Download Lecture 17- Antifungal Agents

Novel Antifungal Agents
Dr. Khaled H. Abu-Elteen
Dr. Mawieh A. Hamad
Department of Biological Sciences
Faculty of Science- Hashemite University
Most Important Antifungal Agents
Used in Treatment of Fungal Infections
Griseofulvina
1947
Amphotericin B
1957
Fluocytosine
1971
Miconazole
1978
Ketoconazole
1981
Fluconazole
1989
Itraconazole
1990
Voriconazole
2003
Caspofungin
2003
Polyene Macrolide Antibiotics
The discovery of nystatin (fungicidin) by Rachel
Brown and Elizabeth Hazen in the early 1950s
had led to the isolation and characterization of
numerous antibiotics. Amphotericin B
(fungizone), first isolated in the 1957 from
Streptomyces nodosus, an actinomycete
cultured from the soil of the Orinoco Valley in
Venezuela, was the first commercially available
systemic antifungal drug; so far, about 200
antifungal agents of this class exist. However,
problems associated with the stability, solubility,
toxicity and absorption of most such compounds,
cut down the number of polyenes approved for
therapeutic use to only a few.
Polyenes are characterized by a large macrolide ring of
carbon atoms closed by the formation of an internal ester
of lactone (Figure 1). The macrolide ring contains 12- 37
carbon atoms, the conjugated double bond structure is
contained exclusively within the cyclic lactone. A number
of hydroxyl groups (6-14) are distributed along the
macrolide ring on alternate carbon atoms. Amphotericin
B has a free carboxyl group and a primary amine group
that confer amphoteric properties on the compound,
hence the drug’s name. Being amphoteric, amphotericin
B tends to form channels through the cell membrane
causing cell leakage.
POLYENES
Amphotericine B
O
CH3
HO
O
CH3
H3C
OH
CH3
NH2
OH
OH
OH
O
COOH
O
OH
OH
OH
OH
O
OH
Nystatin
O
CH3
HO
O
CH3
H3C
OH
OH
OH
O
COOH
O
OH
OH
OH
OH
O
OH
FLUCYTOSINE
NH2
F
HN
O
N
H
Figure 6.1
CH3
NH2
OH
Although amphotericin B remains the preferred
compound for treating systemic mycoses, problems
associated with solubility in water, toxicity and
ineffectiveness against mold diseases in
immunocompromised patients limit its therapeutic
potential. Three lipid formulations of amphotericin B
(amphotericin B lipid complex, amphotericin B
cholesteryl sulfate and liposomal amphotericin B)
have been developed and approved for use in the
US. These drug delivery systems offer several
advantages over conventional amphotericin B. The
parent drug can be introduced in much higher
doses (up to 10-fold) compared with conventional
amphotericin B.
Mechanism of Action of Polyenes
Polyene antibiotics increase cell membrane permeability,
which causes leakage of cellular constituents (amino
acids, sugars and other metabolites), cell lysis and death.
Inhibition of aerobic and anaerobic respiration observed
in cells treated with polyenes is though to be a
consequence of leakage of cellular constituents.
Polyenes could also cause oxidative damage to the
fungal plasmalemma, which may contribute to the
fungicidal activity of the drug. Inhibition of fungal growth
by polyenes depends, to a large extent, on the binding of
the drug to the cell; only cells that bind appreciable
amounts of the drug are sensitive. Bacterial cells and
protoplasts do not take up polyenes; therefore, they are
resistant to the drug.
Polyene antifungals selectively bind to membrane sterols;
ergosterol in fungal cells and cholesterol in mammalian
cells.
The interaction of larger polyenes like amphotericin B
with fungal membrane sterols results in the production
of aqueous pores consisting of an annulus of eight
amphotericin B molecules linked hydrophobically to
membrane sterols (Figure 6.2). This leads to the
formation of pores in which the hydroxyl residues of
the polyene face inwards to give an effective pore
diameter of 0.4 to 1.0 nm. Leakage of vital
cytoplasmic components and death of the cell follows.
The selective mode of action of polyenes is also
related to the differential affinity of different polyenes to
membrane sterols on target cells. Amphotericin B
binds with high affinity to ergosterol in fungal cell
membrane.
PHARMACOKINETICS
PHARMACODYNAMICS
dose
absorption
SERUM
LEVELS
high
serum
or tissue
levels
MIC
KILLING
TISSUE
distribution
LEVELS
metabolism
low or
absent
serum
or tissue
levels
elimination
PK
PD
CORRELATION
(T>MIC AUC/MIC Cmax/MIC)
dose optimisation
TOXICITY
EFFICACY
RESISTANCE (?)
PAFE
PAFSE
Sites of action of antifungal agents
(A) Mechanism of azole action
(B) Mechanism of polyene
Endocellular
space
Lanosterolo
Zymosterolo Ergosterolo
Cytoplasm
Cytoplasm
Pore/channel formed by AmB
results in cell death
FLU inhibits ergosterol biosynthesis , resulting
In depletion of this sterol in the cell membrane
Ergosterolo
Cell membrane
Amphotericin B
ergosterolo
fluconazolo
Cell wall
(C) Mechanism of 5-fluorocytosine action
5-FUMP
Cytosine
5-FC permease 5-FC
Inhibition
of protein
synthesis
-(1,6)-D-glucan
Candin
Cell wall
Cell
membrane
5-FU
5-FdUMP
Cell membrane
(D) Mechanism of echinocandin action
-(1,3)-D-glucan
cytoplasm
Inhibition
of DNA
synthesis
Candins inhibit
fungal glucan synthase
-(1,6)-D-glucan synthase
Mukherjee PK et al.,
Clin Microbiol Rev, 2005
Membrane Antifungal Agents
SQUALENE
ALLYLAMINES:
Naftifine
Terbinafine
Squalene epoxidase
Squalene epoxide
LANOSTEROL
Lanosterol 14-α demethylase
14-α-demethyl lanosterol
Zymosterol
Fecosterol
ERGOSTEROL
POLYENES:
Amphotericin B
Nystatin
AZOLES:
Ketoconazole
Fluconazole
Itraconazole
Voriconazole
Structural features of Amphotericin B
Hydrophilic stretch
Hydrophobic stretch
Mycosamine ring with
Carbohydrate moiety
AMPHOTERICIN B DEOXYCOLATE
Main side effects
Nausea and vomiting
Fever, chills
Artralgia, myalgia
Headaches
Thrombophlebitis





Nephrotoxicity
Hypotension
Cardiotoxicity
Bronchospasm




Main pharmacokinetic parameters (0.5-1.0 mg/kg)
Cmax 1.2-2.4 mg/ml
T1/2 initial phase: 24-48 h
T1/2 terminal phase: 15 days
Protein binding: 91 - 95 %




Elimination: biliary-renal 
Fu 24 h: 3 - 5 % 
CSF/serum: 2 - 4 % 
Modified from Como and Dismuekes, 1994; Groll et al., 1998
Physicochemical information on the lipid formulations of
AmB in comparison to AmB deoxycholate
c-AmB
Brand name
Fungizone
L-AmB
ABLC
Liposomal AmB
AmB lipid complex
AmBisome
Abelcet
Lipids (molar ratio) Deoxycholate HPC/CHOL/DSPG
(2:1:0.8)
Mol% AmB
Lipid configuration
Diameter (mm)
DMPC/DMPG (7:3)
34%
10%
35%
Micelles
Liposomes
Lipid-sheets
< 0.4
0.08
1.6 - 11.0
The first Liposomal formulation of AMB is made up by a
mixture of two phospholipid : Dimyristoyl
phosphatidylcholine [ DMPC] and dimyristoyl
phosphatidylglycerol [ DMPG] in a 7:3 molar ratio with 510% of the mixture being AMB.
Mechanisms of action
Amphotericin B (AMB)
Amphotericin B lipid
formulations
(formation of transmembrane pores)
Host cell
Fungal cell
endosome
a
b
Free
AmB
lysosome
AmB-LDL
a
c
AmB-lipid
complex
Slow release of free
AmB
d
d
c
b
endosome
Fungalcell
cell
Fungal
LDL=low density lipoproteins
cholesterol
endosome
parasite
lipid peroxidation
ergosterol
macrophages
Hartsel S and Bolard J, TiPS, 1996
Antifungal activity of amphotericin B
VERY ACTIVE
AVERAGE ACTIVITY
Candida spp
Criptococcus neoformans
Blastomyces dermatitidis
Histoplasma capsulatum
Sporothrix schenckii
Coccidioides immitis
Paracoccidioides braziliensis
Aspergillus spp
Penicillium marneffei
Candida lusitaniae
Candida tropicalis
Candida parapsilosis
Scedosporium boydii
Fusarium spp
Malassezia furfur
Trichosporon beigelii
Physicochemical and pharmacokinetic information on the lipid
formulations of AmB in comparison to AmB deoxycholate
AmB
L-AmB
ABLC
Brand name
Lipids (molar ratio)
Fungizone
Deoxycholate
Mol% AmB
Lipid configuration
34%
Micelles
< 0.4
1 mg/kg
----+++
High
AmBisome
HPC/CHOL/DSPG
(2:1:0.8)
10%
SUVs
0.08
3-5 mg/kg
Increased
Increased
Decreased
Decreased
±
Mild
Abelcet
DMPC/DMPG
(7:3)
35%
Ribbon-like
1.6 - 11.0
5 mg/kg
Decreased
Decreased
Increased
Increased
±
Moderate
Diameter (mm)
Standard dosage (mg AmB/kg)
Cmax (relative to AmB)
AUC (relative to AmB)
Vd (relative to AmB)
Cl (relative to AmB)
Relative nephrotoxicity
Infusion-related toxicity
AmB, amphotericin B deoxycholate; L-AmB, liposomal AmB; ABLC, AmB lipid complex; HPC, hydrogenated
phosphatidylcholine; CHOL, cholesterol; DSPG, disteaorylphosphatidylglycerol; DMPC, dimyristoyl
phosphatidylcholine; DMPG, dimyristoyl phosphatidylglycerol; SUV, small unilamellar vesicles (liposomes)
Azole derivatives
• FIRST GENERATION
– Ketoconazole
• SECOND GENERATION
– Fluconazole
– Itraconazole
• THIRD GENERATION
– Voriconazole ( most widly used)
– Ravuconazole (BMS-207147)
– Posaconazole (SCH-56592)
R-120758
VR-9746
SYN-2869
VR-9751
T-8581
(D0870)
The inhibition of fungal growth by azole derivatives was
described in the 1940s and the fungicidal properties of Nsubstituted imidazoles were described in the 1960s. Clotrimazole
and miconazole have proven very important in combating human
fungal infections. More than 40 of the β-substituted 1phenethylimidazole derivatives are known to be potent against
fungi, dermatophytes and Gram-positive bacteria. Imidazoles
and triazoles are available for treatment of systemic fungal
infections. Imidazoles are five –membered ring structures
containing two nitrogen atoms with a complex side chain
attached to one of the nitrogen atoms. The structure of triazoles
is similar but they contain three nitrogen atoms in the rings
(Figure). Imidazoles in clinical use are clotrimazole, miconazole,
econazole and ketoconazole. Triazole compounds approved for
clinical use are itraconazole, fluconazole, voriconazole,
lanconazole, ravuconazole and posaconazole
Mechanism of Action
Antifungal activity of azoles is mediated mainly through the
inhibition of a cytochrome P450–dependent enzyme involved in
the synthesis of ergosterol. In eukaryotic cells, these are integral
components of the smooth endoplasmic reticulum and the inner
mitochondrial membrane. They contain an iron protoporphyrin
moiety located at the active site and play a key role in metabolic
and detoxification reactions. They interact with sterols, steroids,
bile acids, phenols, alkenes, epoxides, sulfones, and soluble
vitamins. Azoles activity is also manifested in inhibiting
cytochrome C oxidative and peroxidative enzymes, influencing
cell membrane fatty acids causing leakage of proteins and amino
acids, inhibiting catalase systems, decreasing fungal adherence
and inhibiting germ tube and mycelia formation
The principle molecular target of azoles (fluconazole,
itraconazole and voriconazole) is a cytochrome P450–Erg 11P or
Cyp 51P according to gene–based nomenclature. Cytochrome
P450–Erg 11P catalyses the oxidative removal of the 14 αmethyl group in lanosterol and/or eburicol by P450 mono–
oxygenase activity. As cytochrome P450–Erg 11P contains an
iron protoporphyrin moiety located at the active site, the drug
binds to the iron atom via a nitrogen atom in the imidazole or
triazole ring. Inhibition of 14 α–demethylase leads to depletion
of ergosterol and accumulation of sterol precursors including 14
α–methylated sterol as shown in figure 6.5. With ergosterol
depleted and replaced by unusual sterols, permeability and
fluidity of the fungal cell membrane is altered. Miconazole and
ketoconazole can inhibit the ATPase system in the cell
membrane of C. albicans and other yeasts, which may account
for the rapid collapse of the electrochemical gradient and the fall
in intracellular ATP.
Additionally, at growth inhibitory concentrations, miconazoles
and ketoconazoles tend to inhibit the activity of C. albicans
plasma membrane glucan synthase, chitin synthase,
adenylcyclase and 5-nucleotidase enzymes.
Incubation of C. albicans and other yeasts at fungistatic
concentrations with clotrimazole, miconazole, econazole,
voriconazole, posaconazole or ketoconazole results in extensive
changes in the cell envelope especially the plasma membrane;
for example, the appearance of holes in the nuclear membrane.
At fungicidal concentrations however, changes in the membrane
are more pronounced and include the disappearance of
mitochondrial internal structures and the complete loss of the
nuclear membrane. Ketoconazole can affect the transformation
of C. albicans from the budding form to the pseudomycelial
form, the prevailing type found in infected individuals.
Antifungal activity of triazoles
Candida albicans
Candida non albicans
Aspergillus spp
Criptococcus neoformans
Fusarium spp
Scedosporium spp
Blastomyces dermatitidis
Histoplasma capsulatum
Sporothrix schenckii
Coccidioides immitis
Zygomycetes spp
Paracoccidioides braziliensis
Dermatophytes spp
Malassezia furfur
Fluconazole
Itraconazole
Voriconazole
+++
+ +*
+++
+++
++
++
+/+++
+++
+++
+++
+++
+ +**
+++
+++
++
+++
+++
+++
+/+++
++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+ + + very active + + average activity +/- low activity - no activity
* except C.krusei and C.glabrata ** +/- for C.krusei
Membrane Antifungal Agents
SQUALENE
ALLYLAMINES:
Naftifine
Terbinafine
Squalene epoxidase
Squalene epoxide
LANOSTEROL
Lanosterol 14-alpha demethylase
14-alpha-demethyl lanosterol
Morpholines
Zymosterol
Fecosterol
ERGOSTEROL
POLYENES:
Amphotericin B
Nystatin
AZOLES:
Ketoconazole
Fluconazole
Itraconazole
Voriconazole
Pharmacokinetic properties of
oral azole agents
Dose Ketoconazole
200 mg po
Itraconazole
200 mg po
Fluconazole
200 mg po
Voriconazole
400mg 200mg po
Bioavailability (%)
75
< 70
> 90
96
Plasma Cmax (mg/l)
1.5 - 3.1
0.2 - 0.4
10.2
2-4
Tmax (h)
1-4
4-5
2-4
1-2
t ½ (h)
7 - 10
24 - 42
27 - 37
6-9
Protein binding (%)
99
> 99
11
58
CSF penetration (%)
< 10
<1
> 70
> 60
>1-2
>1-2
>3
4 .6
2-4
<1
80
-
Vd (l/kg)
Urinary recovery* (%)
*as active drug
Dismukes W.E., CID, 2000
Itraconazole
Combination with cyclodextrine in the oral solution
Bioavailability increased of 30-60% in HIV and/or
neutropenic patients
IV formulation as a complex with cyclodextrine
De Beule K. & Van Gestel J.V., Drugs, 2001
TRIAZOLES
Fluconazole
Itraconazole
PK-PD correlation = AUC/MIC
Current data suggest an exposure-dependent 
pharmacodynamics
Both compounds may be most effective when 
adequate levels are maintained at target site
Groll AH et al., 1999, 2001
Side Effect
Itraconazolo
Fluconazolo
(n=3446)
(n=3648)
Dispepsia
0,7 - 2,3% Nausea
2,1%
Dolori addominali
1,2 - 2%
Dolori addominali
1,4%
Nausea
1,2 - 1,8% Cefalea
1,2%
Diarrea
0,3 - 1,6% Diarrea
0,8%
Vertigini
0,2 - 1,2% Vomito
0,65%
Cefalea
Prurito
0,5 - 1%
Vertigini
0,2 - 0,5% Rash-cutanei
Prurito
0,5%
0,4%
0,3%
Azole derivatives
(Ketoconazole, Itraconazole, Fluconazole)
Drug interactions
Decreased absorption of azole
Antacids, H2 antagonists, Omeprazole
KETOCONAZOLE
ITRACONAZOLE
Increased metabolism of azole
Rifampin
Phenytoin
Carbamazepine
ALL
KETOCONAZOLE, ITRACONAZOLE
ITRACONAZOLE
Decreased metabolism of other drugs
Cyclosporine
Tacrolimus
Phenytoin
Warfarin
Sulphonylureas
Benzodiazepines
Statins
Terfenadine, Loratadine
Cisapride
Digoxin
ALL
FLUCONAZOLE
ALL
ALL
ALL
ALL
ITRACONAZOLE
ITRACONAZOLE
ALL
ITRACONAZOLE
Voriconazole
Metabolism
Voriconazole is metabolised in vitro via three
CYP450 isozymes
CYP2C19
CYP2C9
CYP3A4
In vivo the major isozyme is CYP2C19
Genetic polymorphism in CYP2C19
HL Hoffman & R Chris Rathbun, Expert Opin Investig Drugs, 2002
FDA - Briefing document for Voriconazole - Pfizer, October 2001
courtesy of Dr. N. Wood, Pfizer Central Research
Voriconazole metabolism
• CYP2C19 genetic polymorphism results in significant
inter-subject variation in plasma levels and drug
exposure
– Poor metabolizers (3-5% Caucasians and Blacks, 15-20%
Asians) have 3-4 fold increase in systemic exposure
Heterozygous poor metabolizers have about a 2-fold
increase in exposure
• Genotype, age and gender result in wide intersubject variability in exposure
Flucytosine
Flucytosine or 5–fluorocytosine (5-FC) is a synthetic
fluorinated pyrimidine used as an oral antimycotic agent.
It was first synthesized in the 1950s as a spin off of work
in cytostatic and antineoplastic agents. 5-FC lacks such
activities but it has noticeable antifungal activity.
Currently, 5-FC is used as an adjunct to amphotericin B
therapy because amphotericin B potentate the uptake of
5-FC through increasing fungal cell membrane
permeability. The activity of 5-FC is enhanced when used
in combination with fluconazole against C. neoformans
and C. albicans
Mechanism of Action
The antifungal activity of 5-FC is mediated through one of two mechanisms:
(i) disruption of DNA synthesis and /or (ii) alteration of the amino acid pool.
Initially, 5-FC enters susceptible cells by means of cytosine permease,
which is usually responsible for the uptake of cytosine, adenine, guanine,
and hypoxanthine. Once inside the cell, 5-FC is converted to 5- fluorouracil
(5FU) by cytosine deaminase. Inside target cells, 5-FU is then converted by
uridine monophosphate pyrophosphorylase to 5-fluorouridylic acid (FUMP),
which is phosphorylated further and incorporated into RNA resulting in
disruption of protein synthesis. Extensive replacement of uracil by 5-FC in
fungal RNA can lead to alterations in the amino acid pool. Some 5-FU can
be converted to 5-fluorodeoxyuridine monophosphate, which functions as a
potent inhibitor of thymidylate synthase, one of the enzymes involved in
DNA synthesis and nuclear division.
Inhibition of DNA synthesis in C. albicans can take place before 5-FU
incorporation into RNA or inhibition of protein synthesis. Resistant
strains of C. neoformans incorporate 5-FC into RNA at levels
comparable with sensitive strains. This could mean that resistance
inhibition of DNA synthesis is more important than the production of
aberrant RNA in mediating the effects of 5-FC. The drug incorporates
in large quantities into the 80S ribosomal subunits in C. albicans. The
number of ribosomes synthesized in the presence of high
concentrations of 5-FC is greatly reduced.
Morphological and ultrastructural changes that occur in C. albicans
cells include increased cell diameter if growing at sub-inhibitory
concentrations of 5-FC
Echinocandins
The fungal cell wall contains compounds, such as mannan,
chitin, and α– and β-glucans, which are unique to the
kingdom Fungi. A number of compounds that have the ability
to affect the cell wall of fungi have been discovered and
described over the past 30 years. Of the three groups of
compounds (aculeacins, echinocandins, and papulacandins)
that are specific inhibitors of fungal 1-3 β-glucan synthase.
Echinocandins are actively pursued in clinical trials to
evaluate their safety, tolerability and efficacy against
candidiasis. Discovered by random screening in the 1970s,
echinocandins are fungal secondary metabolites comprising a
cyclic hexapeptide core with a lipid side chain responsible for
antifungal activity. A modified form of echinocandin B,
cilofungin, was developed to the point of phase 2 trials, but
then abandoned due to increased toxicity. In the late 1990s,
three
echinocandin
compounds
(anidulafungin,
caspofungin and micafungin) entered clinical development
and evaluation.
Caspofungin: Mechanism of Action
Ergosterol
Caspofungin specifically inhibits beta (1-3)-D-glucan synthesis, essential to
the cell-wall integrity of many fungi, including Aspergillus and Candida spp,
thereby compromising the integrity

As a result, the fungal cell wall becomes permeable, and cell lysis

Beta (1-3)-D-glucan synthesis does not occur in human cells

Antifungal activity of the echinocandins
HIGHLY ACTIVE
VERY ACTIVE
SOME ACTIVITY
INACTIVE
Candida albicans
Candida glabrata
Candida tropicalis
Candida krusei
Candida kefyr
Pneumocystis carinii*
Candida parapsilosis
Candida gulliermondii
Aspergillus fumigatus
Aspergillus flavus
Aspergillus terreus
Candida lusitaniae
Coccidioides immitis
Blastomyces dermatididis
Scedosporium spp
Paecilomyces variotii
Histoplasma capsulatum
Zygomycetes
Cryptococcus neoformans
Fusarium spp
Trichosporon spp
* Only active against cyst form, and probably only useful for prophylaxis
Denning DW, Lancet, 2003
CASPOFUNGIN
Pharmacokinetic parameters in healthy adults
Variable
Parameter
Peak (mg/l)
12.1
Trough (mg/l)
1.3
Volume of distribution (l)
9.7
AUC0-24h (mg·h/l)
93.5
Half-life (h)
a Phase
 Phase
g Phase
1-2
9-11
40-50
Protein binding (%)
96.5
Clearance (ml/min)
12
Renal clearance (ml/min)
0.15
Deresinski SC & Stevens DA, CID, 2003
Chitin synthesis is inhibited competitively by polyoxin and
nikkomycin, nucleoside–peptide antibiotics produced by
soil strains of Streptomycetes. These agents specifically
inhibit chitin synthase by acting as mimics or decoys of
the enzyme substrate (uridine diphosphate-Nacetylglucosamine). In vitro susceptibility testing of
nikkomycins X and Z against various fungi show
moderate susceptibility of C. albicans and C. neoformans
to these compounds. Activity against C. albicans and C.
neoformans improves significantly when nikkomycin Z is
used in combination with fluconazole and itraconazole
Allylamines and Thiocarbamates
Naftifine and terbinafine are the two major allylamines in
clinical use and tolnaftate is the only thiocarbamate
available for use. Naftifine is used as a topical agent while
terbinafine is administered orally. These are two synthetic
compounds with a chemical structure similar to
naphthalene ring substituted at 1 position with an aliphatic
chain. Both allylamines thiocarbamates function as
noncompetitive inhibitors of squalene epoxidase, an
enzyme involved in the conversion of squalene to
lanosterol, which is an essential step in the synthesis
fungal cell membrane.
Cell death is dependent on the accumulation of squalene
rather than ergosterol deficiency as high levels of
squalene increase membrane permeability leading to
disruption of cellular organization. Terbinafine inhibits the
growth of dermatophytic fungi in vitro at concentrations of
0.01 µg/ml or lower.