SUSCEPTIBILITY PROFILE OF EMERGING FUNGAL PATHOGENS

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1 SUSCEPTIBILITY PROFILE OF EMERGING FUNGAL PATHOGENS Professor Lia Monica JUNIE, Department of Microbiology, University of Medicine and Pharmacy, Cluj Napoca, Romania

2 The most common fungal pathogens are: Candida species, Aspergillus, Cryptococcus, Coccidioides, Histoplasma; Scedosporium spp., Trichosporon spp.,. Fungal infections

3 Over the past 2 decades, The incidence of systemic fungal infections has increased dramatically Fungal infections Increase in number of immunocompromised patients transplant recipients

4 Newly developed antifungal drugs Antifungal susceptibility testing Resistance to antifungal drugs FUNGAL INFECTIONS

5 INVASIVE FUNGAL INFECTIONS - IFI Invasive Candida infections 4th most common nosocomial bloodstream infections Pathogen (%) Candida No. of Isolates/ Incidence Coagulase-negative staphylococci Staphylococcus aureus Enterococci Candida species

patients receiving chemotherapy, HIV infected patients AIDS Increased

6 Invasive candida infections the increase in the number of atrisk individuals, Immunocompromised patients transplant recipients, cancer patients receiving chemotherapy, HIV infected patients AIDS Increased use of invasive procedures urinary catheters CVC=central venous catheter

7 Invasive Candidiasis Candidemia in Neutropenic Patients with Cancer: Clinical Characteristics Patients at High Risk Neutropenic (n=217) Broad-spectrum antibiotics in previous 2 weeks Corticosteroids within previous 2 weeks Chemotherapy within previous 30 days Abdominal surgery within previous 2 months Intravenous hyperalimentation within previous 30 days Concomitant infection within previous week CVC in place at time of positive blood culture CVC=central venous catheter *In univariate analysis Adapted from Anaissie EJ et al Am J Med 1998;104: % 39% 56% 63% % with clinical characteristic* 7 90% 89% 98%

9 Species of Candida most commonly isolated in bloodstream infections C. tropicalis 8% C. parapsilosis 15% C. glabrata 16% C. krusei 2% other Candida spp. 5% C. albicans 54% The frequency of non-c. albicans species has increased over the last decade (46% of isolates).

10 Percent of patients Invasive candidiasis Mortality associated with candidemia % Patients with candidal bloodstream infections -significant morbidity and mortality - up to 90% mortality in immunocompromised patients despite treatment 25% Patients with bacterial (non-candidal) bloodstream infections

11 Effective treatment requires: an early diagnosis, to facilitate prompt initiation of therapy, broad-spectrum therapeutic agents (Antifungal drugs) with activity against both common and "emerging" pathogens. Until now, the drugs available to treat invasive fungal infections were limited by: their spectrum of activity, the development of resistance, optimal tolerability drug interaction profiles Invasive fungal infections

12 Overview of Antifungal Drugs Mechanisms of Action & of Resistance

13 Also called antimycotic drugs Used to treat two types of fungal infection: Superficial fungal infections skin or mucous membrane Systemic fungal infections lungs or central nervous system Antifungal drugs Groups: Polyenes (amphotericin B, nystatin) The antimetabolic antifungal: Flucytosine Imidazoles: ketoconazole, miconazole, clotrimazole and others Allylamines Echinocandins Griseofulvin Other drugs

14 Classification - by their site of action in fungal cells Antifungals Polyenes Imidazoles Triazole Nystatin Amphotericin B miconazole clotrimazole ketoconazole fluconazole Terbinafine itraconazole voriconazole posaconazole ravuconazole naftifine butenafine Allylamines caspofungin micafungin anidulafungin β-3-glucan synthase inhibitors ECHINOCANDINS griseofulvin Other Nucleoside analogs: Flucytosine flucytosine tolnaftate

15 Current treatment options Amphotericin B The gold standard for efficiency Wide acute and chronic side effects Azoles broad-spectrum azoles as treatment of IFIs Increased use for prophylaxis may promote the development of antifungal resistance Increasing resistance in Candida infections caused by nonalbicans species

16 Current treatment options The concomitant use of amphotericin B and an azole, principally fluconazole, is now-a-days common in clinical practice Toxicity Including nephrotoxicity, even with lipid formulations Low efficiency rates Drug resistance

18 New antifungal agents Lipid-based formulations of the polyene: amphotericin B (AmB) Improving their effect in invasive fungal infections extended-spectrum triazoles: Posaconazole (POS), Voriconazole (VRC), Ravuconazole (RAV), These agents have potent broad-spectrum activity both systemic and superficial fungal infections favorable pharmacokinetic profiles Echinocandins a newer class of agents high potential in the treatment of many fungal infections

19 RESISTANCE is.. IN VITRO MECHANISMS OF RESISTANCE CLINICAL MOLECULAR

20 Clinical Resistance is a Multifactorial Issue FUNGUS HOST Immune status Site of infection Severity of infection Foreign devices Noncompliance with drug regimen Initial MIC Cell type: Yeast/hyphae.. Genomic stability Biofilm production Population bottlenecks DRUG Fungistatic nature Dosing Pharmacokinetics Drug-drug interactions

A resistant strain may be present due to: Intrinsic resistance Replacement with: a more resistant species a more resistant strain epigenetic resistance:

21 A resistant strain may be present due to: Intrinsic resistance Replacement with: a more resistant species a more resistant strain epigenetic resistance: (Transitory gene expressions that cause temporary resistance) Alterations in cell type (?) Genomic instability within a single strain (population bottleneck)

22 Present in Mechanism Bacteria Fungi Drug inactivation X Drug modification MECHANISMS OF RESISTANCE Target mutation X X Target over expression X X Efflux pumps X X X Drug 22

Efflux-Mediated Antifungal Drug Resistance Efflux pumps all azoles appear to be substrates for the ATPdependent pumps, the level of the efflux pump expression can strongly influence the

23 Efflux-Mediated Antifungal Drug Resistance Efflux pumps all azoles appear to be substrates for the ATPdependent pumps, the level of the efflux pump expression can strongly influence the susceptibility of a cell to azoles Molecular pumps that actively push the drug out of a cell. In cells with clinically important resistance to azole drugs, high-level transcription of PDR efflux-pump genes

24 Multidrug Resistance to Antifungals

25 MODES of ACTION

26 Antifungal drugs & targets Every component of the cell wall and membrane can be targeted

27 What are the targets for antifungal therapy? Cell membrane Fungi use principally ergosterol instead of cholesterol DNA Synthesis Some compounds may be selectively activated by fungi, arresting DNA synthesis. Cell Wall Unlike mammalian cells, fungi have a cell wall

28 Cell Membrane Active Antifungals Cell membrane Polyene antifungals - Amphotericin B, lipid formulations - Nystatin (topical) Azole antifungals - Ketoconazole - Itraconazole - Fluconazole - Voriconazole - Miconazole, clotrimazole (and other topicals)

29 DNA/RNA synthesis Cell membrane Polyene antibiotics Azole antifungals DNA/RNA synthesis Pyrimidine analogues - Flucytosine Cell wall Echinocandins -Caspofungin acetate (Cancidas)

30 HOW DO THEY WORK? Mannoproteins are another potential target

Targets for antifungal activity Ergosterol (Cell membrane) Drug-ergosterol interaction Inhibition of ergosterol synthesis RNA/EF3 (Nucleic acid/protein synthesis)

31 Targets for antifungal activity Ergosterol (Cell membrane) Drug-ergosterol interaction Inhibition of ergosterol synthesis RNA/EF3 (Nucleic acid/protein synthesis) Incorporation of 5-FU in RNA Inhibition of EF3 Glucan/Chitin (Cell wall) Inhibition of glucan/chitin synthesis block the production of the β- (1,3)-glucan protein damaging the cell wall

32 Site of action of selected antifungal agents Cell membrane Membrane disrupting agents Polyenes - Amphotericin B, nystatin Ergosterol synthesis inhibitors Azoles -Allylamines -Morpholine Cell wall Glucan synthesis inhibitors β-3-glucan synthesis inhibitor - Echinocandins Chitin synthesis inhibitor - target chitin synthesis Nikkomycin and Polyoxin Nucleic acid inhibitor - Flucytosine: inhibit DNA/RNA synthesis RNA/EF3 (Nucleic acid/protein synthesis) - Incorporation of 5-FU in RNA - Inhibition of EF3 Protein synthesis inhibitors - Sordarins, Anti-mitotic (spindle disruption) - Azasordarins - Griseofulvin: inhibit fungal cell mitosis preventing cell proliferation and function

33 Polyenes 1.Amphotericin B 2. Nystatin (Mycostatin)

Fungicidal Increasing the permeability of the cell membrane by targeting ergosterol in the membrane Polyenes Amphotericin B (Fungizone) AmB Fermentation product of

34 Fungicidal Increasing the permeability of the cell membrane by targeting ergosterol in the membrane Polyenes Amphotericin B (Fungizone) AmB Fermentation product of Streptomyces nodusus Nystatin Significant nephrotoxicity Has not been developed to treat systemic fungal infections Chemical properties - amphoteric aqueous insolubility at neutral ph

35 Amphotericin B -the most widely used antifungal for systemic infections - is the most potent and broad-spectrum antifungal of all the drugs discovered in more than a century of global efforts -Active against most fungi except Aspergillus terreus, Scedosporium spp. High level of toxicity

36 FUNGISOME i.v. has the highest efficacy against all pathogenic fungi

37 Binds sterols (ergosterol) in fungal cell membrane Modify the permeability selectively to K + and Mg 2+ Creates transmembrane channel and electrolyte leakage generates pores in the membrane It is cidal Amphotericin B Mechanism of action

38 + a polyene Resistance may develop from altered sterols or decreased sterols ergosterol Amphotericin B ergosterol with pore

39 Ribbon-like particles Carrier lipids: DMPC, DMPG Particle size (µm): Lipid Amphotericin B Formulations Abelcet ABLC Amphotec ABCD Ambisome L-AMB DMPC-Dimyristoyl phospitidylcholine DMPG- Dimyristoyl phospitidylcglycerol - AmB lipid complex - AmB colloidal dispersion - Liposomal AmB Disk-like particles Carrier lipids: Cholesteryl sulfate Particle size (µm): HSPC-Hydrogenated soy phosphatidylcholine DSPG-Distearoyl phosphitidylcholine Unilaminar liposome Carrier lipids: HSPC, DSPG, cholesterol Particle size (µm) : 0.08

40 Lipid formulations of polyenes invasive fungal infections in patients refractory or intolerant to standard AmB The Liposomal formulation Improve the therapeutic index for polyene macrolides from 33% to 76% bring down nephrotoxicity from 60% to 20% Effective in treating Candida spp grow as biofilms Broad spectrum Candida spp. C neoformans Aspergillus spp. Less toxicity in vivo Disadvantages Increased cost Not active for dermatophytes infections

Resistance to Amphotericin B does not emerge during treatment for invasive aspergillosis Aspergillus nidulans is frequently resistant to amphotericin B In vivo resistance

41 Resistance to Amphotericin B does not emerge during treatment for invasive aspergillosis Aspergillus nidulans is frequently resistant to amphotericin B In vivo resistance is possible for: C. lusitaniae, C. krusei C. neoformans Trichosporon spp. A. terreus S. apiospermum Fusarium spp. Technical difficulties in detection of resistance in vitro

Mechanisms of Amphotericin B Resistance Reduced ergosterol content (defective ERG2 or ERG3 genes) Alterations in sterol content (fecosterol, episterol: reduced affinity) in sterol to

42 Mechanisms of Amphotericin B Resistance Reduced ergosterol content (defective ERG2 or ERG3 genes) Alterations in sterol content (fecosterol, episterol: reduced affinity) in sterol to phospholipid ratio Reorientation or masking of ergosterol Stationary growth phase Previous exposure to azoles (?) Disruption of Ergosterol Biosynthesis - Resistance to Amphotericin B in Candida lusitaniae

43 Antimetabolites Restricted spectrum of activity. F H N N NH 2 Flucytosine O

44 FLUCYTOSINE (5-fluorocytosine) 1.taken up into the fungal cell by means of permease Cytosine permease 5-FC cytosine deaminase 5-FU 5-FU 5-FU uracil phosphoribosyl FUMP 5-fluorodeoxyuridine monophosphate thymidylate synthase inhibitor inhibits DNA synthesis transferase (UPRTase) phosphorylation 2. converted to 5- fluorouracil (5-FU) by cytosine deaminase 5-fluorouridilic acid (FUMP) 3. synthesized to 5-FUTP 5-fluoro-UTP incorporated in the RNA inhibits the protein synthesis

5-flucytosine (outside) 1) Decreased uptake (permease activity) Loss of permease activity 2) Altered 5-FC metabolism Loss of cytosine deaminase activity Decrease in the activity of UMP

45 5-flucytosine (outside) 1) Decreased uptake (permease activity) Loss of permease activity 2) Altered 5-FC metabolism Loss of cytosine deaminase activity Decrease in the activity of UMP pyrophosphorylase activity (UPRTase) Molecular Aspects FCY genes (FCY1, FCY2) encode for UPRTase low UPRTase activity Mechanisms of Resistance to Flucytosine permease 5-flucytosine (inside) 5-FU eventually inhibits thymidylate synthetase RNA 5dUMP (inhibits thymidylate synthase) 5-FUMP Acquired Resistance. Cytosine deaminase 5-fluorouracil Phosphoribosyl transferase FLUCYTOSINE works by inhibiting protein and DNA what? It has two mechanisms of action.

inhibit specific enzymes The target site: 14-α-sterol demethylase lanosterol demethylase cytochrome P-450 - dependent enzyme CYP450 3A-dependent C14-alpha-demethylase, This enzyme is critical

46 inhibit specific enzymes The target site: 14-α-sterol demethylase lanosterol demethylase cytochrome P dependent enzyme CYP450 3A-dependent C14-alpha-demethylase, This enzyme is critical for the synthesis of ergosterol Inhibits CYP450 enzyme responsible for ergosterol synthesis; Azole, allylamines & morpholines mechanism of action damages cytoplasmic membrane Ergosterol synthesis

47 Squalene monooxygenase 14-a-demethylase Acetyl CoA Squalene Squalene-2,3 oxide Lanosterol (ergosterol) Mechanism of action Allylamine drugs Azoles

Monistat, Micatin) Triazoles: (a type of azole)

48 AZOLES: Imidazoles: Fluconazole (Diflucan) Itraconazale (Sporanox) Ketoconazole ( Sporanox) Miconazole nitrate ( Monistat, Micatin) Triazoles: (a type of azole) Voriconazole, Posaconazole, Ravuconazole Chemistry Ketoconazole Fluconazole

inhibit the synthesis of ergosterol by blocking

ergosterol - Accumulation of toxic sterols -

Action TERB Weaker effects in human than in

50 inhibit the synthesis of ergosterol by blocking demethylation (14-demethylase) of lanosterol - inhibit fungal demethylase also inhibit cytochrome activity resulting in: - Depletion of ergosterol - Accumulation of toxic sterols - Damage to cytoplasmic membrane Mechanism of Action TERB Weaker effects in human than in fungal cells contribute to the favorable tolerability of the triazole antifungals

51 Well-known particularly for fluconazole Data available also for other azoles A significant clinical problem Resistance to Azoles

Molecular mechanisms of azoles resistance The azoles inhibit lanosterol 14-a demethylase (ERG11) blocking the formation of ergosterol Lanosterol 14-a demethylase is encoded by the gene ERG11

52 Molecular mechanisms of azoles resistance The azoles inhibit lanosterol 14-a demethylase (ERG11) blocking the formation of ergosterol Lanosterol 14-a demethylase is encoded by the gene ERG11 Several genetic alterations have been identified that are associated with the ERG11 gene of C. albicans, Target enzyme modification The azoles then inhibit Erg11, blocking the formation of ergosterol

Molecular mechanisms of azole resistance Single point mutation of ERG11 gene (in the coding region), Altered lanosterol (14- alpha) demethylase gene

53 Molecular mechanisms of azole resistance Single point mutation of ERG11 gene (in the coding region), Altered lanosterol (14- alpha) demethylase gene amplification (which leads to overexpression) Overexpression of ERG11 gene Increased production of lanosterol demethylase gene conversion or mitotic recombination

The CDR proteins are ABC transporters (ABCT) with both a membrane pore and two ABC domains The MDR protein is an Major Facilitator transport protein (MF) with a membrane pore ABC transporters use ATP

54 The CDR proteins are ABC transporters (ABCT) with both a membrane pore and two ABC domains The MDR protein is an Major Facilitator transport protein (MF) with a membrane pore ABC transporters use ATP as their energy source, whereas MF transporters use the proton motive force the azoles are removed from the cell by overexpression of the CDR genes (ABCT) and MDR (MF) Decreased accumulation of the azole in fungal cell concentrations within the cell In a susceptible cell, azole drugs enter the cell through an unknown mechanism, perhaps by passive diffusion

55 Drug import (decreased permeability) Alterations in ERG3 or ERG5 genes Production of low affinity sterols Changes in sterol and/or phospholipid composition of fungal cell membrane Altered membrane sterol composition methylated sterols, such as methylfecosterol replacing ergosterol an azole-resistant and polyene-resistant C albicans mutant The more efficient removal of the azoles means that the drugs never reach their therapeutic effect

56 Mechanisms of resistance to drug action Modification of the drug itself in quantity or quality of the drug target Reduced binding to the target The resistance may result from a combination of these mechanisms Azole resistance

Heterogeneity in susceptibility to the azoles differences in activity of azoles differing binding affinities of azoles Some species of Candida

57 Heterogeneity in susceptibility to the azoles differences in activity of azoles differing binding affinities of azoles Some species of Candida different mechanisms of resistance to the azoles azole drugs - Factors in resistance In cells with clinically important resistance to azole drugs,

58 Fluconazole - spectrum Good activity against C. albicans and Cryptococcus neoformans Primary resistance Aspergillus Non-albicans Candida species more likely to exhibit primary resistance C. krusei C. glabrata C. norvegensis... Selection of resistant species or subpopulations Replacement with more resistant strain Always resistant Sometimes resistant C. krusei > C. glabrata > C. parapsilosis C. tropicalis C. kefyr

59 Efflux of fluconazole by increased expression of the multidrug resistance transporter proteins (especially MDR1) development of fluconazole resistance in some Candida species Fluconazole - resistance - altered demethylase or by enhanced removal from the fungal cell

60 Energy-dependent efflux systems (pumps) overexpression of genes regulating efflux pumps high-level transcription of PDR efflux-pump genes involves the recruitment of RNA polymerase II, which depends on a druginduced interaction between the ScPdr1p/Pdr3p and mediator complexes. The efflux pumps reduce the intracellular concentration of the drug below that required to inhibit the azole target Erg11p, allowing normal cell growth

Secondary resistance seen in patients with AIDS received long-term fluconazole therapy Genetic mutation Upregulation of efflux pumps Increase in mrna levels of CDR1 or MDR1 genes The CDR

61 Secondary resistance seen in patients with AIDS received long-term fluconazole therapy Genetic mutation Upregulation of efflux pumps Increase in mrna levels of CDR1 or MDR1 genes The CDR pumps are effective against many azole drugs, while MDR appears to be specific for fluconazole Fluconazole - resistance Secondary Resistance to Fluconazole in C. albicans, C. dubliniensis...

Voriconazole A synthetic derivative of fluconazole Substitution of a triazole group with a fluoropyrimidine moiety increase potency and in vivo efficacy Addition of a methyl group to the propyl

62 Voriconazole A synthetic derivative of fluconazole Substitution of a triazole group with a fluoropyrimidine moiety increase potency and in vivo efficacy Addition of a methyl group to the propyl backbone increasing the affinity of the drug for the target enzyme (14-α-sterol demethylase) Broad-spectrum in vitro activity Ravuconazole is similar to fluconazole with a thiazole in the place of a second triazole

Dermatophytes Voriconazole & Ravuconazole Voriconazole In vivo studies Effective in various

63 The similarity of MIC values similar modes of action similar mechanisms of resistance Broad spectrum of activity against Aspergillus spp. C neoformans Candida spp. Trichosporon spp. Dermatophytes Voriconazole & Ravuconazole Voriconazole In vivo studies Effective in various animal models disseminated aspergillosis invasive pulmonary aspergillosis systemic candidiasis

64 Azole cross-resistance cross-resistance between some azoles despite apparent structural similarities Reduced susceptibility of fluconazole-resistant isolates of Candida spp. to voriconazole and itraconazole an indication that azole cross-resistance is developing specific to isolates of C. tropicalis cross-resistance can be species-specific Cross-resistance to itraconazole, miconazole, and voriconazole Potential cross-resistance of itraconazole with fluconazole isolates of C neoformans Further studies into azole susceptibility patterns are required

Posaconazole An analogue of itraconazole with a 1,3-dioxolone backbone In comparative studies against subsets of the isolates, broad antifungal spectrum of activity a useful agent for patients with

66 Posaconazole An analogue of itraconazole with a 1,3-dioxolone backbone In comparative studies against subsets of the isolates, broad antifungal spectrum of activity a useful agent for patients with severe systemic mycoses Aspergillus spp. Candida spp. - Invasive candidiasis, including strains resistant to fluconazole C. neoformans Trichosporon spp. Zygomycetes Dermatophytes more effective against yeast and nondermatophyte fungi

67 Not show cross-resistance with other four azoles May have a mechanism of action or resistance that are different from the other azoles not show elevations of MIC in conjunction with increased MIC values of the azoles itraconazole, miconazole, and voriconazole

from both changes in intracellular accumulation the target site

68 The molecular basis for the activity of Pos in vitro Several lines of evidence suggest that decreased susceptibility to azoles results from both changes in intracellular accumulation the target site Inhibits CYP3A4 Mapping of C. albicans mutations in azole-resistant isolates

The Extent of Cross-Resistance among POS, FLU and VOR the long side chain of POS, a side chain that is absent in VRC and FLC, helps stabilize binding to CYP51 this appears to be particularly true for

69 The Extent of Cross-Resistance among POS, FLU and VOR the long side chain of POS, a side chain that is absent in VRC and FLC, helps stabilize binding to CYP51 this appears to be particularly true for CYP51 proteins with mutations close to the active site Isolates of C. albicans that are resistant to FLU or VOR may be susceptible to POS No cross-resistance between FLU and POS or VOR among isolates of C. krusei 69 Pfaller MA et al. J Clin Microbiol. 2008;46:

70 Effect of the extended side chain on cross resistance In clinical isolates, mutations that involve the MIC of fluconazole and voriconazole (no side chain) do not give cross-resistance to posaconazole (extended side chain) For Candida albicans, even multiple mutations in the target have less of an effect on posaconazole than on fluconazole or voriconazole Clinical isolate Number of mutations MIC (µg/ml) Posaconazole Fluconazole Voriconazole Candida albicans Control strain C C > C >256 >16 C C >64 4 Xiao L et al. Antimicrob Agents Chemother. 2004;48:568. Li X et al. J Antimicrob Chemother. 2004;53:74.

71 The broad activity spectrum of Posaconzole prevents resistance The broad activity spectrum of posaconazole prevent - the colonization by resistant organisms Low MIC values for posaconazole against many fungi also prevent resistance, as pathogens cannot persist in the presence of drug A flavus A fumigatus A niger A terreus B dermatitidis C albicans C krusei C glabrata Other Candida spp C neoformans F solani Coccidioides spp H capsulatum S apiospermum Fluconazole X X +/- X X X X Itraconazole X X X X X +/- X X X X X X Voriconazole X X X X X X X X X X X +/- X X ND Posaconazole X X X X X X X X X X X +/- X X X X Trichophyton spp Zygomycetes ND, not determined. Sabatelli F et al. Antimicrob Agents Chemother. 2006;50:2009.

Resistance to an Antifungal Agent Does Not Indicate Cross-Resistance to an Antifungal Class Most clinical isolates of itraconazole-resistant Aspergillus spp.

72 Resistance to an Antifungal Agent Does Not Indicate Cross-Resistance to an Antifungal Class Most clinical isolates of itraconazole-resistant Aspergillus spp. retained sensitivity to posaconazole Posaconazole MIC (µg/ml) > > Itraconazole MIC (µg/ml) Pfaller MA et al. J Clin Microbiol. 2008;46:2568.

73 Why is this important? 36% of drugs are metabolized by CYP 3A4 and antifungals are largely 3A4 inhibitors Antifungals can effect up to 60% of all drugs due to inhibition of 3A4, 2C9, 2C19, 1A2.

74 Cell Wall Active Antifungals Cell membrane Polyene antibiotics Azole antifungals DNA/RNA synthesis Pyrimidine analogues - Flucytosine Cell wall Echinocandins -Caspofungin acetate (Cancidas)

The Fungal Cell Wall b1,3 b1,6 glucans Cell membrane Glucan/Chitin (Cell wall) Inhibition of glucan / chitin synthesis antifungals interfere with fungal cell wall synthesis by

75 The Fungal Cell Wall b1,3 b1,6 glucans Cell membrane Glucan/Chitin (Cell wall) Inhibition of glucan / chitin synthesis antifungals interfere with fungal cell wall synthesis by inhibition of ß-(1,3) D-glucan synthase Loss of cell wall glucan results in osmotic fragility TARGETS for antifungal activity b1,3 glucan synthase mannoproteins ergosterol chitin

76 Echinocandins and pneumocandins In vitro susceptibility testing Fungicidal activity Candida species including nonalbicans isolates resistant to fluconazole C albicans C tropicalis C glabrata Aspergillus spp. Caspofungin limited activity against C. neoformans contains little or no β-(1,3)- D-glucan synthase In vivo studies Caspofungin in animal models Candida Aspergillus Histoplasma Pneumocystis carinii

77 ECHINOCANDINS Cyclic lipopeptide antibiotics Inhibition of the fungal cell wall β-(1-3) glucan synthesis Inhibitors of β-(1,3)-dglucan synthase Secondary reduction in ergosterol & lanosterol Increase in chitin

Echinocandins and pneumocandins β-glucan synthase inhibitors Caspofungin (MK-0991) Mycafungin (FK463) Substrate 3A4 minor; weak inhibitor of 3A4 Increased levels of nifedipine Cmax and AUC 42% and

78 Echinocandins and pneumocandins β-glucan synthase inhibitors Caspofungin (MK-0991) Mycafungin (FK463) Substrate 3A4 minor; weak inhibitor of 3A4 Increased levels of nifedipine Cmax and AUC 42% and 18% and sirolimus AUC 21% Anidulafungin (LY303366) Phase III trials for esophageal candidiasis Phase II studies for invasive candidiasis Not inhibitor/inducer/substrate of CYP Cyclosporine induced AUC 22% Monitor effectiveness in antifungal treatment

80 Echinocandins act at the apical tips of Aspergillus hyphae - Kills hyphae at their growth tips and branching points - Buds fail to seperate from the mother cell - Yields osmotically sensitive fungal cells Bowman et al. Antimicrob Agent Chemother 2002;46:

81 Echinocandins and pneumocandins have the potential to provide a superior efficacy versus current agents This novel mechanism of action may have particular value in the treatment of resistant fungal strains The unique, specific action mechanism of caspofungin results in a low potential for mechanism-based toxicities O H 2 N OH H HO H 3 C H H HO HO O H H H O N NH OH H H H HO NH O NH H O NH O OH H NH O H N H CH 3 OH H H OH

82 PRIMARY C. neoformans Fusarium spp. SECONDARY (?) Resistant mutants due to therapy are not available. Resistance to Echinocandins They show potent MIC and epidemiological cutoff values against susceptible Candida and Aspergillus isolates, and the frequency of resistance is low

83 FKS1 encodes glucan synthase GNS1 encodes an enzyme involved in fatty acid elongation mutations in FKS1 or GNS1 Other mechanisms (?) Echinocandin Resistance Molecular Aspects

84 Caspofungin (MK-0991) the first of the echinocandins to receive FDA approval in January 2001 Metabolized by hydrolysis and N-acetylation Not inhibitor/inducer/substrate of CYP

85 invasive aspergillosis Aspergillus infection responded to caspofungin treatment,

86 . is an anti-fungal drug that can effectively treat different types of fungal infection. This drug can be administered intravenously Caspofungin - MSD

87 Evaluation of caspofungin treatment of invasive fungal infections caspofungin was an effective treatment in invasive aspergillosis after thoracic transplantations.

90 Nıkkomycın competitive inhibitors of fungal chitin-synthase enzymes Yet investigational

91 New antifungal agents Pradimicinsbenanomicins bind to cell wall mannoproteins causing osmotic sensitive lysis and cell death Allylamines/thiocarbam ates non-competitive inhibitors of squalene epoxidase Cationic peptides bind to ergosterol and cholesterol and lead to cell lysis Sordarıns, Azasordarıns inhibit protein synthesis, i.e. elongation factor 2 EF3: A target in protein synthesis machinery unique to FUNGI GM (sordarins) GW (azasordarins) Yet investigational

92 Agent Mechanism of action of current therapies and implications for efficacy Fungal Cell Target Activity Clinical Implications Polyenes Membrane Binds to ergosterol; causes cell death Azoles Membrane Inhibits CYP450 enzyme responsible for ergosterol synthesis; damages cytoplasmic membrane Caspofungin Wall Inhibits glucan synthesis; disrupts cell-wall structure Potent, broad-spectrum activity Activity of variable potency and spectrum Broad-spectrum antifungal activity; potential for additive effects in combination therapy

93 Mechanism of action of current antifungal drugs

94 Multidrug Resistance to Antifungals Although several drugs are available to combat oftendeadly fungal infections, many of these pathogens have acquired multidrug resistance.

95 Summary of Treatments Pathogen Primary Secondary Aspergillus fumigatus Blastomyces dermatidis Voriconazole Posaconazole Itraconazole or Amphotericin B Candida albicans Fluconazole Amphotericin B Caspofungin Posaconazole Anidulafungin Coccidioides immitis Itraconazole, Fluconazole or Amphotericin B Itraconazole, Caspofungin Amphotericin B Fluconazole Voriconazole, Itraconazole, Ketoconazole (topical many)

96 Summary of Treatments Pathogen Primary Secondary Cryptococcus neoformans Histoplasma capsulatum Amphotericin B ± Flucytosine followed by Fluconazole Itraconazole or Amphotericin B Itraconazole or Amphotericin B Fluconazole Mucomycosis Amphotericin B Posaconazole Sporothrix schenckii Amphotericin B Itraconazole * Saturated solution of potassium iodide SSKI*

97 Conclusions Cross-resistance of fungal species to antifungal drugs A potential problem to future antifungal treatment Determination of susceptibility of fungal species to antifungal agents Standardization of MIC value determination Heterogeneity in susceptibility of species to azole antifungals Differences in activity of azoles Different mechanisms of resistance to the azoles

98 Final word Currently, use of standard antifungal therapies can be limited Toxicity Low efficacy rates Drug resistance Antifungal resistance is a complex, gradual and multifactorial issue An increased understanding of antifungal drug resistance should allow the development of new diagnostic strategies to identify resistant clinical isolates, introduction of new treatment and prevention strategies to treat these resistant infections. Several uncertainties remain Molecular assays to detect resistance are not simple

99 Future Directions to Avoid Development of Resistance Proper dosing strategies Restricted and well-defined indications for prophylaxis with azoles Fungi will continue to develop NEW resistance mechanisms!..

100 Thanks for your attention

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