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L 4: ANTIFUNGAL THERAPEUTIC

AGENTS

Clinical Mycology

(MLAB 474)

Dr. Mohamed A. El-Sakhawy

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Antifungal agent

• An antifungal agent is a drug that selectively eliminates

fungal pathogens from a host with minimal toxicity to the

host.

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Fungal Infection in Humans = Mycosis

• Major Types of Mycoses

– superficial

– cutaneous

– subcutaneous

– systemic

– opportunistic

• Symptoms vary from cosmetic to life threatening

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Fungal infections = mycoses

• Opportunistic or primary

• Systemic or local

• Slow onset

• Long duration of therapy

• Difficult to diagnose & eradicate

• Symptoms vary from cosmetic to life threatening 4

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Wide-spectrum antibiotics

surgery

Immunosuppressant agents &

chemotherapy AIDS

Increase risk

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MOST COMMON FUNGAL PATHOGENS

• Dermatophytes

• Candida • Aspergillus • Cryptococcus • Rhizopus

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ANTIFUNGAL DRUGS

• Antıfungal drugs classified according to structure or by mechanism of action.

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Keep in mind: • We need to target differences between our cell and the

fungal cell. • Antibiotics will not work because of both cells are

eukaryotic and the medication could kill off the good gut flora that competes with the fungus for nutrients.

• Treatments usually include many combinations of different antifungals.

ANTIFUNGAL DRUGS --by structure

• POLYENES

are poly-unsaturated organic compounds that contain one or more sequences of alternating double and single carbon-carbon bonds.

Organic compounds with two carbon-carbon double bonds are dienes; those with three such double bonds are trienes; those with four are tetraenes, etc.

Amphotericin B, nystatin

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Amphotericin B

Note the alternating double and single bonds in the center

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ANTIFUNGAL DRUGS --by structure

• AZOLES

An azole is a class of five-membered nitrogen heterocyclic ring compounds containing at least one other noncarbon atom, nitrogen, sulfur or oxygen.

Imidazoles: Ketoconazole..

Triazoles: Fluconazole, itraconazole, voriconazole, posaconazole, ravuconazole

Fluconazole Clotrimazole Miconazole 9

ANTIFUNGAL DRUGS --by structure

• ALLYLAMINES

Allylamine is an organic compound with the formula C3H5NH2. This colorless liquid is the simplest stable unsaturated amine.

Terbinafine, butenafine , Amorolfine

Terbinafine 10

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ANTIFUNGAL DRUGS --by structure

• FLUORINATED PYRIMIDINE

Pyrimidine is a heterocyclic aromatic organic compound similar to benzene and pyridine, containing two nitrogen atoms at positions 1 and 3 of the six-member ring

Flucytosine

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ANTIFUNGAL DRUGS --by structure

• ECHINOCANDINS

Caspofungin, anidulafungin, micafungin

• PEPTIDE-NUCLEOSIDE

Nikkomycin Z

• TETRAHYDROFURAN DERIVATIVES

Sordarins, azasordarins

• OTHER

Griseofulvin

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MODES of ACTION

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ANTIFUNGAL DRUGS (--by mode of action) • A) Drugs that disrupt fungal cell

membrane • i) Polyenes

Amphotericin Nystatin Natamycin

• ii) Azoles a) Imidazole

Ketoconazole Butaxonazole Clotrimazole Econazole Miconazole Oxiconazole Sulconazole

b) Triazole Fluconazole Itraconazole Tioconazole

iii) Allylamines

Terbinafine Naftifine Butenafine

vi) Echinocandins caspofungin, •

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B) Drugs that inhibits mitosis Griseofulvin

C) Drugs that inhibits DNA synthesis

flucytosine

D) Drugs that inhibits Protein synthesis

Sordarins, azasordarins

E) Drugs that inhibits Cell wall

i) Glucan synthesis

Echinocandins ii) Chitin synthesis

Nikkomycin

TARGETS for antifungal activity

• Ergosterol (Cell membrane) Drug-ergosterol interaction Inhibition of ergosterol synthesis

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AMPHOTERICIN B

• Isolated 1955, in clinical use since 1960

• Produced by Streptomyses nodosum

• Broad-spectrum polyene macrolide antibiotic is the most potent antifungal agent for systemic mycosis,

• Fungicidal drug at higher concentrations & static at lower levels.

• Highest concentrations in liver, spleen, bone marrow with less in kidneys and lungs.

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MECHANISM OF ACTION

• High affinity for fungal ergosterol, forms “micropore”

in fungal cell membrane through which ions, amino

acids, & other water soluble substances move out.

• Markedly increases cell permeability.

• Cholestrol, present in host cell membranes, closely

resembles fungal ergosterol & thus explains the high

toxicity of AMB in humans

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Amphotericin B

Mech of Action!

Associates with the membrane and

causes leakage of Na, K, and Ca

across membrane. But how does it

differentiate between fungal cells

and human cells??

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Mechanism of Action

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AMPHOTERICIN B generates pores in the membrane

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• Instead of cholesterol in the human cell membrane, fungal cells have ergosterol. The heptaene portion of the ring interacts strongly with ergosterol instead of cholesterol.

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Why Amphotericin B inhibits fungal

cells instead of human cells?

Clinical use • Treatment of nearly all life threatening mycotic infections.

• For systemic disease: slow IV

• Local: rarely (Drug concentration achieved in infected skin is very low, & hence ineffective against superficial fungal infections).

o Keratitis& corneal ulcers: drops, conjunctival irrigation,

• AMB is not absorbed integrally; hence can be given orally for intestinal candidiasis.

• Penetration in brain & CSF is poor (but extremely effective in fungal meningitis when combined with 5-FC).

• Liposomal Amphotericin B (New lipid formulations): Amphotericin B is incorporated into lipid formulations to reduce toxicity & enhance efficacy. This allows higher dose to be used without increasing the toxicity.

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Side effects

• Infusion related Fever & chills,

Dyspnea,

Nausea &vomiting,

Hypotension,

Convulsions

• Cumulative toxicity

Nephrotoxicity K & Mg wasting

Anemia

NYSTATIN

• It is polyene macrolide, similar in structure to amphotericin B and with same mechanism of action

• Too toxic for systemic use

• Not absorbed from GIT, skin or vagina, therefore administered orally to

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• Prevent or treat superficial candidiasis of mouth, esophagus or intestinal tract, oral suspension 4 times a day and tablets are used to decrease GIT colonization with Candida

• For vaginal candidiasis in form of pessaries used for 2 weeks

• In Cutaneous infection available in cream, ointment or powder form and applied 2-3 times a day

• Can be used in combination with antibacterial agents and corticosteroids

TARGETS for antifungal activity

• Ergosterol (Cell membrane)

Drug-ergosterol interaction

Inhibition of ergosterol synthesis

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Azoles

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Azoles

Imidazoles

Ketoconazole

Miconazole

Clotrimazole

Butaxonazole

Econazole

Oxiconazole

Sulconazole

Triazoles

Itraconazole

Fluconazole

Voriconazole

Posaconazole

Tioconazole

MECHANISM OF ACTION of Azoles

Inhibition of fungal cytochrome P450 enzymes

Reduction of ergosterol synthesis

http://www.youtube.com/watch?v=T-dwE11AhqA

Specifically inhibits the cytochrome P450 fungal enzymes. This enzymes is require in the 20 steps pathway from lanosterol (intermediate in cholesterol synthesis) to ergosterol.

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Azoles, allylamines & morpholines inhibit specific ENZYMES

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Clinical use of Azoles BROAD SPECTRUM OF ACTIVITY:

Candida, Cryptococcus, Blastomyces,

Histoplasma, Coccidiodes, Dermatophytes

ADVERSE EFFECTS

Relatively nontoxic.

Minor GI upset

Abnormalities in liver enzymes (inhibit cytochrome P450 enzymes)

Very rarely, clinical hepatitis

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KETOCONAZOLE • (older, more toxic, replaced by itraconazole, but less

costly)

• The first oral azole introduced into clinical use.

• It is less selective for fungal P450 than are the

newer azoles.

• Absorption variable (better in acidic medium)

• Penetration in brain & CSF is poor

• In high doses inhibits adrenocortical steroids

and testosterone synthesis, resulting in

gynecomastia in some males.

ITRACONAZOLE

• Broad-spectrum antifungal with fungistatic

action

• MOA: Inhibits fungal ergosterol synthesis like other

azoles

• Penetration of drug in brain & CSF is poor.

• Much more selective than ketoconazole

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FLUCONAZOLE

Broad-spectrum Fungicidal drug;

• It is also somewhat effective against some Gram-

positive & anaerobic bacteria

• Of the orally administered fluconazole 94% is

absorbed;

• Penetration in brain & CSF is good, hence

used for cryptococcal meningitis

POSACONAZOLE

The newest triazole

It is the broadest spectrum member of the

azole family.

It is the only azole with significant

activity against the agents of zygomycosis

and mucormycosis.

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TARGETS for antifungal activity

• Ergosterol (Cell membrane)

Drug-ergosterol interaction

Inhibition of ergosterol synthesis

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TERBINAFINE (lamisil)

Belongs to class of antifungals – the allylamines (Synthetic allylamine).

Discovered by accident in 1974.

Treats mainly superficial infections.

• Orally Active.

• Dermatophytoses, especially onychomycosis .

• Keratophilic , fungicidal.

Terbinafine

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• Like the azole drugs, it interferes with ergosterol

biosynthesis, but rather than interacting with the

P450 system, terbinafine inhibits the fungal enzyme

squalene epoxidase.

MOA of TERBINAFINE

• This leads to the

accumulation of the

sterol squalene, which is

toxic to the organism.

It interferes with ergosterol biosynthesis by:

Inhibiting the fungal enzyme squalene epoxidase

Accumulation of the sterol squalene,

MOA of TERBINAFINE

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TARGETS for antifungal activity

• RNA/EF3 (Nucleic acid/Protein synthesis) Incorporation of 5-FU Inhibition of EF3

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FLUCYTOSINE (5-fluorocytosine)

5-fluoro-UTP Inhibition of DNA synthesis

incorporated into RNA

Mechanism of action

• It is converted to antimetabolite 5-florouracil in a fungal but not

human cell. This 5-FU inhibits thymidylate synthetase

enzyme and thus DNA synthesis. Resistant mutants may occur,

should never be used alone.

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Mechanism of action of Flucytosine

Fdump (fluorodeoxyuridne monophosphate)

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Thymidylate synthetase is an enzyme that catalyzes the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP). dTMP is one of the three nucleotides (dTMP, dTTP, and dTDP) that form thymine

• Flucytosine is taken up by fungal cells via the enzyme cytosine

permease.

• It is converted intracellularly first to 5-FU and then to 5-

fluorodeoxyuridine monophosphate (FdUMP) and

fluorouridine triphosphate (FUTP), which inhibit DNA and RNA

synthesis, respectively.

• 5-FC (outside)

Cytosine permease enzyme

• 5-FC (inside)

• 5-FU (inside)

Inhibits thymidylate

synthase

• Inhibits DNA & RNA

synthesis

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Human cells are unable to convert the parent

drug to its active metabolites.

FLUCYTOSINE

• Has useful activity against Candida and Cryptococcus.

• Since this is a narrow-spectrum fungistatic, it is mainly used as an adjuvant drug & not used as a sole therapy.

• CSF penetration is excellent, hence it is combined with AMB in fungal meningitis.

• Clinical use at present is confined to combination therapy, either with:

Amphotericin B for cryptococcal meningitis , or

Itraconazole for chromoblastomycosis

• Water soluble • Oral only,

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ADVERSE EFFECTS

• Bone marrow toxicity with anemia, leukopenia,

thrombocytopenia, (Mammalian bone marrow

cell have the capacity to convert 5-FC to 5-FU)

• GI disturbances

• Mild & reversible liver dysfunction

TARGETS for antifungal activity

• RNA/EF3 (Nucleic acid/protein synthesis) Incorporation of 5-FU into RNA Inhibition of EF3

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SORDARINS, AZASORDARINS

• EF3: A target in protein synthesis machinery unique to FUNGI

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sordarin acts on elongation factor 2 (EF2). This enzyme catalyzes the translocation of the ribosome along mRNA during elongation of the emerging polypeptide chain

Strong activity against Saccharomyces cerevisiae , Candida albicans , and a number of pathogenic fungi make sordarin a promising antimycotic agent.

TARGETS for antifungal activity

• Glucan/Chitin (Cell wall) Inhibition of glucan / chitin synthesis

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ECHINOCANDINS Caspofungin

Micafungin

Anidulafungin

The newest class of antifungal .

Active against Candida and Aspergillus, but not C.

neoformans or the agents of zygomycosis and

mucormycosis.

MOA of ECHINOCANDINS

Inhibit the synthesis of B glucan in the fungal cell wall

Disruption of the fungal cell wall and cell death.

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ECHINOCANDINS Caspofungin is licensed

• Inhibition of β-(1-3) glucan synthesis (of glucan synthase ??)

• Secondary reduction in ergosterol & lanosterol

• Increase in chitin • Kills hyphae at their

growth tips and branching points

• Buds fail to seperate from the mother cell

• Yields osmotically sensitive fungal cells

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ADVERSE EFFECTS

• Extremely well tolerated,

• Minor GI side effects

• Flushing

• Elevated liver enzymes (caspofungin +

cyclosporine).

• Histamine release during IV infusion.

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TARGETS for antifungal activity

• Glucan/Chitin (Cell wall) Inhibition of glucan / chitin synthesis

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NIKKOMYCIN

• Competitive inhibition of chitin synthase

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TARGETS for antifungal activity

Inhibition of mitosis

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GRISEOFULVIN

• Very insoluble, fungistatic

• Derived from a species of Penicillium.

• Better absorption when given with fatty foods.

• It is deposited in newly forming skin where it

binds to keratin, protecting the skin from new

infection.

• Interferes with spindle formation in dividing cells

and therefore with mitosis

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Adverse effects of griseofulvin

• Allergic reaction

• photosensitivity

• Hepatitis

• Teratogenesis

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Conclusion

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Antimycotic drugs

Systemic antifungal agents

• Polyene macrolide

• Flucytosine

• Azoles

• Morpholine derivatives

• Griseofulvin

Topical antifungalagents

• Polyene macrolide

• Azole

• Griseofulvin

• Allyamines

• Thiocarbamate

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A resistant strain may be present due to:

• Intrinsic resistance

• Replacement with a more resistant species

• Replacement with a more resistant strain

• Transient gene expressions that cause temporary resistance (epigenetic resistance)

• Alterations in cell type

• Genomic instability within a single strain (population bottleneck)

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Clinical Resistance is a Multifactorial Issue

• FUNGUS Initial MIC

Cell type: Yeast/hyphae..

Genomic stability

Biofilm production

• DRUG Fungistatic nature

Dosing

Pharmacokinetics

Drug-drug interactions

• HOST Immune status

Site of infection

Severity of infection

Foreign devices

Noncompliance with drug regimen

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• Antifungal resistance is a complex, gradual and multifactorial issue

• Several uncertainties remain

• Molecular assays to detect resistance are not simple

• The best way to improve the efficacy of antifungal therapy is to improve the immune status of the host

• Superficial fungal infections may take a LONG time to effectively treat (weeks to months)

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MOST COMMON INDICATIONS Tinea Infections (1-4 wks)

• Ketoconazole • Terbinafine

Onychomycosis (6wks-1yr)

• Itraconazole • Terbinafine Vaginal Candidiasis (1d-2wks) • Fluconazole • Nystatin

Oropharyngeal Candidiasis (7-14d) o Fluconazole o Itraconazole o Nystatin

Esophageal Candidiasis (14-21d) o Fluconazole o Itraconazole o Voriconazole Systemic Infections • Fluconazole • Voriconazole • Nystatin

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Administration Superficial fungal infections may take a LONG time to effectively treat (weeks to months) Exception-Fluconazole for vaginal yeast infection Important to counsel on adherence and time to effect

Onychomycosis