetic compounds with a chemical structure similar to naphthalene ring substituted at 1 position with an aliphatic chain. Both allylamines thiocarbamates function as noncompetitive inhibitors of squalene epoxidase, an enzyme involved in the conversion of squalene to lanosterol, which is an essential step in the synthesis fungal cell membrane. Cell death is dependent on the accumulation of squalene rather than ergosterol deficiency as high levels of squalene increase membrane permeability leading to disruption of cellular organization. Terbinafine inhibits the growth of dermatophytic fungi in vitro at concentrations of 0.01 µg/ml or lower.