L22: Antifungal Agents

Five major classes of antifungal agents (PAAPE)

Polyenes

Azoles

Allylamines

Pyrimidine analogues

Echinocandins

Types of fungal infections based on severity

Non-life threatening and life-threatening

Examples of non-invasive or superficial fungal infections

Vaginal candidiasis, oral candidiasis, tinea, nail infections (onychomycosis), dandruff

Examples of life-threatening fungal infections

Bloodstream infections, fungal meningitis, fungal pneumonia

Risk factors for Candida infections

Antibiotic therapy and severely compromised immunity

Risk factors for dermatophyte infections

Contact with sources

Three main morphological categories of medically important fungi

Moulds, yeasts, and dimorphic fungi

Examples of medically important moulds

Aspergillus spp., Fusarium, and dermatophytes

Examples of medically important yeasts

Candida spp. and Cryptococcus

Example of a dimorphic fungus

Histoplasma capsulatum

Definition of dimorphic fungi

Fungi that have both yeast and mould growth forms

Four genera causing ~90% of fungal mortality

Candida, Cryptococcus, Pneumocystis, and Aspergillus

Primary causes of superficial fungal diseases

Candida, Malassezia, and dermatophytes

Reason fungi share many similarities with human cells

Fungi are eukaryotic

3 major components of the fungal cell wall

Chitin, glucan, and mannoproteins

Unique sterol found in the fungal cytoplasmic membrane

Ergosterol

Ribosome type found in fungi

80S ribosomes

Antifungal classes that target the cell membrane

Polyenes

Azoles

Allylamines

Antifungal classes that target cell wall by inhibiting ergosterol synthesis

Azoles and allylamines

Antifungal class that inhibits nucleic acid synthesis

Pyrimidine analogues

Antifungal class that inhibits synthesis of beta(1->3) glucans in the cell wall

Echinocandins

First broad spectrum antifungal class of drugs on the market

Polyenes

Most commonly used polyenes

Amphotericin B, Nystatin, and Natamycin

Bacterial genus that polyenes are derived from

Streptomyces

Discoverers of Nystatin

Rachel Fuller Brown and Elizabeth Lee Hazen

Cellular target of polyenes

Cell membrane / ergosterol

Original theory of polyene mechanism of action

Pore theory: polyenes bind to ergosterol in cell memb → pores form

Pores dissipate membrane potential and promote intracellular ion leakage

Recent theory of polyene mechanism of action

Sterol sponge theory: polyenes binds and extracts ergosterol directly from cell memb, self-assembling into an extramembranous sponge

=disrupts CM function

Spectrum of activity for polyenes

Broad (most yeasts and moulds)

Reason polyene resistance is relatively rare

Difficult for mutations to overcome the specific mechanism of action

Mechanism of polyene resistance in some organisms

Change in membrane lipid composition

Gold standard first-line therapy for cryptococcal meningitis

Amphotericin B

Route of administration for Amphotericin B

Intravenous infusion only

Primary toxicity concern with Amphotericin B

Nephrotoxicity and hepatotoxicity due to slight affinity for human cholesterol

Way to reduce Amphotericin B toxicity and increase bioavailability

Lipid formulations

Largest class of antifungal drugs

Azoles

Origin type of azole antifungals

Synthetic compounds

Examples of first generation azoles (imidazoles)

Clotrimazole, miconazole, econazole

Example of a second generation azole

Ketoconazole

Examples of third generation azoles (triazoles)

Fluconazole, voriconazole, itraconazole, posaconazole

Mechanism of action of azoles

Block ergosterol biosynthesis by inhibiting CYP51A (lanosterol 14-alpha-demethylase)

=increased membrane permeability

Gene that encodes lanosterol 14-alpha-demethylase

ERG11

Mechanisms of azole resistance

Vary by spp. examples:

Efflux by multidrug transporters

ERG11 gene mutations

alteration of the ergosterol biosynthetic pathway

CYP51A mutation → reduced azalea affinity

General antifungal activity type of azoles

Mostly fungistatic

Spectrum of fluconazole + what class is it

Azoles (3rd gen)

Good for yeasts but inactive against moulds

Azoles with the broadest spectrum

Posaconazole and isavuconazole (triazoles - third generation azoles)

Recommended first-line treatment for invasive aspergillosis

Voriconazole (3rd gen azole)

Main toxicity associated with all azoles

Hepatotoxicity

Most common causative agent of vulvovaginal candidiasis

Candida albicans

Common topical treatments for vaginal candidiasis

Clotrimazole, miconazole (azoles)

or nystatin (polyene) creams

Common oral treatment for vaginal candidiasis

Fluconazole 150 mg single dose

(triazole - 3rd gen azole)

First discovered allylamine in 1974

Naftifine

Two drugs in Allylamine class

Naftifine

Terbinafine (derivative of terbinafine)

First and only agent in the benzylamine class of antifungals

Butenafine

Mechanism of action of allylamines

Inhibit squalene epoxidase enzyme = key enzyme in sterol biosynthesis

→ cells accumulate squalene & become ergosterol deficient

Allylamines mechanism of action: Result of squalene epoxidase inhibition

Squalene epoxidase = key enzyme in sterol biosynthesis

→ Cells accumulate squalene and become ergosterol deficient

Mechanism of resistance to allylamines

Mutation on squalene epoxidase gene alleles → conformation change in enzyme/terbinafine binding pocket

Primary clinical use of allylamines

Treating dermatophyte infections of skin and nails (topical and oral)

Fungicidal against dermatophytes

Fungistatic against Candida spp.

Pharmacokinetic property of terbinafine that allows it to accumulate in skin and nails

Highly lipophilic

Recommended first-line topical therapy for tinea pedis

Terbinafine 1% cream (Allylamine)

Narrower spectrum than topical azoles

Treatment for tinea pedis if topical therapy fails

Oral terbinafine 250 mg once daily

Original development purpose of flucytosine (pyrimidine analogue)

Anti-cancer drug (antimetabolite)

Another name for flucytosine

5-fluorocytosine or 5FC

Two anti fungal mechanisms of Flucytosine

1. fungal cell imports 5FC through energy dependent process

2. inside fungal cell 5FC deaminated by cytosine deaminase and converted into active form, 5-fluorouracil (5FU)

=> inhibits RNA and DNA synthesis

Reason flucytosine is safe for human cells at normal doses

Humans lack the cytosine deaminase enzyme

This enzyme converts 5FC into active form 5-fluorouracil (5FU)

Mechanism by which 5FU inhibits protein synthesis

5FU (5-fluorouracil) metabolised into 5-fluorouridine triphosphate → incorporated into fungal RNA & replaces uridylic acid: inhibits protein synthesis

Mechanism by which 5FU inhibits DNA synthesis

5FU (5-fluorouracil) converted into fluoro-deoxyuridine monophosphate which blocks action of thymidylate synthetase: inhibits DNA synthesis

Rate of resistance development for flucytosine monotherapy

Develops rapidly

speed varies between spp.

Flucytosine resistance:

Gene mutation causing decreased flucytosine uptake

FCY2 (purine-cytosine permease)

Flucytosine resistance:

Gene mutation causing decreased conversion of 5FC to 5-FU

FCY1 (cytosine deaminase)

Flucytosine resistance:

Gene mutation causing decreased conversion of 5-5FC to 5-FUMP

FUR1 (uracil phospho-ribosyltransferase)

Common clinical practice to prevent flucytosine resistance

Usually combined with another antifungal agent like amphotericin B

Spectrum of activity for flucytosine

Limited; active against Candida sp., Cryptococcus sp., and some dematiaceous molds

Fungi that flucytosine is notably inactive against

Aspergillus, Mucorales, or endemic primary pathogens

Main clinical uses of flucytosine

Cryptococcal meningitis

specified candida infections

dematiaceous fungi infections

Cause of flucytosine toxicity at high plasma concentrations

Mediated by gut microflora that convert 5FC into toxic 5-FU

safe at pharmacological doses

First discovered echinocandin with strong haemolytic effects

Echinocandin B

Nature of echinocandin molecules

Cyclic lipopeptides synthesised by filamentous fungi

Three echinocandins currently available for clinical use in Australia

Caspofungin, micafungin, and anidulafungin

Mechanism of action of echinocandins

Block synthesis of cell wall beta-(1,3)-d-glucan

Consequence of blocking beta-(1,3)-d-glucan synthesis

Cell wall structural abnormalities and osmotic instability

Specific enzyme subunit targeted by echinocandins

Fks1p (catalytic subunit of glucan synthase)

Mechanism of echinocandin resistance

Mutations in Fks altering the binding of the drug

Primary spectrum of activity of echinocandins

Fungicidal against Candida spp. and fungistatic against Aspergillus spp.

Reason echinocandins are ineffective against Mucorales and Cryptococcus

Their cell walls mostly contain beta-(1,6)-d-glucans instead

Route of administration for echinocandins

Intravenous (poorly absorbed in GI tract)

Pharmacokinetic limitation of echinocandins

Bind strongly to proteins limiting bioavailability and cannot penetrate CNS or eyes

Synthetic thiocarbamate used topically for dermatophyte infections

Tolnaftate

Synthetic hydroxypyridine used topically for nail infections

Ciclopirox

Morpholine that inhibits ergosterol biosynthesis used as a nail lacquer

Amorolfine

Polyketide discovered from Penicillium griseofulvum that inhibits mitosis

Griseofulvin

First-line oral therapy for tinea capitis in children caused by Microsporum

Griseofulvin

Three main ways reduced sensitivity to antimycotics can occur

Genetic mutations, tolerance/hetero-resistance, and intrinsic resistance

Example of tolerance/hetero-resistance mechanism

Upregulation of efflux pumps

Example of intrinsic resistance

Type of cell wall glucans (e.g. lack of target)

Existing challenges in antifungal therapy

Early diagnosis, limited drugs, cytotoxicity, increasing resistance, and emerging pathogens