Antifungal Drugs • Antifungal Susceptibility Testing • Resistance to Antifungal Drugs

SUSCEPTIBILITY PROFILE OF EMERGING FUNGAL PATHOGENS

Professor Lia Monica JUNIE, Department of Microbiology, University of Medicine and Pharmacy, Cluj Napoca, Romania

ESCMID Online Lecture Library Fungal infections • The most common fungal pathogens are: – Candida species, – Aspergillus, – Cryptococcus, – Coccidioides, – Histoplasma; – Scedosporium spp., – Trichosporon spp.,.

ESCMID Online Lecture Library Fungal infections • Over the past 2 decades, • The incidence of systemic fungal infections has increased dramatically • Increase in number of immunocompromised patients • transplant recipients © by author

ESCMID Online Lecture Library FUNGAL INFECTIONS • Newly developed antifungal drugs • Antifungal susceptibility testing • Resistance to antifungal drugs

ESCMID Online Lecture Library INVASIVE FUNGAL INFECTIONS - IFI

• Invasive Candida infections • 4th most common nosocomial bloodstream Candida infections

Pathogen (%) No. of Isolates/ Incidence

Coagulase-negative staphylococci© by author 3908 31.9 Staphylococcus aureus 1928 15.7 Enterococci 1354 11.1 CandidaESCMID species Online Lecture 934 Library 7.6

Invasive candida infections • the increase in the number of at- risk individuals, • Immunocompromised patients – transplant recipients, – cancer patients receiving chemotherapy, – HIV infected patients • AIDS – Increased use of invasive procedures © by author • urinary catheters • CVC=central venous ESCMIDcatheter Online Lecture Library

7 Invasive Candidiasis Candidemia in Neutropenic Patients with Cancer: Clinical Characteristics Patients at High Risk

Neutropenic (n=217) Broad-spectrum antibiotics 90% in previous 2 weeks Corticosteroids within 56% previous 2 weeks Chemotherapy within 98% previous 30 days Abdominal surgery within 3% previous 2 months Intravenous hyperalimentation 39% within previous 30 days Concomitant infection 63% within previous week © by author CVC in place at time 89% of positive blood culture 0 50 100 % with clinical characteristic* CVC=central venous catheter *In univariate analysis ESCMID Online Lecture Library Adapted from Anaissie EJ et al Am J Med 1998;104:238–245.

ESCMID Online Lecture Library Species of Candida most commonly isolated in bloodstream infections

C. krusei other Candida spp. 2% 5% C. tropicalis 8% C. albicans 54% C. parapsilosis 15%

C. glabrata © by author 16%

The frequency of non-C. albicans species has ESCMIDincreased over Online the last decadeLecture (46% Library of isolates). Invasive candidiasis Mortality associated with candidemia -significant morbidity and 45 mortality 40 - up to 90% mortality in 40% 35 immunocompromised 30 patients despite treatment 25

20 15 10 25% Percent of patients of patients Percent 5 © by author 0 Patients with bacterial Patients with (non-candidal) candidal bloodstream bloodstream infections ESCMID Onlineinfections Lecture Library Invasive fungal infections

• 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 © by author – drug interaction profiles

ESCMID Online Lecture Library Overview of Antifungal Drugs

Mechanisms of Action & of Resistance

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

ESCMID Online Lecture Library Classification - by their site of action in fungal cells Antifungals

β-3-glucan Polyenes Imidazoles Triazole Allylamines synthase Other inhibitors ECHINOCANDINS Nystatin miconazole fluconazole naftifine caspofungin griseofulvin

Nucleoside analogs: Amphotericin Terbinafine Flucytosine clotrimazole itraconazole micafungin B © by author flucytosine

voriconazole butenafine tolnaftate ketoconazole anidulafungin

posaconazole ESCMID Onlineravuconazole Lecture Library

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 non- albicans species © by author

ESCMID Online Lecture Library 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

ESCMID Online Lecture Library

ESCMID Online Lecture Library 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 © by author • a newer class of agents • high potential in the treatment of many fungal infections ESCMID Online Lecture Library MECHANISMS OF RESISTANCE

RESISTANCE CLINICAL is..

ESCMID Online Lecture Library Clinical Resistance is a Multifactorial Issue • FUNGUS • Initial MIC • HOST • Cell type: Yeast/hyphae.. • Immune status • Genomic stability • Site of infection • Biofilm production • Severity of infection • Population bottlenecks • Foreign devices • Noncompliance with drug • DRUG regimen © by• author Fungistatic nature • Dosing • Pharmacokinetics ESCMID Online Lecture• Drug-drug Library interactions 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 (?) © by author • Genomic instability within a single strain (population bottleneck) ESCMID Online Lecture Library MECHANISMS OF RESISTANCE Present in Drug Mechanism Bacteria Fungi

Drug inactivation X Drug modification X Target mutation X X Target over expression X X Efflux pumps X X © by author

ESCMID Online Lecture Library 22 Efflux-Mediated Antifungal Drug Resistance Efflux pumps • all azoles appear to be substrates for the ATP- dependent pumps, • the level of the efflux pump expression can strongly influence the susceptibility of a cell to azoles

© by author • Molecular pumps that In cells with clinically actively push the drug out of important resistance to azole a cell. drugs, high-level transcription ESCMID Online Lectureof PDR efflux-pump Library genes Multidrug Resistance to Antifungals

ESCMID Online Lecture Library

MODES of ACTION

ESCMID Online Lecture Library Antifungal drugs & targets

Cell membrane Fungi use principally ergosterol instead of cholesterol

DNA Synthesis Some compounds may be selectively activated by fungi, arresting DNA synthesis.

©Cell by Wallauthor Unlike mammalian cells, fungi have a cell wall

ESCMID Online Lecture Library Cell Membrane Active Antifungals

Cell membrane • Polyene antifungals - Amphotericin B, lipid formulations - Nystatin (topical)

• Azole antifungals - Ketoconazole - Itraconazole - Fluconazole © -by Voriconaz authorole - Miconazole, clotrimazole (and other topicals) ESCMID Online Lecture Library DNA/RNA synthesis

Cell membrane • Polyene antibiotics • Azole antifungals DNA/RNA synthesis • Pyrimidine analogues - Flucytosine

• Mannoproteins are another potential target ESCMID Online Lecture Library

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© by author synthesis block the production of the β- (1,3)-glucan protein damagingESCMID the cell wall Online Lecture Library

Site of action of selected antifungal agents Cell membrane Membrane disrupting agents • Polyenes Cell wall - Amphotericin B, nystatin Glucan synthesis inhibitors Ergosterol synthesis inhibitors β-3-glucan synthesis inhibitor Azoles - Echinocandins -Allylamines Chitin synthesis inhibitor -Morpholine - 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 © by - Inhibition author of EF3 Protein synthesis inhibitors - Sordarins, Anti-mitotic (spindle disruption) - Azasordarins - Griseofulvin: inhibit fungal cell mitosis preventingESCMID cell proliferation andOnline function Lecture Library Polyenes

1.Amphotericin B 2. Nystatin (Mycostatin) © by author

ESCMID Online Lecture Library Polyenes • Fungicidal • Increasing the permeability of the cell membrane by • targeting ergosterol in the membrane • Amphotericin B (Fungizone) AmB • Fermentation product of Streptomyces nodusus • Nystatin – Significant nephrotoxicity – Has not been developed to treat systemic fungal © by authorinfections

ChemicalESCMID properties - amphoteric Online aqueous Lecture insolubility atLibrary neutral pH

Amphotericin B

© by author -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 againstESCMID most fungi except Online Aspergillus Lecture terreus, Scedosporium Library spp . –High level of toxicity

FUNGISOME™ESCMID i.v. has Online the highest Lecture efficacy against Library all pathogenic fungi Amphotericin B

• Binds sterols (ergosterol) in fungal cell membrane Mechanism of action • Modify the permeability selectively to K+ and Mg2+ • Creates transmembrane channel and electrolyte leakage • generates pores in the membrane • It is cidal © by author

ESCMID Online Lecture Library ergosterol Amphotericin B

ergosterol with pore

+ a polyene

• Resistance • may develop from • altered sterols or • decreased sterols © by author

ESCMID Online Lecture Library Lipid Amphotericin B Formulations

Abelcet ® ABLC Amphotec ® ABCD Ambisome ® L-AMB - AmB lipid complex - AmB colloidal dispersion - Liposomal AmB

© by author Ribbon-like particles Disk-like particles Unilaminar liposome Carrier lipids: DMPC, Carrier lipids: Cholesteryl Carrier lipids: HSPC, DMPG sulfate DSPG, cholesterol Particle size (µm): 1.6-11 Particle size (µm): 0.12-0.14 Particle size (µm) : 0.08

DMPC-Dimyristoyl phospitidylcholineESCMID HSPC-Hydrogenated Online soy phosphatidylcholineLecture Library DMPG- Dimyristoyl phospitidylcglycerol DSPG-Distearoyl phosphitidylcholine Lipid formulations of polyenes • invasive fungal infections in • Effective in treating Candida patients refractory or spp intolerant to standard AmB • grow as biofilms • The Liposomal • Broad spectrum formulation • Candida spp. • Improve the therapeutic • C neoformans index for polyene macrolides from 33% to • Aspergillus spp. 76% – Less toxicity in vivo • bring down nephrotoxicity© by• Disadvantagesauthor from 60% to 20% – Increased cost – Not active for dermatophytes infections ESCMID Online Lecture Library 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© by author Fusarium spp.

•Technical difficulties in detection of resistance in vitro ESCMID Online Lecture Library 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 Disruption of Ergosterol ergosterol Biosynthesis - Resistance to • Stationary growth phase © by Amphotericinauthor B in Candida • Previous exposure to azoles lusitaniae • (?) ESCMID Online Lecture Library Antimetabolites

• Restricted spectrum of activity. H N O

N F

NH2

Flucytosine

ESCMID Online Lecture Library 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-fluorodeoxyuridine 2. converted to 5- monophosphate fluorouracil (5-FU) by thymidylate synthase inhibitor cytosine deaminase inhibits DNA synthesis

5-FU uracil phosphoribosyl 5-fluorouridilic acid (FUMP) transferase (UPRTase)© by author3. synthesized to 5-FUTP FUMP phosphorylation 5-fluoro-UTP incorporated in the RNA ESCMID Online Lectureinhibits the Libraryprotein synthesis

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

Azole, allylamines & morpholines mechanism 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 for the synthesis of ergosterol • Inhibits CYP450 enzyme responsible © by author for ergosterol synthesis; • damages cytoplasmic membrane ESCMID Online Lecture Library Ergosterol synthesis Acetyl CoA Mechanism of action

Squalene Allylamine Squalene monooxygenase drugs Squalene-2,3 oxide

Lanosterol 14-a-demethylase Azoles (ergosterol) © by author

ESCMID Online Lecture Library AZOLES: • Imidazoles: Chemistry • Fluconazole (Diflucan) • Itraconazale (Sporanox) Ketoconazole • Ketoconazole ( Sporanox) • Miconazole nitrate ( Monistat, Micatin) Triazoles: (a type of azole) © by author •Voriconazole, Fluconazole •Posaconazole, •Ravuconazole ESCMID Online Lecture Library

ESCMID Online Lecture Library Mechanism of Action inhibit the synthesis of ergosterol by  blocking demethylation TERB (14-demethylase) of lanosterol - inhibit fungal demethylase  also inhibit cytochrome activity  resulting in: - Depletion of ergosterol - Accumulation of toxic © by author sterols - Damage to cytoplasmic Weaker effects in human than in membrane fungal cells contribute to the favorable ESCMID Onlinetolerability Lecture of the triazole Library antifungals Resistance to Azoles • Well-known particularly for fluconazole • Data available also for other azoles • A significant clinical problem

ESCMID Online Lecture Library 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 Target enzyme modification • have been identified • that are associated with the ERG11 gene of C. albicans, © by author

The azoles then inhibit Erg11, blocking the formation of ESCMID Onlineergosterol Lecture Library 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 © by author recombination

ESCMID Online Lecture Library • The CDR proteins are ABC transporters (ABCT) with In a susceptible cell, azole drugs enter the – both a membrane pore and cell through an unknown mechanism, – two ABC domains perhaps by passive diffusion • 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 © by author accumulation of the azole in fungal cell concentrations within the cell

ESCMID Online Lecture Library 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 by author effect

ESCMID Online Lecture Library Azole resistance • 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 © by author these mechanisms

ESCMID Online Lecture Library azole drugs - Factors in resistance

• 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 © by author

In cells with clinically important ESCMID Onlineresistance Lecture to azole drugs,Library 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 © by Sometimes author resistant C. krusei > C. glabrata > C. parapsilosis C. tropicalis ESCMID Online LectureC. kefyr Library Fluconazole - resistance - altered demethylase or by enhanced removal from the fungal cell

• Efflux of fluconazole – by increased expression of the multidrug resistance transporter proteins (especially MDR1) – development of fluconazole resistance in some Candida species © by author

ESCMID Online Lecture Library 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 drug- induced interaction between the ScPdr1p/Pdr3p and © by author mediator complexes. • The efflux pumps • reduce the intracellular concentration of the drug below that requiredESCMID to inhibit the Online azole target Lecture Erg11p, allowingLibrary normal cell growth

Fluconazole - resistance • 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© by author drugs, while • MDR appears to be specific for fluconazole Secondary Resistance to ESCMID OnlineFluconazole Lecture inLibrary C. albicans , C. dubliniensis...

Voriconazole Ravuconazole • A synthetic derivative of fluconazole • is similar to fluconazole with • Substitution of a triazole group a thiazole in the place of a with a fluoropyrimidine moiety second triazole – 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 © by author

ESCMID Online Lecture Library Voriconazole & Ravuconazole • The similarity of MIC values • similar modes of action • similar mechanisms of resistance • Broad spectrum of activity against • Voriconazole – Aspergillus spp. • In vivo studies – C neoformans • Effective in various animal models – Candida spp. © by• disseminatedauthor aspergillosis – Trichosporon spp. • invasive pulmonary – Dermatophytes aspergillosis • systemic candidiasis

ESCMID Online Lecture Library 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© by author • Further studies into azole susceptibility patterns are required

ESCMID Online Lecture Library

ESCMID Online Lecture Library 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 © by author • Dermatophytes • more effective against yeast and nondermatophyte fungi

ESCMID Online Lecture Library • 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 © by author – voriconazole

ESCMID Online Lecture Library 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 © by authorisolates

ESCMID Online Lecture Library 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” © by author • “No cross-resistance between FLU and POS or VOR among isolatesESCMID of C. krusei” Online Lecture Library Pfaller MA et al. J Clin Microbiol. 2008;46:551-559. 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

MIC (µg/mL) Number of Clinical isolate mutations Posaconazole Fluconazole Voriconazole Candida albicans Control strain 0 0.03 0.125 0.03 C438 2 © by0.5 author128 2 C439 3 1 >256 16 C440 4 4 >256 >16 C369 5 0.25 32 2 C373 6 2 >64 4 ESCMID OnlineXiao L etLecture al. Antimicrob Agents Library Chemother . 2004;48:568. Li X et al. J Antimicrob Chemother. 2004;53:74. 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

spp spp spp

Candida

A flavus A fumigatusA niger A terreus B dermatitidisC albicansC krusei C glabrataOther C neoformansCoccidioidesF solani H capsulatumS apiospermumTrichophyton Zygomycetes Fluconazole X X© by +/- author 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 PosaconazoleESCMID X X X X Online X X X Lecture X X X X +/-Library X X X X ND, not determined. Sabatelli F et al. Antimicrob Agents Chemother. 2006;50:2009. 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

>8 1 8 1 4 1 2 3 6 5 1 6 5 12 1 1 0.5 3 28 80 28 1 0.25 2 7 129 157 21 8 0.12 2 53 80 20 3 5 0.06 12 31 31 4 3 0.03 4 14 ©1 by author Posaconazole MIC (µg/mL) 0.015 2

0.12 0.25 0.5 1 2 4 8 >8 Itraconazole MIC (µg/mL) ESCMID Online Lecture Library Pfaller MA et al. J Clin Microbiol. 2008;46:2568. 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. © by author

ESCMID Online Lecture Library Cell Wall Active Antifungals

Cell membrane • Polyene antibiotics • Azole antifungals DNA/RNA synthesis • Pyrimidine analogues - Flucytosine

ESCMID Online Lecture Library The Fungal Cell Wall TARGETS for antifungal activity

mannoproteins

b1,3 b1,6 glucans Cell b1,3 glucan chitin membrane synthase • Glucan/Chitin (Cell wall) ergosterol • Inhibition of glucan / chitin synthesis • antifungals interfere with fungal cell wall synthesis © by author • by inhibition of ß-(1,3) D-glucan synthase • Loss of cell wall glucan results in osmotic fragility ESCMID Online Lecture Library Echinocandins and pneumocandins • In vitro susceptibility testing • Fungicidal activity • In vivo studies • Candida species including non- • Caspofungin albicans isolates resistant to • in animal models fluconazole • Candida • C albicans • Aspergillus • C tropicalis • C glabrata • Histoplasma – Aspergillus spp. • Pneumocystis • Caspofungin carinii – limited activity against C. neoformans © by author – contains little or no β-(1,3)- D-glucan synthase ESCMID Online Lecture Library ECHINOCANDINS • Cyclic lipopeptide antibiotics • Inhibition of the fungal cell wall β-(1-3) glucan synthesis • Inhibitors of β-(1,3)-D- glucan synthase • Secondary reduction in ergosterol & lanosterol © by• author Increase in chitin

ESCMID Online Lecture Library 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© by author 22% • Monitor effectiveness in antifungal treatment ESCMID Online Lecture Library

ESCMID Online Lecture Library 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.ESCMID Antimicrob Agent Chemother Online 2002;46:3001- Lecture12 Library Echinocandins and pneumocandins HO OH • have the potential to provide a H H O superior efficacy versus current agents HO H NH H NH H H C • This novel mechanism of action may 3 O N H NH CH have particular value in the treatment HO O 3 of resistant fungal strains O H H H NH O H OH H N O N • The unique, specific action 2 mechanism of caspofungin HO H NH OH H H H H • results in a low potential for OH O mechanism-based toxicities © by authorOH

ESCMID Online Lecture Library Resistance to Echinocandins

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

ESCMID Online Lecture Library Echinocandin Resistance Molecular Aspects • FKS1 encodes glucan synthase • GNS1 encodes an enzyme involved in fatty acid elongation • mutations in FKS1 or GNS1 • Other mechanisms (?) © by author

ESCMID Online Lecture Library 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

ESCMID Online Lecture Library

ESCMID Online Lecture Library Caspofungin - MSD

• is an anti-fungal drug that can effectively treat different types of fungal infection. • This drug can be administered intravenously © by author .

ESCMID Online Lecture Library Evaluation of caspofungin treatment of invasive fungal infections

ESCMID Online Lecture Library

ESCMID Online Lecture Library Nıkkomycın

• competitive inhibitors of fungal chitin-synthase enzymes • Yet investigational

ESCMID Online Lecture Library • New antifungal agents • Pradimicins- Sordarıns, benanomicins Azasordarıns – bind to cell wall • inhibit protein synthesis, i.e. mannoproteins causing osmotic elongation factor 2 sensitive lysis and cell • EF3: A target in protein death synthesis machinery unique to • Allylamines/thiocarbam FUNGI ates • GM 237354... (sordarins) – non-competitive inhibitors of squalene GW 471558...(azasordarins) epoxidase • Yet investigational • Cationic peptides

– bind to ergosterol and © by author cholesterol and lead to cell lysis ESCMID Online Lecture Library Mechanism of action of current therapies and implications for efficacy

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

ESCMID Online Lecture Library Mechanism of action of current antifungal drugs

ESCMID Online Lecture Library Multidrug Resistance to Antifungals

• Although several drugs are available to combat often- deadlyESCMID fungal infections, Online many Lectureof these pathogens Library have acquired multidrug resistance. Summary of Treatments Pathogen Primary Secondary

Aspergillus Voriconazole Itraconazole, fumigatus Posaconazole Caspofungin Amphotericin B Blastomyces Itraconazole or Fluconazole dermatidis Amphotericin B Candida albicans Fluconazole Voriconazole, Amphotericin B Itraconazole, Caspofungin Ketoconazole Posaconazole© by author (topical – many) Anidulafungin Coccidioides Itraconazole, immitis Fluconazole or ESCMID AmphotericinOnline Lecture B Library Summary of Treatments

Pathogen Primary Secondary

Cryptococcus Amphotericin B ± Itraconazole or neoformans Flucytosine Amphotericin B followed by Fluconazole Histoplasma Itraconazole or Fluconazole capsulatum Amphotericin B Mucomycosis Amphotericin B Posaconazole © by author Sporothrix Amphotericin B SSKI* schenckii Itraconazole * SaturatedESCMID solution of potassiumOnline iodide Lecture Library 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 © by author

ESCMID Online Lecture Library 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© by author • Molecular assays to detect resistance are not simple

ESCMID Online Lecture Library Future Directions to Avoid Development of Resistance

• Proper dosing strategies

• Restricted and well-defined indications for prophylaxis with azoles

ESCMID Online Lecture Library Thanks for your attention

ESCMID Online Lecture Library