Antifungal Pharmacology

Antifungal PharmacologyAntifungal PharmacologyDeborah Fox, Ph.D.Children’s Hospital3219 [email protected]

Special thanks to:John Perfect, MDAndy Alspaugh, MDSevtap Arikan, MDJohn Rex, MDwww.doctorfungus.org

Objectives

• To recognize the clinical significance offungal infections

• To identify the antifungal agents and theirmechanisms of action

• To evaluate potential antifungal druginteractions and toxicities

• To differentiate the mechanisms ofantifungal drug resistance

Lecture outlineLecture outline

• Clinical significance of fungal infection• Fungal cell structure and targets• Antifungal agents and mechanism of action• Antifungal drug interactions & nephrotoxicity• Mechanisms of antifungal resistance• Summary

Mycology ResourcesMycology Resources

• http://www.doctorfungus.org• http://mycology.adelaide.edu.au/mycoses• http://mycology.cornell.edu/• http://www.mycology.net/

Medical Problems Caused by FungiMedical Problems Caused by Fungi

1) Allergic disease

2) “Mushroom” poisoning

3) MycotoxinsSecondary metabolites

--many have industrial uses--Fusarium mycotoxin -- USSR after WWII--A. flavus “aflatoxin”

4) Mycoses -- infection and resulting disease cause by fungi

100,000Validly described species

of fungi

150primary human

fungal pathogens

Fungi yet to be discovered

The FungiThe Fungi

-Kwon-Chung andBennett, 1992

100,000Validly described species

of fungi

150primary human

fungal pathogens

Fungi yet to be discovered

The FungiThe Fungi

-Kwon-Chung andBennett, 1992

Candida, Aspergillus,

Crypto, Blasto, Histo,Cocci, Dermatophytes

Candida, Aspergillus,

Crypto, Blasto, Histo,Cocci, Dermatophytes

Recent events in fungal diseasesRecent events in fungal diseasesProblems of today:a. Growing population of immunocompromised

i. Modern medical practices: transplantations, indwelling catheters,surgeries, anti-cancer therapies, broad-spectrum antibacterials,immunosuppressants; mycoses: especially candidiasis and aspergillosisii. Natural diseases: AIDS; mycoses: especially candidiasis andcryptococcosisiii. Mean age of population is increasing; mycoses: especially candidiasis

b. Special problems associated with immunocompromised.i. Mycoses often are more severe, difficult to treat and diagnose.ii. Number of disease-causing fungi has increased.

c. Mobile population.i. People commonly travel through areas of endemic mycoses, whichpresents diagnostic challenges.

Advances in research:a. New antifungal agents and treatment options

b. Tremendous increase in understanding of molecular basis of pathogenesis

Fungal cell

Mannoproteins

β-(1,6)-glucanβ-(1,3)-glucan

Chitin

Phospholipid bilayerof cell membrane

Cell membrane and cell wall

Ergosterolβ-(1,3)-glucan synthase

Squalene

ErgosterolSynthesisPathway

DNA/RNA Synthesis

ANTIFUNGAL DRUGStargets

• Membrane disrupting agentsAmphotericin B, nystatin

• Ergosterol synthesis inhibitorsAzoles, allylamines, morpholine

• Nucleic acid inhibitorFlucytosine

• Anti-mitotic (spindle disruption)Griseofulvin

• Glucan synthesisinhibitorsEchinocandins

• Chitin synthesisinhibitorNikkomycin

• Protein synthesis inhibitorsSordarins, azasordarins

• POLYENESAmphotericin B, nystatin

• AZOLESImidazoles: Ketoconazole..Triazoles: Fluconazole,

itraconazole, voriconazole, posaconazole, ravuconazole• ALLYLAMINES

Terbinafine, butenafine• MORPHOLINE

Amorolfine• FLUORINATED PYRIMIDINE

Flucytosine

• ECHINOCANDINSCaspofungin,

anidulafungin, micafungin

• PEPTIDE-NUCLEOSIDENikkomycin Z

• TETRAHYDROFURANDERIVATIVES

Sordarins, azasordarins

• OTHERGriseofulvin

ANTIFUNGAL DRUGSclasses

PolyenesPolyenes

CH3O

O

OH OH OH O

H3C

HO

H3C

OHOH

COOHH

OH

OH

O CH3OH

NH2

O

OH

amphotericin B

CH3

O

O

OH OH OH OH O

H3C

HO

H3C

OHOH

OH

COOH

O CH3

OH

NH2OH

O

Nystatin A1

Cell membrane

ErgosterolAmphotericin B

Binding to ergosterol,Intercalation of cell membrane

K+K+

Na+Na+Ca++Ca++ Ca++Ca++

Na+Na+

K+K+

Leakage of intracellular cationsand proteins

Amphotericin BAmphotericin B• Mechanism: binds sterols, preferentially ergosterol, and disrupts osmotic integrity of cell membrane

• Complications: fever, chills, myalgia, nephrotoxicity, thrombophlebitis

• Pharmacokinetics: poorly soluble in water•rapid uptake by RES, then redistributed•four formulations

• ampho B colloidal dispersion (ABCD; Amphotec)• amphotericin B lipid complex (ABLC; Abelcet)• liposomal amphotericin B (L-AMB; Ambisome)• oral amphotericin B (poor absorption)

• Indications: broad range of activity, ABCD is mainstay of antifungal therapy

Azoles, allylamines & morpholinesAzoles, allylamines & morpholinesergosterol synthesis inhibitorsergosterol synthesis inhibitors

Clin Microbiol RevClin Microbiol Rev 1998; 11: 382 1998; 11: 382

NN

H3C

O

O

OO

Cl

N

N

ClH

ketoconazole

NNNN

N

OCH3

H3CO

OO

Cl

N

NN

ClH

itraconazole

N

N

N

F

FOH

N

N N

fluconazole

N N

N N

N

CH3F

OHF

F

voriconazoleNNN

N

N

OH3C

O

O

F

N

NN

FHHO

H3C

posaconazole

AzolesAzoles

Cell membrane

Ergosterol

Azole

Squalene

ErgosterolSynthesisPathway

Toxic sterols

Accumulation oftoxic sterols incell membrane

Inhibition of14-alpha-demethylase

AzolesAzoles• Mechanism: block ergosterol synthesis via inhibition of cytochrome P450 dependent 14α-demethylase (Erg11)

• Complications: well-tolerated, hepatotoxicity, hypertension, headache, visual disturbances, resistance

• Formulations: poorly soluble in water, fungistatic• Fluconazole (Diflucan)• Voriconazole (Vfend)• Ravuconazole• Itraconazole (Sporanox)• Posaconazole• Ketoconazole (Nizoral)

• Indications: Candida, Cryptococcus, Coccidioides,Histoplasma, Blastomyces, some Aspergillus spp.

Azoles, allylamines & morpholinesAzoles, allylamines & morpholinesergosterol synthesis inhibitorsergosterol synthesis inhibitors

Clin Microbiol RevClin Microbiol Rev 1998; 11: 382 1998; 11: 382

N

CH3 C(CH3)3

terbinafine

CH3

N

OCH3

CH3H3CCH3

H3C

butenafine

Allylamines, morpholinesAllylamines, morpholines

Allylamines, morpholinesAllylamines, morpholines• Mechanism: block ergosterol synthesis via inhibition of squalene epoxidase (allylamines), sterol reductase and isomerase activity (morpholines)

• Complications: mild gastrointestinal and skin reactions

• Formulations: poorly soluble in water, oral and topical, fungicidal

• Terbinafine (Lamisil)• Amorolfine (Loceryl)• Butenafine (Mentax)

• Indications: dermatophytes, Candida (Mentax)

HO

O

NH

O

HO

OH

HN

O

N

HO

H2N

O

H3C

HO

NH

O

HN

CH3

OH

N

O

OOH

O

NH

O

O

N

HO OH

O

H3C

S

OH

O

O

micafunginN

O

NH

OHO

HO

NH

O

OH

HN

H2N

OH

H2N

O

OH

HN

OH

HO

H

H

H HNH O

H

CH3

OH

ONH

O

H3C

CH3 CH3

caspofungin

HO

NH

O

HO

OH

HN

O

N

H3C

HO

H3C

HO

NH

O

HN

CH3

OH

N

O

OOH

O

NH

O

HO OH

O

H3C

anidulafungin

EchinocandinsEchinocandins

Mannoproteins

ß(1,6)-glucanß(1,3)-glucan

Chitin

Phospholipid bilayerof cell membrane

ß(1,3) glucan synthaseglucan synthase inhibitor

Inhibition ofß(1,3) glucan synthase

Depletion of ß(1,3) glucansin cell wall

EchinocandinsEchinocandins• Mechanism: block cell wall synthesis via β-1,3 glucan synthesis inhibition

• Complications: well-tolerated, histamine release, no activity against Cryptococcus, Fusarium spp.

• Formulations: poorly soluble in water, fungicidal• Caspofungin (Cancidas)• Micafungin• Anidulafungin (Eraxis)

• Indications: Candida, Aspergillus spp.

N

HN

F

NH2

O

flucytosine

O

O

H3C

OCH3

H3CO

Cl

O OCH3

griseofulvin

AntimetabolitesAntimetabolites

5-FC

5-FC

5-FU

Cytosine permeaseCytosine permease

Cytosine deaminaseCytosine deaminase

PhosphorylationPhosphorylation

Inhibition of thymidylate synthaseInhibition of thymidylate synthase

FdUMP

Conversion todeoxynucleosidesConversion todeoxynucleosides

dUMP

dTMP

Inhibition of DNA synthesisInhibition of DNA synthesis

Inhibition of Protein SynthesisInhibition of Protein Synthesis

FdUMP

FUTP

Substitution for uracilSubstitution for uracil

5-FC, 5-fluorocytosine; 5-FU, 5-fluorouracil; FdUMP, 5-fluorodeoxyuridine;FUMP, 5-fluorouridine monophosphate; FUDP, 5-fluorouridine diphosphate;FUTP, 5-fluorouridine triphosphate; dUMP, deoxyuridine monophosphate;dTMP, deoxythymidine monophosphate

5-FC

AntimetabolitesAntimetabolites• Mechanism: block fungal DNA and protein synthesis (Flucytosine), fungal mitosis (Griseofulvin)

• Complications: GI intolerance, bone marrow suppression, hepatotoxicity, headache, hallucinations, sedation, nausea

• Formulations: poorly soluble in water• Flucytosine (Ancobon)• Griseofulvin (Grifulvin V, Fulvicin U/F, Grisactin, Peninol)

• Indications: (Flucytosine): for resistant Candida, Aspergillus spp. and in combination with Ampho B for Cryptococcus; (Griseofulvin): dermatophytes

Antifungal drug interactionsAntifungal drug interactions• Pharmacokinetic interactions: changes in absorption or

elimination of interacting drug and the antifungal• Interactions of drug absorption

• Ketoconazole, itraconazole require low pH for absorption (avoid antacids, vitaminsupplements)

• Pre-systemic clearance via membrane transporters (P-gp) & metabolic enzymes.Azoles can be both substrates and inhibitors of P-gp

• Interactions of drug metabolism• Oxidation, reduction, hydrolysis, conjugation of lipophilic

compounds• Interactions with cytochrome P450

• Azoles are metabolized by CYP P450 system• Azoles are also reversible inhibitors of P450 enzymes• Co-administered metabolites are a concern

Azoles: Interactionsin the GI Tract

Drug-pH

Portal vein

Gut wallTo feces

Metabolism

Metabolism

Absorption

Bioavailability

Liver

CYP

P-gpefflux

CYP CYP

CYP

CYP

CYP CYP CYP CYP



• Ketoconazole, itraconazole require low pH for absorption (avoid antacids,vitamin supplements)

• Pre-systemic clearance via membrane transporters (P-gp) & metabolicenzymes. Azoles can be both substrates and inhibitors of P-gp




Proportion of Drugs Metabolized by CYPP450



• Ketoconazole, itraconazole require low pH for absorption (avoid antacids,vitamin supplements)

• Pre-systemic clearance via membrane transporters (P-gp) & metabolicenzymes. Azoles can be both substrates and inhibitors of P-gp




Inducers of CYP 3A4

3A4

2C19 2D6 2C9

1A2 2E1 2A6 2B6 2C8

Ketoconazole (>90%)Fluconazole (~ 50%)Itraconazole (> 90%)Voriconazole (~ 90%)

RifampinPhenytoinCarbamezepinePhenobarbital

Ritonovir?

Reduction in levels

Azole Inhibition of CYP P450Increased serum concentration ofco-administered drug or metabolite Oral hypoglycemics S-warfarin R-Wafarin Cyclosporin Tacrolimus Sirolimus Phenytoin Carbamezepine Triazolam, alprazolam, midazolam Diltiazem Lovastatin Isoniazid Rifabutin Quinidine Protease inhibitors (saquinavir, ritonavir) Busulfan Vincristine Cyclophosphamide Digoxin Loratidine and others…

NephrotoxicityNephrotoxicity

• Primarily due to Amphotericin B• Two mechanisms:

• Effects of ampho B on renal blood flow andglomerular filtration

• Constriction of afferent arterioles decreases renalblood flow and GFR

• Subsequent increase in serum creatinine and BUN• Direct toxic effect on distal tubules via membrane

disruption• Cholesterol target

Afferent arteriole

Efferent arteriole

Proximaltubule Distal

tubule

Glomerulus

Direct damage of distal tubularmembranes leading to wastingof Na+, K+, and Mg++

Constriction of the afferentarterioles leading to decreasedglomerular filtration

Tubular-glomerular feedback:Further constriction of arterioles

Clinical Resistance is aClinical Resistance is aMultifactorial IssueMultifactorial Issue

• FUNGUSInitial MICCell type: Yeast/hyphae..Genomic stabilityBiofilm productionPopulation bottlenecks

• DRUGFungistatic natureDosingPharmacokineticsDrug-drug interactions

• HOSTImmune statusSite of infectionSeverity of infectionForeign devicesNoncompliance with drug

regimen

Anderson JA. (2005) Nature Reviews. 3:547-556

Resistance to Amphotericin BResistance to Amphotericin B

• Technical difficulties in detection ofresistance in vitro

• In vivo resistance is rareC. lusitaniae, C. kruseiC. neoformansTrichosporon spp.A. terreusS. apiospermumFusarium spp.

Mechanisms of Amphotericin BMechanisms of Amphotericin Bresistanceresistance

• Reduced ergosterol content (defective ERG2 or ERG3genes)

• Alterations in sterol content (fecosterol, episterol:reduced affinity)

• Alterations in sterol to phospholipid ratio• Reorientation or masking of ergosterol• Stationary growth phase• Previous exposure to azoles• (?)

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

RESISTANCE TO FLUCONAZOLEPRIMARY C. krusei

AspergillusC. glabrataC. norvegensis...

SECONDARY C. albicans

C. dubliniensis...

Azole ResistanceAzole Resistance• Single point mutation of ERG11 gene

Altered lanosterol demethylase

• Overexpression of ERG11 gene Increased production of lanosterol demethylase

• Alterations in ERG3 or ERG5 genes Production of low affinity sterols

• Increase in mRNA levels of CDR1 or MDR1 genes Decreased accumulation of the azole in fungal cell

• Changes in sterol and/or phospholipid composition offungal cell membrane (decreased permeability)

If your fungus is susceptibleIf your fungus is susceptibleto azoles..to azoles..

Clin Microbiol Rev 1998; 11: 382

If it is azole-resistant..If it is azole-resistant..

Clin Microbiol Rev 1998; 11: 382

Resistance to FlucytosineResistance to Flucytosine

• PRIMARY non-albicans CandidaC. neoformansAspergillus (highest)

• SECONDARY C. albicansC. neoformans

Secondary resistance develops following flucytosineMONOtherapy.

Mechanisms of Resistance toMechanisms of Resistance toFlucytosineFlucytosine

• Loss of permease activity

• Loss of cytosine deaminase activity

• Decrease in the activity of UPRTase

Resistance to EchinocandinsResistance to Echinocandins

PRIMARY C. neoformansFusarium spp.

SECONDARY (?) Candida spp.

Echinocandin ResistanceEchinocandin ResistanceMolecular AspectsMolecular Aspects

• FKS1 encodes glucan synthase• GNS1 encodes an enzyme involved in fatty acid

elongation• Resistance is observed following laboratory

derived mutations in FKS1 or GNS1

• Other mechanisms (?)

Future Directions to AvoidFuture Directions to AvoidDevelopment of ResistanceDevelopment of Resistance

• Proper dosing strategies• Combination therapies• Restricted and well-defined indications for

prophylaxis with azoles

Fungi will continue to develop NEWresistance mechanisms!..

Yeast in blood culture

Immunocompromised(transplant, BMT, AIDS) “Non-immunocompromised”

Start (lipid) polyene and wait for ID

Endemic mycosis Candida

Continue (lipid) polyene Until stable, then consider

fluconazole or itraconazole as appropriate

Hemodynamically stable, no previous azoles

Hemodynamically unstable,previous azoles

Start fluconazole, wait for ID and

monitor response

Start echinocandin or (lipid) polyene, wait

for ID and monitor response

If good response complete 14 days from

first negative culture

No response or clearly resistant

isolate

If good response complete 14 days from first

negative culture, may Switch to fluconazole or

voriconazole if stable and susceptible

If no response switch to other agent

from the above classes

Ostrosky-Zeichner & Pappas, Crit Care Med 2006

Acompletealgorithm,includingmouldsasaconcern

Baseline After caspofungin

This AIDS patient failed fluconazole,amphotericin B, and itraconazole…

Courtesy of John Rex, MD

Echinocandins: No cross-resistance

RecommendationsRecommendations

Ideal antifungal?Ideal antifungal?Not yetNot yet……

The future ofThe future of antifungals antifungals

Sample Exam Question

The effectiveness of the azole class ofantifungals is based upon what structuralfeature of the fungus?

A. the chitin composition of the cell wallB. the cholesterol composition of the cell membraneC. the protein components of the cell membraneD. the ergosterol composition of the cell membrane

Antifungal Pharmacology

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