ArticlePDF AvailableLiterature Review

Antifungal agents of use in animal health - Chemical, biochemical and pharmacological aspects

March 2003
Journal of Veterinary Pharmacology and Therapeutics 26(1):5-29

DOI:10.1046/j.1365-2885.2003.00456.x

Source
PubMed

Authors:

Hugo Vanden Bossche

Janssen Pharmaceutica

Marc Engelen

Janssen Pharmaceutica, Johnson & Johnson

Frans Rochette

Janssen Pharmaceutica Beerse Belgium

A limited number of antifungal agents is licensed for use in animals, however, many of those available for the treatment of mycoses in humans are used by veterinary practitioners. This review includes chemical aspects, spectra of activity, mechanisms of action and resistance, adverse reactions and drug interactions of the antifungals in current use.

. Antifungal drugs

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Chemical structures of azole antifungal agents.

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Chemical structures of azole antifungal agents.

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Chemical structures of terbinafine, tolnaftate, thiabendazole and flucytosine.

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Chemical structures of terbinafine, tolnaftate, thiabendazole and flucytosine.

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Content uploaded by Hugo Vanden Bossche

Content may be subject to copyright.

Content uploaded by Hugo Vanden Bossche

Content may be subject to copyright.

Antifungal agents of use in animal health – chemical, biochemical

and pharmacological aspects

H. VANDEN BOSSCHE*

M. ENGELEN



F. ROCHETTE



*Steenweg op Gierle, Turnhout, Belgium;



Janssen Animal Health B.V.B.A.,

Turnhoutsebaan, Beerse, Belgium

Vanden Bossche, H., Engelen, M., Rochette, F. Antifungal agents of use in

animal health – chemical, biochemical and pharmacological aspects. J vet.

Pharmacol. Therap. 26, 5–29.

A limited number of antifungal agents is licensed for use in animals, however,

many of those available for the treatment of mycoses in humans are used by

veterinary practitioners. This review includes chemical aspects, spectra of

activity, mechanisms of action and resistance, adverse reactions and drug

interactions of the antifungals in current use.

(Paper received 12 July 2002; accepted for publication 29 November 2002)

Hugo Vanden Bossche, Steenweg op Gierle, 68, B2300 Turnhout, Belgium. E-mail:

hugo.vdbossche@skynet.be

INTRODUCTION

For most mycoses veterinary practitioners have a variety of

antifungal agents at their disposal. An outlook of the different

classes is given in Table 1. The earliest known compound with

proven antifungal activity is griseofulvin – the product of

Penicillium species. The spectrum of activity of this fungicidal

agent is limited to dermatophytes (reviewed by Polak, 1990).

The polyene macrolides, natural products of Streptomyces

species, are a class of poorly absorbed, large macrocyclic polyke-

tides that interact with membrane sterols. The antifungal polyenes

were the earliest broad-spectrum antifungal agents introduced for

clinical use (Odds, 1996). Amphotericin B (AmB), candicidin,

nystatin and natamycin (pimaricin) have found use in the topical

treatment of superﬁcial infections of men and ⁄or animals. AmB is

mainly formulated for intravenous administration.

An amazing number of antifungal agents are found within the

group of azole-containing compounds. A great number were

breakthroughs in antifungal therapy in both human and

veterinary medicine, and some of them show excellent activity

against phytopathogenic fungi. Examples are the imidazole

derivatives bifonazole, clotrimazole, econazole, enilconazole

(imazalil), ketoconazole, miconazole and parconazole, and the

triazole derivatives terconazole, itraconazole, ﬂuconazole and

propiconazole. Most of the imidazole derivatives (e.g. miconazole,

clotrimazole and econazole) are available for topical application

only, enilconazole and ketoconazole are the exceptions. Enil-

conazole has been developed as a smoke generator and as a

spray formulation (Desplenter, 1988). The dioxolane imidazole

derivative ketoconazole is the ﬁrst compound of the azole class

that is systemically absorbed after oral administration (Thien-

pont et al., 1979; Daneshmend & Warnock, 1988), and is used

both orally and topically. Fluconazole and itraconazole are used

orally and intravenously. The antifungal activity of the azole

derivatives arises from interaction with the sterol-14a-demethy-

lase, involved in the biosynthesis of ergosterol.

Other groups of antifungal agents, the allylamines (naftiﬁne

and terbinaﬁne) (Ryder, 1988; Ryder & Mieth, 1992) and the

thiocarbamates (e.g. tolnaftate) (Barrett-Bee et al., 1986; Ryder

et al., 1986) perturb fungal sterol synthesis by inhibiting the

squalene epoxidase. The use of the allylamines and the much older

tolnaftate is essentially restricted to dermatophyte infections.

The benzimidazoles, such as carbendazim, fuberidazole, and

thiabendazole are effective at relatively low doses for the

inhibition of a broad range of plant pathogenic fungi (literature

reviewed by Delp, 1995).

This review includes chemical, biochemical and pharmacolo-

gical aspects of the most interesting antifungal agents registered

for the treatment of fungal infections in animals as well as those

used off label. In the next issue of this journal the clinical aspects

of the most interesting antifungal agents and their applications

in animals are reviewed.

GRISEOFULVIN

Although griseofulvin (Fig. 1) was isolated from Penicillium

griseofulvum as early as 1939 (Oxford et al., 1939), its action

against experimental ringworm in guinea-pigs was ﬁrst des-

cribed in 1956 (Gentles, 1956). The use of griseofulvin as an

orally active antifungal against ringworm in man was ﬁrst

described by Williams et al. (1958).

Griseofulvin is practically insoluble in water, slightly soluble in

ethanol and soluble in dimethylformamide (DMF). It is effective

after oral ingestion and reaches the stratum corneum. Fat and

reduction of the particle size enhances the absorption of

griseofulvin (Lin et al., 1973). The compound is metabolized by

the liver. Chang et al. (1975) compared the metabolism of

C-

griseofulvin in rat liver microsomes, isolated perfused rat livers,

and rats with bile duct cannulas. In all three models,

4-desmethylgriseofulvin and 6-desmethylgriseofulvin were the

major metabolites. In isolated perfused liver, most of the drug

J. vet. Pharmacol. Therap. 26, 5–29, 2003. REVIEW

2003 Blackwell Publishing Ltd 5

(59% of dose) was excreted into bile within 4 h, primarily as

4-desmethylgriseofulvin and 6-desmethylgriseofulvin. In animals

with bile duct cannulas, 65% of the dose was excreted into bile

and 18% into urine.

Mode of action

Griseofulvin inhibits the growth of dermatophytes by preventing

microtubules from sliding and thus inhibits mitosis (Crackower,

1972; Gull & Trinci, 1973; Langcake et al., 1983). Griseofulvin

also affects the cytoplasmic microtubules. These are involved in

intracellular transport of endogenous compounds. Polak (1990)

hypothesized that this interaction results in disturbance of the

transport of newly synthesized cell wall constituents and may

lead to the distortion of the hyphae, the ‘curling’ effect. In a more

recent study, Chaudhuri and Luduena (1996) proved that

griseofulvin interacts with tubulin and changes its conformation,

but this mycotoxin ‘binds’ to another site than colchicine, a well-

known inhibitor of tubulin polymerization.

Spectrum of activity

The spectrum of activity of griseofulvin is limited to Trichophyton,

Epidermophyton and Microsporum species. This is due to the fact

that dermatophytes possess a prolonged energy-dependent

transport system for this antibiotic, whereas in insensitive cells,

such as Candida albicans, this is replaced by a short energy-

independent transport system (Polak, 1990).

Differences, depending on the test method used, have to be

taken into account when comparing minimum inhibitory

concentration (MIC) data in the literature. For example,

Niewerth et al. (1998) compared the susceptibility of dermato-

phyte strains to griseofulvin, itraconazole, sertaconazole, terbin-

aﬁne and cyclopiroxolamine by using an agar macrodilution and

broth microdilution test. For all ﬁve antimycotics, MIC data were

three- to seven-fold lower in the microdilution test system.

Korting et al. (1995) used the microdilution test to compare

the susceptibility of T. rubrum and T. mentagrophytes. The

CH3CH3

NH2

CH3

H3C

H2N

OGriseofulvin Natamycin

HO OH

OHO

COOH

OHOH OH

COOH

Nystatin A1

OHOH

CH3

COOH

Amphotericin B

Fig. 1. Chemical structures of griseofulvin

and the polyenes.

Table 1. Antifungal drugs

Chemical classes Drugs* Targets

Griseofulvin (o) Inhibits microtubule sliding

Polyenes Amphotericin B (iv, t, o) Membrane barrier function

Natamycin (t)

Nystatin (t, o)

Azoles Clotrimazole (t) Ergosterol biosynthesis

Imidazoles Econazole (t) 14a-demethylase

Enilconazole (t) CYP51

Ketoconazole (t, o)

Miconazole (t)

Parconazole (o)

Triazoles Fluconazole (o, iv)

Itraconazole (o, iv)

Allylamines Terbinaﬁne (t, o) Squalene epoxidase

Thiocarbamates Tolnaftate (t)

Benzimidazoles Thiabendazole (o, t) Microtubule assembly

Pyrimidine Flucytosine (o, iv) DNA and RNA synthesis

*o, oral; t, topical; iv, intravenous.

6H. Vanden Bossche et al.

2003 Blackwell Publishing Ltd, J. vet. Pharmacol. Therap. 26, 5–29

concentration of griseofulvin that inhibited 90% of isolates of

T. mentagrophytes was 10 mg ⁄L, whereas for T. rubrum this was

achieved at 3 mg ⁄L. More recently Jessup et al. (2000a) used

oatmeal cereal agar medium to determine the antifungal

susceptibilities of T. rubrum to ﬂuconazole, itraconazole, terbin-

aﬁne and griseofulvin. The mean ± SE of the mean MICs

were 2.07 ± 0.29, 0.13 ± 0.01, 0.002 ± 0.0003 and 0.71 ±

0.05 mg ⁄L, respectively. Using a broth macrodilution method,

Perea et al. (2001a) found for M. canis and M. gypseum an

MIC50 of 0.5 mg ⁄L, whereas for T. equinum and T. verrucosum

MIC50s of 8 and 5.66 mg ⁄L were found.

Resistance of dermatophytes to griseofulvin is rare. However,

in the study of Korting et al. (1995) 11 of the 32 T. rubrum

strains tested showed an MIC of 3 mg ⁄L or greater. According to

Artis et al. (1981) such MIC values indicate resistance to

griseofulvin.

Drug interactions ⁄adverse reactions

Griseofulvin has been shown to induce drug-metabolizing

enzymes (Okey et al., 1986). For example, it increases the rates

of warfarin metabolism. Serum levels of griseofulvin are

decreased by coadministration of phenobarbital. It has been

reported that administration of this antibiotic markedly stimu-

lates the activity of 5-aminolevulinate synthase, the rate-limiting

enzyme of porphyrin synthesis, and causes a rapid inhibition of

mitochondrial porphyrin-metal chelatase and may thereby

inhibit the synthesis of haem in the liver (De Matteis, 1982).

Embryotoxic and teratogenic effects of griseofulvin have been

demonstrated in pregnant rats exposed during organogenesis

(De Carli & Larizza, 1988).

POLYENES

Polyene macrolides are a class of poorly absorbed, large

macrocyclic polyketides that interact with membrane sterols.

They are produced by species of Streptomyces, and characterized

by large lactone rings, containing three to eight conjugated

double bonds, which are generally combined with one sugar

moiety (Hamilton-Miller, 1973; Ryley et al., 1981). The polyenes

can be subdivided into trienes (rapamycin), tetraenes (natamy-

cin), pentaenes (pentamycin), heptaenes (AmB and candicidin)

and a pseudo-heptaene ⁄tetraene, nystatin. They are effective

against eukaryotic organisms and those prokaryotic organisms

containing sterols in their membranes (Kerridge & Whelan,

1984). The antifungal polyenes AmB, candicidin, nystatin and

natamycin (pimaricin) have found use in the topical treatment of

superﬁcial infections of men and ⁄or animals.

Mechanism of action

The toxicity of the polyenes to fungal cells is thought to occur as a

result of its binding to sterols incorporated into cell membranes

changing as such the physical state of the membrane, causing an

impairment of membrane functions, resulting in an enhanced

permeability to protons and leakage of internal constituents such

as K

,Ca

and PO

3–

(Bolard, 1986; Vanden Bossche et al.,

1987a; Brajtburg et al., 1990; Vanden Bossche, 1995). The

addition of AmB to yeast induced an increased permeability for

protons resembling the increased proton inﬂux with the proton

conductor dinitrophenol. This collapses the proton gradient and

therefore the uptake of nutrients. The effects of polyenes on the

proton gradient may result from an interaction with the proton-

translocating adenosine triphosphatase (H

-ATPase) found to be

present in the plasma membrane of, for example, Saccharomyces

cerevisiae (Goffeau & Slayman, 1981), C. albicans (Hubbard et al.,

1986), Neurospora crassa (Rao et al., 1988), Fusarium oxysporum

(Brandao et al., 1992), C. neoformans (Soteropoulos et al., 2000)

and Aspergillus fumigates (Burghoorn et al., 2002). After solubi-

lization and puriﬁcation, the H

-ATPase requires phospholipids

to provide sufﬁcient ﬂuidity for maximal activity (Goffeau &

Slayman, 1981). Therefore, it is tempting to speculate that the

effects of polyenes on the proton gradient may result from a

speciﬁc interaction with components that determine the ﬂuidity

of the membrane and thus the activity of the H

-ATPase.

At concentrations >0.3 mg ⁄L AmB is lethal to, for example,

C. albicans and C. neoformans and is lytic to erythrocytes and

protoplasts (Brajtburg et al., 1990).

The polyenes have been divided into groups: those provoking

potassium leakage and cell death in S. cerevisiae and haemolysis

in erythrocytes at comparable concentrations, and a second

group in which haemolysis occurs at much higher concentra-

tions than yeast potassium leakage. The topically active

natamycin belongs to the ﬁrst group, whereas AmB and nystatin

belong to the second group (Ryley et al., 1981). This selectivity

may be due to the greater interaction of, for example, AmB with

ergosterol as compared with cholesterol (Readio & Bittman,

1982; Fournier et al., 1998). It should be noted that lethality is

not a simple consequence of changes in permeability of cell

membranes (Brajtburg et al., 1990). Evidence for the role of

reactive forms of oxygen in the lethal or lytic action of AmB was

obtained from studies which showed that AmB injury to cells can

be modulated by extracellular scavengers, such as 1-phenyl-3-

pyrazolidone, and prooxidants (e.g. ascorbic acid). Exposure of

erythrocytes or protoplasts of C. albicans to AmB under hypoxic

conditions reduced the polyene-induced lysis, and cells with

higher catalase activity are less sensitive to lysis by AmB

(reviewed by Brajtburg et al., 1990, 1992).

Thus, the mechanism of action of polyenes is much more

complex than just binding to sterols.

Resistance

Resistance to polyenes is unusual, but known (for reviews see

Vanden Bossche et al., 1994a, 1998; White et al., 1998; Espinel-

Ingroff et al., 2000; Ellis, 2002). Resistance of C. albicans,

C. tropicalis and C. glabrata strains to AmB and to nystatin was

associated with loss or marked depression of ergosterol in the cell

membranes (Dick et al., 1980). Uchida et al. (1994) studied the

membrane composition in polyene antibiotic-sensitive and

resistant strains of Malassezia pachydermatis. The wild-strains

Antifungal agents 7

2003 Blackwell Publishing Ltd, J. vet. Pharmacol. Therap. 26, 5–29

contained more than 0.8 mg of sterols (79% ergosterol) per

gram freeze-dried mycelium, whereas the resistant mutants

contained less than 0.35 mg ⁄g (76% ergosterol).

Ziogas et al. (1983) analysed the sterol content of pimaricin-

resistant A. nidulans mutants and found a correlation between

the resistance level and the ergosterol content of 17 mutants,

with low ergosterol contents leading to elevated levels of

resistance. However, in a number of mutants, resistance was

not related to the ergosterol content. Diverse factors inﬂuence

the sensitivity of fungi to polyene antibiotics. Therefore, it is not

surprising that a diversity of mechanisms may cause resistance.

Intrinsic resistance to AmB is common for C. lusitaniae (Pfaller

et al., 1994; Peyron et al., 2001), C. guilliermondii (Dick et al.,

1985) and Trichosporum beigelii (Walsh et al., 1990) and some

isolates of Pseudallescheria boydii (Scedosporium apiospermum) are

resistant to AmB in vitro with MICs well in excess of 2 mg ⁄L

(Vanden Bossche et al., 1994b; Ellis, 2002). The pseudofungus

Pythium insidiosum differs from the true fungi in many aspects,

for example the plasma membrane is free from sterols. Therefore,

polyenes (and also ergosterol biosynthesis inhibitors) cannot be

used in the treatment of pythiosis (Pier et al., 2000).

Polyenes of interest

Natamycin

Natamycin (pimaricin, Fig. 1) is a polyene antifungal antibiotic

produced by Streptomyces natalensis. A complex multienzyme

system encoded by ﬁve polyketide synthase genes is involved in

the biosynthesis of this 26-membered tetraene macrolide (Apar-

icio et al., 1999, 2000).

The natamycin molecule contains a large lactone ring of 25

C-atoms which is linked to the deoxysugar mycosamine (Raab,

1972). Natamycin is insoluble in water and most organic

solvents. It is not absorbed from the gastrointestinal tract.

Spectrum of activity

Natamycin is active against fungal organisms pathogenic for

animals and plants, and is important in the food industry as a

preservative to prevent mould contamination of foods. Natamy-

cin is effective against C. albicans,A. niger,Trichophyton spp.,

Epidermophyton ﬂoccosum,Acremonium spp., Cunninghamella spp.,

Fusarium spp. and Pseudallescheria boydii (Raab, 1972, 1978;

Reuben et al., 1989; Rotowa et al., 1990). Although it has been

advocated for use as an aerosol in pulmonary candidosis and

aspergillosis, Aspergillus spp., and also dermatophytes are

frequently resistant to natamycin. Its most important place as

an antifungal agent is in mycotic keratitis. Although natamycin

was found to control experimental A. niger keratitis, some

investigators did not ﬁnd subconjunctival pimaricin therapy to

be of use in the treatment of experimental keratitis, in rabbits,

caused by A. fumigatus (reviewed by Thomas & Rajasekaran,

1988).

Nystatin

Nystatin (Fig. 1) was discovered in the early 1950s (Hazen &

Brown, 1950). This polyene macrolide antibiotic is produced by

S. noursei (ATCC 11455). The nystatin molecule contains a

polyketide moiety represented by a 38-membered macrolactone

ring to which the deoxysugar mycosamine is attached.

Molecular cloning and characterization of the genes governing

the nystatin biosynthesis resulted in the identiﬁcation of genes

encoding the polyketide synthase, genes for thioesterase,

deoxysugar biosynthesis, modiﬁcation, transport and regulatory

proteins (Brautaset et al., 2000). At pH 6–7, nystatin is slightly

soluble in water (4 mg ⁄mL). Nystatin is not absorbed appreci-

ably from the gastrointestinal tract.

Spectrum of activity

Nystatin is active against a broad spectrum of fungi in vitro and

in vivo, including Candida spp., A. fumigatus,C. neoformans and

T. beigelii (Raab, 1978; Johnson et al., 1998). It has poor activity

against dermatophytes. For C. albicans the MIC90 and the

minimum lethal concentration (MLC90) ¼2mg⁄L, the MIC90

and MLC90 values for A. fumigatus were both 8 mg ⁄L (Johnson

et al., 1998). The use of nystatin is limited to superﬁcial

cutaneous and mucosal mycoses.

Adverse reactions

Parenteral administration of nystatin resulted in dose-limiting

toxicities and infusion-related reactions, this precluded for a

long time its development as a parenteral therapeutic agent

(Hamilton-Miller, 1973). A less toxic parenteral preparation of

nystatin has been formulated. It is a multilamellar liposomal

formulation containing dimyristoylphosphatidylcholine and

dimyristoylphosphatidylglycerol in a 7:3 ratio (Mehta et al.,

1987; Groll et al., 1999). In vitro, liposomal nystatin appeared to

be as active as free nystatin (Johnson et al., 1998).

Amphotericin B

Amphotericin B (Fig. 1) contains eight free hydroxyl groups in

the macrocyclic lactone ring and possess a glycosidically linked

carbohydrate moiety: 3-amino-3,6-dideoxy-

-mannopyranose

(mycosamine). This polyene is produced by S. nodosus. The

molecule is rod-shaped with an opposing hydrophobic and

hydrophilic face, and a length of 2.4 nm or about half the

thickness of a gel-phase phospholipid bilayer (Andreoli, 1974).

At pH 6–7 AmB is almost insoluble in water.

Due to its extremely poor intestinal absorption, for systemic

infections AmB is administered intravenously. Normally AmB is

formulated for intravenous administration as a complex with

sodium deoxycholate. Topical preparations may be used for the

treatment of superﬁcial Candida infections; they are of no value in

dermatophytoses (Odds, 1996).

Spectrum of activity

Amphotericin B-deoxycholate has, depending on the concen-

tration used, fungistatic and fungicidal properties and is

active in vitro against Candida species including C. albicans

(MIC90 ¼0.25 mg ⁄L; MLC90 ¼0.5 mg ⁄L), C. glabrata,C. kefyr,

C. krusei,C. tropicalis and C. parapsilosis (Johnson et al., 1998). It

is active against C. neoformans (MIC90 ¼0.5 mg ⁄L; MLC90 ¼

0.5 mg ⁄L), H. capsulatum,Sporothrix schenckii,Blastomyces

8H. Vanden Bossche et al.

2003 Blackwell Publishing Ltd, J. vet. Pharmacol. Therap. 26, 5–29

dermatitidis,Coccidioides immitis,Phialophora sp. and strains of

A. fumigatus (MIC90 ¼1mg⁄L; MLC90 ¼4mg⁄L) and A. ﬂavus

(MIC90 ¼1mg⁄L; MLC90 ¼1mg⁄L) (see for example, Shad-

omy & Espinel-Ingroff, 1974; Troke, 1993; Johnson et al., 1998;

Goldberg et al., 2000; Moore et al., 2001). An MIC value of

0.09 mg ⁄L has been reported for Absidia corymbifera (Troke,

1993). Recent data indicate that the sensitivity of

S. schenckii to AmB is strain dependent; the MIC and minimal

fungicidal concentrations (MFC) were 0.125–2 mg ⁄L

(McGinnis et al., 2001). Troke (1993) determined MIC values

(mg ⁄L) for Microsporum canis (0.04), M. gypseum (0.3),

T. quinckeanum (0.6), T. mentagrophytes (0.6), T. rubrum (0.3),

T. soudanense (0.09), T. tonsurans (0.05) and T. verrucosum (0.1).

AmB is also active against F. solani and other Fusarium species

(Reuben et al., 1989). Using the microdilution method recom-

mended by the National Committee for Clinical Laboratory

Standards (1998) (NCCLS) the MICs found for Scedosporium

apiospermum,S. proliﬁcans,F. oxysporum and F. solani were:

2–4 mg ⁄L, 0.5–16 mg ⁄L, 0.25–2 mg ⁄L and 0.5–2 mg ⁄L,

respectively (Meletiadis et al., 2000).

Adverse reactions

The multiplicity of interactions with both fungal and mamma-

lian cells may be the reason why AmB is such an effective, but

not a well tolerated, antifungal agent (for reviews see Armstrong

& Schmitt, 1990; Sarosi, 1990; Bennett, 1992). Nephrotoxicity

is the most common toxic effect, but according to Bennett

(1992), ‘the litany of toxicity would eventually include anaemia,

phlebitis, renal tubular acidosis, hypokalaemia, hypomagnesae-

mia, nausea, vomiting, weight loss and headache’. To produce

less toxic formulations AmB has been formulated in liposomes

and other lipid complexes. Examples are Ambisome (unilamellar

liposome), Amphocyl (ABCD, lipid disc), Abelcet (ABCL, lipid

ribbon) (for an overview of the lipid formulations see Dupont,

2002).

AZOLE ANTIFUNGAL AGENTS

The class of the antifungal imidazoles and triazoles is generally

the largest and most widely used. Examples of imidazole

derivatives are bifonazole, butoconazole, clotrimazole, econazole,

enilconazole, fenticonazole, isoconazole, miconazole, parcona-

zole and ketoconazole. Triazole derivatives are ﬂuconazole,

itraconazole, terconazole and voriconazole. According to Burt

(2001), there are 24 new triazoles that have been patented in

the past several years.

Imidazole derivatives

Clotrimazole

Clotrimazole (Fig. 2) was one of the ﬁrst imidazole-based

compounds to be developed as a broad spectrum agent for the

topical treatment of fungal infections (Plempel et al., 1969;

¨chel et al., 1972). Clotrimazole is a derivative of 1-methylim-

idazole with lipophilic substituents at the methyl group. It is

slightly soluble in water and soluble in DMF. Pharmacokinetic

investigations of clotrimazole in mice, rats and men revealed that

the compound had an inductive effect on microsomal liver

enzymes, which led to rapid metabolism and inactivation of the

active substance. Therefore, this imidazole derivative was

unsuitable as an oral antimycotic (Plempel et al., 1988).

Spectrum of activity

In vitro clotrimazole is active against dermatophytes, Candida

species, Aspergillus spp., C. immitis and C. neoformans.Allescheria

boydii and Phialophora spp. are less sensitive.

Using both the macrodilution and microdilution methods,

Pelletier et al. (2000) analysed 87 isolates of C. albicans for their

susceptibility to clotrimazole. The MIC for 90% of the strains

tested was £0.5 mg ⁄L. Among the isolates used in this study, 10

were recovered from patients who fulﬁlled the deﬁnition of

clinical resistance. For six of these 10, the MIC was ‡0.5 mg ⁄L.

Pelletier et al. (2000) suggest that a breakpoint of 0.5 mg ⁄L may

be useful in deﬁning clotrimazole resistance in C. albicans.

Using a modiﬁed NCCLS method Ferna

´ndez-Torres et al.

(2000) determined the MIC value for 100 isolates of T. rubrum,

the MIC50 and MIC90 were 0.03 and 0.125 mg ⁄L, respectively.

When cultured on Sabouraud’s dextrose agar medium an MIC

value of 0.267 mg ⁄L was found (Fukushiro et al., 1992). Using

the same medium, the MIC values for T. mentagrophytes and

M. canis were 0.255 and 0.266 mg ⁄L, respectively (Fukushiro

et al., 1992). For M. pachydermatis the MICs ranged between

<0.06 and 32 mg ⁄L (mean ¼4mg⁄L) (Schmidt, 1997).

Miconazole, econazole and enilconazole

Imidazoles with an ether group form an important class of

antifungal agents. Miconazole (Fig. 2), the ﬁrst member of the

group, was introduced around the same time as clotrimazole

(Godefroi et al., 1969; Van Cutsem & Thienpont, 1972). Several

other ether compounds have been synthesized; examples are

econazole, isoconazole and orconazole (Godefroi et al., 1969).

These compounds form the basis for further synthesis in many

pharmaceutical companies. Examples are tioconazole, sulcona-

zole, butoconazole and cloconazole. Another ether compound is

imazalil. This imidazole derivative is used in plant protection

(imazalil) and as a veterinary antimycotic (enilconazole).

Enilconazole has also been developed as an antimycotic disin-

fectant (Van Cutsem et al., 1985). In this review we will focus on

miconazole, econazole and enilconazole.

Miconazole nitrate is a white microcrystalline or amorphous

powder, very slightly soluble in water and moderately soluble in

most organic solvents. Its log partition coefﬁcient in a system of

n-octanol and an aqueous buffer is 5.9. The absorption of topical

applied miconazole is extremely low. When given orally to man,

even substantial doses only produce relatively low serum levels

(Boelaert et al., 1976). After intravenous infusion miconazole is

well distributed to the various body tissues, with high drug

concentrations in the lungs, liver, adrenals and kidneys. Poor

levels are observed in the cerebrospinal ﬂuid. Miconazole is

metabolized in the liver, less than 1% of the dose is excreted in

the urine (reviewed in Borgers et al., 1986).

Antifungal agents 9

2003 Blackwell Publishing Ltd, J. vet. Pharmacol. Therap. 26, 5–29

Econazole (Fig. 2) is very slightly soluble in water and most

common organic solvents (for a review of the older literature see

Raab, 1978).

Pharmacologically, enilconazole (Fig. 2) has virtually the

same characteristics as the other imidazole derivatives. Like the

other imidazole antifungal agents, enilconazole is largely broken

down and at least 35 metabolites have been identiﬁed. Enilcon-

azole has a low vapour pressure but evaporates easily in closed

environments to an antifungally active gas phase (Van Gestel

et al., 1981; Van Gestel, 1986).

Spectrum of activity

(1). Miconazole: Fungistatic concentrations [geometric mean

(GM) of MICs] of miconazole in casitone medium were tested

against C. albicans (3.9 mg ⁄L), C. tropicalis (1 mg ⁄L), C. parapsi-

losis (5 mg ⁄L), C. glabrata (5 mg ⁄L), Trichophyton spp. (1 mg ⁄L),

Aspergillus spp. (1 mg ⁄L) and H. capsulatum (mycelial phase,

1mg⁄L) (Polak & Dixon, 1987). Using the microdilution method

recommended by NCCLS the MICs found for A. fumigatus,

A. ﬂavus,S. apiospermum,S. proliﬁcans,F. oxysporum and F. solani

were: 4–16, 1–16, 1–8, 16 to >64, 1 to >64 and 4 to

>64 mg ⁄L, respectively (Meletiadis et al., 2000). Using an other

NCCLS (1992) method Hennequin et al. (1997) found for 13

isolates of S. apiospermum a mean MIC90 value of 0.12 mg ⁄L.

Miconazole was tested for its activity against 23 isolates of

M. furfur by agar dilution. A GM of MIC of 14 mg ⁄L was found

(Van Gerven & Odds, 1995). A similar sensitivity of M. furfur was

found by Hammer et al. (2000). For T. beigelii an MIC90 value of

0.78 mg ⁄L was measured (Perparim et al., 1996). Microsporum

canis (in Sabouraud broth) is also sensitive to miconazole, the

lowest active antifungal concentration found by Van Cutsem

et al. (1986) was 1 mg ⁄L. Using the NCCLS (M38-P) guidelines

for testing ﬁlamentous fungi, Ferna

´ndez-Torres et al. (2001)

found an MIC90 of 0.25 mg ⁄L.

(2) Econazole: Thienpont et al. (1975) determined the fungi-

static activity against human, animal and plant pathogenic

fungi. Complete growth inhibition of Candida spp. was obtained

at 10–100 mg ⁄L (for C. albicans 100 mg ⁄L was needed). More

recently, Arias et al. (1996), using the broth dilution micro-

method, found complete inhibition of C. glabrata growth at

12.5 mg ⁄L. For M. canis (0.1–1 mg ⁄L), M. gypseum (1 mg ⁄L),

T. mentagrophytes (0.01 mg ⁄L), T. rubrum (0.1–1 mg ⁄L) and

NCl

Clotrimazole

Cl Cl

Miconazole

Econazole

Enilconazole

(±) Ketoconazole

ONN

CH3

(±) Parconazole

(±) Itraconazole

ONNN

Fluconazole

Fig. 2. Chemical structures of azole anti-

fungal agents.

10 H. Vanden Bossche et al.

2003 Blackwell Publishing Ltd, J. vet. Pharmacol. Therap. 26, 5–29

E. ﬂoccosum (0.01 mg ⁄L) much lower concentrations were

needed (Thienpont et al., 1975). Econazole was also found to

be a potent inhibitor of the growth of B. dermatitidis (0.1 mg ⁄L),

H. capsulatum (0.01 mg ⁄L), A. fumigatus and A. ﬂavus (1 mg ⁄L)

and S. schenckii (1 mg ⁄L). Whereas, for Absidia corymbifera

(100 mg ⁄L) and Allescheria boydii (10 mg ⁄L) much higher

concentrations were needed (Thienpont et al., 1975). Hammer

et al. (2000) determined the in vitro susceptibilities of M. furfur

and M. sympodialis by a broth dilution assay. MIC90 values of

64 and 1 mg ⁄L were found, respectively. However, using the

agar dilution assay MIC90 values of 16 and 0.5 mg ⁄L were

reported.

(3) Enilconazole: Enilconazole base and different salts were

tested in vitro on various human, animal and plant pathogenic

fungi; as with econazole, dermatophytes were the most sensitive

(Thienpont et al., 1981). Its antimycotic activity is excellent

against M. canis,M. gypseum,T. mentagrophytes,T. verrucosum,

M. pachydermatis and Aspergillus spp. (Thienpont et al., 1981;

de Jaham et al., 2000). For example, growth of A. fumigatus in

Sabouraud broth was inhibited at 0.1–1 mg ⁄L (Van Cutsem &

Janssen, 1988). The in vivo antifungal activity was high for the

dermatophytes and rather low for Candida (Thienpont et al.,

1981).

Ketoconazole and parconazole

Ketoconazole (Fig. 2) belongs to the class of the imidazoles with

ketal groups. Its synthesis has been reported by Heeres et al.

(1979). Ketoconazole is a lipophilic molecule. Its log partition

coefﬁcient (Log P) is 3.84. The pKa of the imidazole ring of

ketoconazole is 6.51. That means that at, for example, pH 5.6

the imidazole ring is more than 90% protonated (Vanden

Bossche et al., 1988). As the antifungal activity of the imidazole

derivatives depends, at least partly, on the binding of the

unprotonated imidazole nitrogen to cytochrome P450 (see

mechanisms of action of azole antifungals), protonation will

affect the in vitro antifungal activity of ketoconazole.

After oral administration, ketoconazole is metabolized in the

liver and is converted into many inactive metabolites. Absorp-

tion of ketoconazole from the gastrointestinal tract is more rapid

in rats and guinea-pigs than in rabbits and dogs. Following a

dose of 10 mg ⁄kg peak plasma levels were 1.0, 3.7, 8.9 and

16.5 mg ⁄L in rabbits, guinea-pigs, dogs and rats, respectively

(Heel, 1982). Limited pharmacokinetic studies in pigeons and

cockatoos are performed. The peak concentration varied

between 0.5 and 5 h and the elimination half-life time was

2–2.8 h in pigeons and 3.8 h in cockatoos (Kollias et al., 1986).

For dissolution and subsequent absorption of ketoconazole the

acidity of the stomach plays an essential role. Therefore,

coadministration of compounds such as cimetidine should be

avoided. After topical application ketoconazole is found in the

stratum corneum (Pershing et al., 1994). Both ketoconazole and

parconazole (Fig. 2) have a dioxalan group.

After a single oral dose of 13 mg parconazole per kg body

weight a blood peak level of 2.28 mL ⁄L was reached after 2 h in

guinea-fowls; the unchanged drug is specially localized in crop

and skin (Levron et al., 1985).

Spectrum of activity

(1) Ketoconazole: The spectrum of antifungal activity of ketocon-

azole resembles qualitatively that of miconazole. Quantitative

results for MICs vary among laboratories, depending on

inoculum size, culture medium, pH, duration and time of

incubation and growth phase of the fungus (see for example,

Galgiani & Stevens, 1976; Thienpont et al., 1979; Granade &

Artis, 1980). Using the NCCLS method (M38-P) Ferna

´ndez-

Torres et al. (2001) determined the in vitro activities of 10

antifungal drugs against 508 strains of dermatophytes. In this

study ketoconazole displayed excellent activity against, for

example, E. ﬂoccosum,M. canis,M. gypseum,T. mentagrophytes

and T. verrucosum. The GM of MICs were 0.08, 0.12, 0.23, 0.46

and 0.5 mg ⁄L, respectively. Trichophyton simii was less sensitive

with a GM of 4 mg ⁄L. When all 508 strains were considered

together the GM was 0.5 mg ⁄L. The tests were performed in

round-bottomed 96 microplates. Using tubes (ﬁnal volume

10 mL) instead of microplates, Perea et al. (2001a) found

somewhat higher GMs. For example, the GMs for E. ﬂoccosum,

M. canis,M. gypseum,T. mentagrophytes and T. verrucosum were

0.61, 1.12, 1.78, 1.55 and 6.72 mg ⁄L, respectively. For

T. equinum these investigators found a GM of 6.35 mg ⁄L.

The MICs of ketoconazole were determined by the macrodi-

lution test for isolates of A. fumigatus,A. ﬂavus,P. boydii,

R. arrhizus and S. schenckii (Espinel-Ingroff et al., 1995). The

MIC ranges were: 2–4 mg ⁄L, 0.5–16, 0.03–2, 1–16 and

0.25–4 mg ⁄L, respectively.

A broth microdilution method was used to determine the

in vitro activities of ketoconazole against Cladosporium spp.,

Exophiala dermatitidis,Cladophialophora spp., Scopulariopsis chart-

arum,Paecilomyces spp., and F. solani (Llop et al., 2000). The

mean MICs were: 1.32, 0.25, 0.09, 2, 0.17 and 32 mg ⁄L,

respectively.

Ketoconazole was tested for its activity against 23 isolates of

M. furfur by agar dilution. A GM of 0.51 mg ⁄L was found (Van

Gerven & Odds, 1995). It was much more potent than

bifonazole (GM ¼8.1 mg ⁄L), miconazole (GM ¼14 mg ⁄L) and

clotrimazole (GM ¼15 mg ⁄L). That ketoconazole is much more

active than miconazole against M. furfur was also found by

Hammer et al. (2000). Of interest is that these investigators

found that the MICs and MFCs of ketoconazole were equival-

ent. Microsporum sympodialis,M. sloofﬁae,M. pachydermatis,

M. globosa,M. obtusa and M. restricta were also susceptible to

ketoconazole with MIC ranging from £0.03 to 0.125 mg ⁄L

(Gupta et al., 2000).

Morace et al. (1995) used a microdilution broth method to

determine the susceptibility of Candida spp. isolates to ketocon-

azole. The MIC ranges for C. albicans,C. krusei,C. glabrata,

C. kefyr,C. tropicalis,C. rugosa and C. norvegensis were 0.04–50,

0.09–12.5, 0.09–12.5, 0.04, 12.5, 0.39 and 0.19 mg ⁄L,

respectively. With the NCCLS M27-A broth microdilution

method lower MIC values were found for C. albicans (0.03–

16 mg ⁄L), C. krusei (0.25–1 mg ⁄L), C. glabrata (£0.03–4 mg ⁄L)

and C. tropicalis (0.03–1 mg ⁄L) (Arthington-Skaggs et al.,

2000). For C. parapsilosis,C. dubliniensis and C. neoformans the

MIC ranges were: 0.03–2, 0.03–8 and 0.06–1, respectively.

Antifungal agents 11

2003 Blackwell Publishing Ltd, J. vet. Pharmacol. Therap. 26, 5–29

Ketoconazole is also active against B. dermatitidis (MIC

range: 0.1–04 mg ⁄L) (Chapman et al., 1998), H. capsulatum

(0.39 mg ⁄L) and some isolates of C. immitis (0.39 mg ⁄L)

(Shadomy et al., 1985). However, for other C. immitis isolates

more than 100 mg ⁄L was needed (Shadomy et al., 1985).

(2) Parconazole: This orally active imidazole derivative has

been developed for veterinary applications. It has a spectrum of

antifungal activity in vitro which quantitatively resembles that of

the other azole antifungal agents. As with the other imidazole

derivatives, the inhibitory concentration depends on the culture

medium used. For example, in casein hydrolysate-yeast extract-

glucose (CYG) medium 50% inhibition of C. albicans growth was

achieved at 30 n

, in yeast-nitrogen base-glucose medium

2000 n

was needed (F. Cornelissen & H. Vanden Bossche,

unpublished result).

The in vitro antifungal activity of parconazole was tested in

Sabouraud liquid medium against dermatophytes, yeasts,

dimorphic fungi, mycetoma agents and phycomycetes (Desplen-

ter & Van Cutsem, 1980). Growth of dermatophytes is

completely inhibited at 1–10 mg ⁄L, Candida spp. at 10–

100 mg ⁄L and the yeast form of B. dermatitidis at 0.01 mg ⁄L.

Growth of mycetoma agents and actinomycetales is inhibited at

0.1–100 mg ⁄L. Absidia ramosa,Mucor spp., Rhizopus spp.,

Aspergillus spp. (except A. nidulans), Fusarium spp., S. schenckii,

Alternaria spp. and S. brevicaulis are less sensitive.

Triazole derivatives

Itraconazole and ﬂuconazole

The orally active 1,2,4-triazole derivative, itraconazole (Fig. 2),

is a highly lipophilic molecule. Its log P (n-octanol ⁄aqueous

buffer of pH 8.1) is 5.66. Itraconazole is a weak base (pKa of the

triazole is <2 and of the piperazine is 3.7) and therefore is only

protonated at low pH, such as in gastric juice. Itraconazole is

soluble in N,N-dimethylformamide (3.5 g ⁄100 mL) and dimethyl

sulphoxide (1.1 g ⁄100 mL), slightly soluble in polyethylene glycol

400 (0.19 g ⁄100 mL), but almost insoluble in water (at pH 7.0

<0.0001 g ⁄100 mL) (for more information see Vanden Bossche

et al., 1993a).

Pharmacokinetics (Heykants et al., 1987, 1989): The distri-

bution of itraconazole was studied in male and female Wistar rats

after an oral 10 mg ⁄kg dose of

H-itraconazole. In most tissues,

peak radioactivity concentrations were achieved within 2–4 h

after dosing. The levels obtained in the adrenals, liver, kidney,

lung, skin and perirenal fat were consistently higher than in

plasma. Only in the brain were levels lower than in plasma.

Tissue levels in dogs were measured 24 h after the last dose of

a 12-month chronic toxicity experiment at daily doses of 5, 20 or

80 mg ⁄kg. Except for the brain, itraconazole tissue levels were

higher than the corresponding plasma levels. Brain tissue levels

were similar to those measured in plasma. Distribution studies

have shown that therapeutic active levels of itraconazole in

humans are maintained much longer in some infected tissues

than in plasma. For instance, active levels persist for 4 days in

the vaginal epithelium after a 1-day treatment and for 3 weeks

in the stratum corneum of the skin after treatment has been

stopped. After oral administration, itraconazole can be detected

in nails 1–2 weeks after the start of therapy (Debruyne &

Coquerel, 2001). Itraconazole is still detectable in nails 27 weeks

after stopping administration.

The recently developed oral hydroxypropyl-b-cyclodextrin

solution of itraconazole showed improved gastric absorption

and high topical drug concentrations in the oral cavity and the

oesophagus (Stevens, 1999). More information on the pharma-

cokinetics and pharmacodynamics of cyclodextrin itraconazole

in patients can be found in Groll et al. (2002).

Itraconazole is extensively metabolized. Main metabolic path-

ways are oxidative scission of the dioxolane ring, oxidative

degradation of the piperazine ring and N-dealkylation of the

1-methylpropyl substituent. One metabolite deserves special

attention. Hydroxy-itraconazole is the antifungally active meta-

bolite of itraconazole formed in the liver by oxidation of

1-methylpropyl substituent of itraconazole (Heykants et al.,

1989). In vitro tests showed the same IC

values for itraconazole

and hydroxy-itraconazole, within a mode ± 1 dilution range of

experimental error, for 90% of 1481 isolates of pathogenic fungi

(Odds & Vanden Bossche, 2000). Some 10–15% of C. glabrata

and T. mentagrophytes isolates were more susceptible to itracon-

azole than hydroxy-itraconazole.

In common with other 1-substituted 1,2,4-triazole derivatives,

the bis-triazole propanol derivative, ﬂuconazole (Fig. 2) is a weak

base. In contrast with most of the available azole antifungal

agents, ﬂuconazole is soluble in water (8.71 mg at 37 C;

2.20 mg ⁄Lat4C) (Hitchcock & Whittle, 1993). Its relatively

high polarity contributes to ﬂuconazole’s low protein binding

and even distribution throughout the body (Troke, 1993).

Fluconazole is rapidly and virtually completely absorbed by the

oral route in mouse, rats, dogs and man (pharmacokinetics are

reviewed by Brammer & Tarbit, 1987). It is extensively

distributed throughout the body and penetrates readily into

cerebrospinal ﬂuid and saliva. Following administration of

50 mg ﬂuconazole per day for 8 days to male cats, ﬂuconazole

concentrations in cerebrospinal ﬂuid exceeded reported MICsof

this triazole for susceptible pathogenic fungi (Vaden et al., 1997).

Marked species differences exist in the elimination half-life of

ﬂuconazole, values ranging from 4 h in rodents, almost 15 h in

dogs to about 25 h in human volunteers. The major route of

excretion of the drug is as unchanged ﬂuconazole in the urine in

all species studied. Excretion ranges between 70% of the dose in

dogs and 80% in rodents and humans. As shown further,

ﬂuconazole has only modest in vitro activity against several

pathogenic fungi. Nevertheless its clinical efﬁcacy in many

systemic and superﬁcially fungal infections is remarkable (Troke,

1993). Fluconazole pharmacokinetics certainly contribute to this

in vivo antifungal activity.

Spectrum of activity

(1) Itraconazole: With the NCCLS M27-A broth microdilution

method (Arthington-Skaggs et al., 2000) the following MIC

ranges were found: C. albicans (0.03 to >16 mg ⁄L), C. krusei

(0.06–0.25 mg ⁄L), C. glabrata (0.25 to ‡16 mg ⁄L), C. tropicalis

(0.125–0.5 mg ⁄L). For C. parapsilosis,C. dubliniensis and

12 H. Vanden Bossche et al.

2003 Blackwell Publishing Ltd, J. vet. Pharmacol. Therap. 26, 5–29

C. neoformans the MIC ranges were 0.03–0.125, 0.03–4 and

0.03–0.125 mg ⁄L, respectively. Itraconazole had MICsof

0.06–2, 0.03–0.5 and 0.03–1 mg ⁄L for C. famata,C. lusitaniae

and C. guilliermondii, respectively (Pfaller et al., 2001). Using the

same method, Li et al. (2000) tested the activity of itraconazole

against the mould forms of three dimorphic fungi. Itraconazole

had MIC90s (MIC at which 90% of the isolates are inhibited) of

0.06 mg ⁄L for H. capsulatum, 0.125 mg ⁄L for B. dermatitidis and

1mg⁄mL for C. immitis. Using the macrobroth dilution suscep-

tibility test to evaluate the activity of itraconazole against

B. dermatitidis isolates, Chapman et al. (1998) found an MIC

range of £0.018–0.07 mg ⁄L. The range for the minimal lethal

concentration was the same. For T. beigelii an MIC90 value of

0.19 mg ⁄L was measured (Perparim et al., 1996). Using the

agar dilution method M. furfur,M. sympodialis,M. sloofﬁae,

M. pachydermatis,M. globosa,M. obtusa and M. restricta were

found susceptible to itraconazole at low concentrations (MICs

ranging from £0.03 to 0.125 mg ⁄L; Gupta et al., 2000).

Itraconazole displayed excellent activity against, for example,

E. ﬂoccosum,M. canis,M. gypseum,T. mentagrophytes,T. rubrum,

T. simii and T. verrucosum. The GM of MICs were 0.03, 0.08,

0.04, 0.17, 0.09, 0.12 and 0.01 mg ⁄L, respectively (Ferna

´ndez-

Torres et al., 2001). The tests were performed in round-bottomed

96 microplates. Using tubes (ﬁnal volume 10 mL) instead of

microplates, Perea et al. (2001a) found for E. ﬂoccosum,M. canis,

M. gypseum,T. equinum,T. mentagrophytes and T. verrucosum the

following GMs: 0.11, 0.10, 0.04, 0.04, 0.04 and 0.35 mg ⁄L,

respectively. Thus with both methods itraconazole was found to

be more active than ketoconazole (vide supra) and, as shown

below, much more active than ﬂuconazole.

Moore et al. (2000) determined the in vitro activity of

itraconazole against clinical isolates of Aspergillus. The GM of

MICs (mg ⁄L) for A. fumigatus,A. terreus,A. ﬂavus and A. niger

were 0.77, 0.33, 0.5 and 0.93 mg ⁄L, respectively.

For isolates of P. boydii,R. arrhizus and S. schenckii the MIC

ranges were 0.03–1, 0.12–16 and 0.03–16 mg ⁄L, respectively

(Espinel-Ingroff et al., 1995).

A broth microdilution method was used to determine the

in vitro activity of itraconazole against Cladosporium spp.,

E. dermatitidis,Cladophialophora spp., S. chartarum,Paecilomyces

spp. and F. solani (Llop et al., 2000). The mean MICs were 0.76,

0.25, 0.04, 32, 0.19, 0.17 and 32 mg ⁄L, respectively.

(2) Fluconazole: As already mentioned, the clinical efﬁcacy of

ﬂuconazole in many systemic and superﬁcial fungal infections is

remarkable, although it has only modest in vitro activity against

several pathogenic fungi (Troke, 1993).

With the NCCLS M27-A broth microdilution method the fol-

lowing MIC ranges were found: C. albicans (0.25 to ‡64 mg ⁄L),

C. krusei (16–64 mg ⁄L), C. glabrata (1 to ‡64 mg ⁄L), C. tropicalis

(0.25–64 mg ⁄L), C. parapsilosis (0.25–1 mg ⁄L), C. dubliniensis

(0.125 to ‡64 mg ⁄L) and C. neoformans (0.25–64 mg ⁄L) (Arth-

ington-Skaggs et al., 2000). Pfaller et al. (2001) found MICsof

£8mg⁄L for 78% of the C. neoformans isolates tested; MICsof

16–32 mg ⁄L were found for 21% of the isolates. Using the same

method, Perparim et al. (1996) found for T. beigelii an MIC90 of

6.25 mg ⁄L. High MIC90 values were also found for E. ﬂoccosum

(4.39 mg ⁄L), M. canis (16 mg ⁄L), M. gypseum (>64 mg ⁄L),

T. mentagrophytes (>64 mg ⁄L) and T. rubrum (16 mg ⁄L) (Fern-

´ndez-Torres et al., 2001). For T. equinum the MIC was

‡64 mg ⁄L (Perea et al., 2001a). An MIC of 64 mg ⁄L was found

for A. fumigatus,A. ﬂavus,R. arrhizus and S. schenckii. The

P. boydii isolates tested were more susceptible; MIC range was

2–32 mg ⁄L (Espinel-Ingroff et al., 1995).

Chapman et al. (1998) found an MIC range of 2.5–4 mg ⁄L for

B. dermatitidis, whereas the MIC range was £0.018–0.07 mg ⁄L

for itraconazole.

Mechanisms of action of azole antifungal agents

The antifungal activity of azole derivatives such as miconazole

(Vanden Bossche et al., 1978), econazole, bifonazole, clotrima-

zole (Vanden Bossche, 1985, 1988), ketoconazole (Vanden

Bossche et al., 1980, 1988), enilconazole (Siegel & Ragsdale,

1978; Vanden Bossche et al., 1987b), parconazole (Pye &

Marriott, 1982), terconazole (Vanden Bossche & Marichal,

1991), itraconazole (Vanden Bossche et al., 1986, 1987a,b,

1988) and ﬂuconazole (Hitchcock, 1991; Hitchcock & Whittle,

1993) arises from a complex multimechanistic process initiated

by the inhibition of two cytochromes P450 involved in the

biosynthesis of ergosterol, namely the P450 that catalyses

the 14a-demethylation step encoded by ERG11 (CYP51) and the

D22-desaturase, encoded by ERG5 (CYP61) (Kelly et al., 1997a).

The sterol-14a-demethylase is the major fungal target for all

azole derivatives studied so far. Interaction with CYP51 results in

a decreased availability of ergosterol and an accumulation of

14-methylsterols, such as 14a-methyl-ergosta-8,24(28)-dien-

3b,6a-diol (3,6-diol) (Vanden Bossche et al., 1993b; Vanden

Bossche, 1995; Marichal et al., 1999a). In C. neoformans

(Vanden Bossche et al., 1993b) and H. capsulatum (Vanden

Bossche et al., 1990) azoles induce the accumulation of

14-methylated 3-ketosteroids such as obtusifolione and ⁄or

14-methylfecosterone, suggesting that azoles affect directly or

indirectly the 3-ketostereductase, a product of the ERG27 gene.

Ergosterol is an essential component of fungal plasma mem-

branes. It regulates membrane permeability and the activity of

membrane-bound enzymes; this sterol is a major component of

secretory vescicles and has an important role in mitochondrial

respiration and oxidative phosphorylation (for reviews see Vanden

Bossche, 1985, 1988, 1990; Daum et al., 1998). Thus, it can be

expected that changes in ergosterol levels and sterol structure

inﬂuence the activity of several metabolic pathways. For example,

in C. albicans grown for 16 h in the presence of 10 n

miconazole,

a shift from oleic acid to palmitic acid was observed suggesting an

inhibition of the D-9 desaturase (Vanden Bossche, 1985). An

increase in palmitate was also found in C. albicans grown in the

presence of 1 l

clotrimazole, econazole, miconazole or ketocon-

azole (Georgopapadakou et al., 1987). As expected from the

important role of ergosterol in mitochondrial activities, inhibition

of ergosterol synthesis by, for example, miconazole (De Nollin

et al., 1977) and econazole (Wilm & Stahl, 1983) affects

cytochrome c oxidase in the mitochondrial membranes of

C. albicans and S. cerevisiae. Chitin is a structural component of

Antifungal agents 13

2003 Blackwell Publishing Ltd, J. vet. Pharmacol. Therap. 26, 5–29

fungal cell walls (see Kollar et al., 1995). Chitin synthesis is inhibited

at a high and stimulated at a low ergosterol content (Pesti et al.,

1981; Chiew et al., 1982). Thus, ergosterol biosynthesis inhibitors

increase chitin synthesis resulting in an irregular distribution of

patches of chitin in, for example, C. albicans (Barug et al., 1983;

Vanden Bossche, 1985) and A. fumigatus (Marichal et al., 1985).

Recently DNA chip technology has been used to study cellular

responses to perturbations of ergosterol biosynthesis caused by

itraconazole. Simultaneous examination of over 6600 C. albicans

gene transcript levels, representing the entire genome, upon

treatment of cells with itraconazole, revealed 296 genes to be

responsive: 116 gene transcript levels were at least 2.5-fold

decreased while 180 were increased (De Backer et al., 2001).

These results further prove that the antifungal activity of azole

derivatives arises from a complex multimechanistic process

initiated by the inhibition of ergosterol biosynthesis.

In addition to their effects on ergosterol biosynthesis high

concentrations of miconazole, econazole and clotrimazole disturb

the lipid organization in membranes (Brasseur et al., 1983;

Vanden Bossche, 1985, 1988). For example, using differential

scanning calorimetry, it has been shown that miconazole shifts the

transition temperature of dipalmitoylphosphatidylcholine (DPPC)

multilamellar vesicles to low values without affecting the enthalpy

of melting (Vanden Bossche et al., 1982, 1984). The conformation

of miconazole inserted in a DPPC monolayer was calculated by

using a procedure of conformational analysis (Brasseur et al.,

1983). The area occupied per miconazole molecule was 9 nm

This is higher than the area occupied per DPCC molecule: 6 nm

Such a conformation should result in a destabilizing effect of high

concentrations of miconazole. This effect may explain, at least

partly, its activity against Gram-positive bacteria. In Gram-

positive bacteria miconazole may also cause an impairment of

cell wall function by inhibiting carrier lipid function (phosphor-

ylated derivative of C-55 isoprenoid alcohol) and peptidoglucan

synthesis (Vanden Bossche et al., 1984). The miconazole induced

impairment of cell wall integrity in Gram-positive bacteria may be

at the origin of the synergism between miconazole and polymyxin

B; miconazole may increase the accessibility of the cell membrane

to polymyxin B (Cornelissen & Vanden Bossche, 1983).

Miconazole alone is inactive against Escherichia coli; but it

enhances the sensitivity of this Gram-negative bacterium to

polymyxin B. This synergism might be due to a direct interaction of

miconazole with membrane components. Miconazole, polymyxin

B together with prednisolone are the components of Surolan



(Janssen Animal Health, Beerse, Belgium), an otic preparation for

treatment of otitis externa in dogs.

Resistance

A comprehensive analysis of the clinical outcome of Candida

infections and of the MIC results obtained with the NCCLS M27-T

broth macrodilution method on Candida isolates suggested that

isolates with MIC >32mg⁄L are regarded as ﬂuconazole

resistant (Rex et al., 1997). Isolates for which the MICs are

£8mg⁄L are susceptible to ﬂuconazole, isolates with ﬂuconazole

MICs of 16–32 mg ⁄L are considered susceptible on the basis of

data indicating a clinical response when >100 mg ﬂuconazole

per day is given. In the same study, isolates with an MIC ‡1

mg ⁄L were considered to be resistant to itraconazole. Examples of

ﬂuconazole and ⁄or itraconazole resistant C. albicans isolates are

shown in Table 2. It should be noted that large-scale surveys of

yeasts isolated from blood cultures, based on standardized

methodology and resistance deﬁnitions, do not support the view

that antifungal resistance in pathogenic yeasts constitutes a

signiﬁcant or growing problem (Sanglard & Odds, 2002).

A signiﬁcant reason for clinical treatment failure is severe

immunosuppression.

Until 1986, resistance of pathogens to azole antifungals was

only sporadically reported and invariably in immunocompro-

mized patients who had received long-term oral treatment with

ketoconazole for chronic mucocutaneous candidosis (Horsburgh

& Kirkpatrick, 1983). An increase in the number of immuno-

compromised patients, partially as a result of the AIDS

pandemic, preceded increasing reports of clinical failure to

ﬂuconazole therapy (see for example Vanden Bossche et al.,

1994a, 1998; Johnson et al., 1995; Klepser et al., 1997; Odds,

1998; White et al., 1998; Stevens & Holmberg, 1999; Espinel-

Ingroff et al., 2000; Sanglard & Odds, 2002). The importance of

a normal immune system can be related to the fungistatic mode

of action of the azoles, which implies that part of the clearance of

the fungal infection must be accomplished by host-related factors

(Marichal, 1999).

Some C. albicans isolates from HIV-positive patients with

oropharyngeal candidosis and from some HIV-negative patients

with recurrent vaginal candidosis, have been reported to be

susceptible to other azole derivatives, such as ketoconazole or

itraconazole (Ruhnke et al., 1994; Johnson et al., 1995; Stevens

& Stevens, 1996). In a recent study on safety, pharmacokinetics

and pharmacodynamics of cyclodextrin itraconazole in HIV-

infected paediatric patients with oropharyngeal candidosis Groll

et al. (2002) showed that all patients with ﬂuconazole-resistant

isolates responded to treatment with cyclodextrin itraconazole;

however, they also found that there was no clear correlation

between the MIC of itraconazole and response to therapy. In

many studies on C. albicans a progressive increase in the MICsof

itraconazole (and also of terbinaﬁne) was found to parallel the

increase in ﬂuconazole MICs (Barchiesi et al., 1994; Sanglard

et al., 1996; Lo

´pez-Ribot et al., 1999).

Like many other moulds, A. fumigatus is intrinsically resistant

to ﬂuconazole (Edlind et al., 2001).

The incidence of resistance to itraconazole in Aspergillus

isolates is still rare (see Denning et al., 1997; Dupont et al.,

2000; Mosquera & Denning, 2002).

As a result of the widespread use of ﬂuconazole Candida species

have been selected that are intrinsically resistant to ﬂuconazole,

such as C. krusei (Marichal et al., 1995), or whose resistance is

easily inducible, such as C. glabrata (Rex et al., 1995; Sanglard

et al., 1999, 2001) and C. tropicalis (Barchiesi et al., 2000).

Fungal pathogens could use several general mechanisms to

become less susceptible to azole antifungals (see for example

Vanden Bossche et al., 1994a,b, 1998; Vanden Bossche, 1997;

White et al., 1998; Marichal, 1999; Sanglard et al., 1999, 2001).

14 H. Vanden Bossche et al.

2003 Blackwell Publishing Ltd, J. vet. Pharmacol. Therap. 26, 5–29

These mechanisms include: failure to accumulate azole

antifungals, changes in the interaction of the drug with the

target enzyme, the 14a-demethylase and changes in other

enzymes of the ergosterol pathway.

Failure to accumulate azole antifungals

Failure to accumulate azole antifungals has been identiﬁed as a

major cause of resistance in several post-treatment fungal isolates

and species that are less sensitive to azole antifungal agents and

other ergosterol biosynthesis inhibitors. These isolates include

C. albicans (Ryley et al., 1984; Venkateswarlu et al., 1995;

Sanglard et al., 1995), C. krusei (Marichal et al., 1995), C. glabrata

(Vanden Bossche et al., 1992; Sanglard et al., 1999, 2001),

C. dubliniensis (Moran et al., 1998), C. neoformans (Venkateswarlu

et al., 1997), A. ﬂavus and A. fumigatus (Tobin et al., 1997).

Failure to accumulate antifungal agents can be the conse-

quence of impaired drug inﬂux or enhanced drug efﬂux. Efﬂux

pumps are now recognized as a common cause of decreased

intracellular content of ergosterol biosynthesis inhibitors.

Two types of efﬂux pumps have been described: membrane

transport proteins belonging either to the major facilitator

superfamily (MFS) or to the ATP-binding cassette (ABC)

superfamily. The MFS are energized by the proton-motive force

(Goffeau et al., 1997). The ABC-type transporters utilize ATP as

a source of energy (Decottignies & Goffeau, 1997).

In C. albicans more than 10 different ABC-type transporters

have been reported (Marr et al., 1998), but only the pumps

encoded by CDR1 and CDR2 are known to be involved in the

mechanisms of azole resistance (Prasad et al., 1995; Sanglard

et al., 1997; Vanden Bossche et al., 1998). CDR1 or CDR2 over-

expression in S. cerevisiae was found to confer cross-resistance to

several antifungal agents, such as ﬂuconazole, ketoconazole,

itraconazole, terbinaﬁne and amorolﬁne (Sanglard et al., 1995,

1996, 1997, 1998a,b; Vanden Bossche et al., 1998).

In ﬂuconazole-resistant C. albicans isolates a multidrug efﬂux

transporter of the MFS has been shown to be responsible for the

low level of accumulation of ﬂuconazole (Sanglard et al., 1995).

The MFS gene, CaMDR1, was found to be overexpressed in

resistant isolates (Sanglard et al., 1995). The product of the

CaMDR1 gene, CaMdr1p, expels terbinaﬁne and ﬂuconazole, but

not itraconazole. This ﬁnding correlates with the observation by

Albertson et al. (1996) that a C. albicans mutant in which the

Table 2. Examples of reported amino acid

polymorphisms in sensitive and resistant

Candida albicans isolates Investigator Strain

Amino acid point

mutations*

GrowthMIC (lg⁄mL)

Fluconazole Itraconazole

White (1997) 1 0.25 0.03

17 R467K >64.00 >2.00

Lo¨fﬂer et al. (1997) l4 F105L 128.00 0.064

17 V488I >256.00 >32.00

18 E266D, G464S >256.00 >32.00

112 F105L, E266D 8.00 0.25

TU3 E266D, G448E >256.00 4.00

TU6 F105L, E266D >256.00 6.00

TU9 K287R, G464S >256.00 >32.00

P2 F105L, G464S >256.00 >32.00

P3 F105L, G450E >256.00 0.75

P4 G450E, V488I >256.00 >32.00

Sanglard et al. (1998a,b) C26 D116E, Y132H, S405F >128 4.00

C34 D116E, E405F 2.00 0.062

C37 G464S, R467K 8.00 0.062

C39 S405F 32.00 0.125

C56 D116E, G129A, G464S 128.00 >2.00

Marichal et al. (1999b) NCPF3363 Y132H 32.00 2.00

B59626àD116E, K128T, A149V >64.00 1.00

B59630àD116E, K128T, A149V 64.00 0.5

J913004 ⁄1§ D116E, K128T, V452A, 64.00 1.00

G464S

ATCC44858 D153E, E266D 0.13 0.016

Kakeya et al. (2000) Darlington Y132H, I471T >64.00–nd

NCPF3310

*A, alanine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleu-

cine; K, lysine; L, leucine; R, arginine; S, serine; T, threonine; V, valine; Y, tyrosine.

MICs determined by a broth microdilution method (Odds et al., 1995) based on the NCCLS M27A

protocol. Results taken from Marichal et al. (1999b).

àB59626 and B59630 were sequentially isolated from an AIDS patient in Germany (provided by

Prof. G. Just).

§J913004 ⁄1 was isolated from an AIDS patient in France (provided by Prof. B. Dupont).

–Result taken from Kakeya et al. (2000).

Antifungal agents 15

2003 Blackwell Publishing Ltd, J. vet. Pharmacol. Therap. 26, 5–29

CaMDR1 gene is overexpressed is speciﬁcally resistant to

ﬂuconazole, but not to the other azole antifungal agents tested.

Lyons and White (2000) showed that CaMDR1 and CDR

mRNAs are transcriptionally overexpressed in a resistant

C. albicans isolate, suggesting that the antifungal drug resistance

in this isolate is associated with the promoter and trans-acting

factors of the CDR1,CDR2 and CaMDR1 genes.

The ATP-binding cassette-type transporters have also been

found in C. krusei (Katiyar & Edlind, 2001), C. dubliniensis

(Moran et al., 1998), C. glabrata (Miyazaki et al., 1998; Sanglard

et al., 1999, 2001), C. tropicalis (Barchiesi et al., 2000),

C. neoformans (Thornewell et al., 1997), A. nidulans (Del Sorbo

et al., 1997), A. fumigatus and A. ﬂavus (Tobin et al., 1997). In

A. nidulans the ABC transporters AtrA and AtrB are involved in

resistance to enilconazole (Del Sorbo et al., 1997).

Bouchara et al. (2000) studied a C. glabrata isolate that

exhibited cross-resistance to ketoconazole and ﬂuconazole. The

results suggested that the resistant isolate had a respiratory

deﬁciency. Further studies showed a partial deletion of mito-

chondrial DNA analogous to that described for the rho

petite

mutants of S. cerevisiae. Sanglard et al. (2000, 2001) observed

up-regulation of the ABC-type transporters CgCDR1 and CgCDR2

in such rho

mutants of C. glabrata. Thus mitochondrial loss may

be one of the pathways leading to the up-regulation of ABC-

transporters.

Members of the MFS have been found in C. dubliniensis (Moran

et al., 1998), C. glabrata (Sanglard et al., 1999), C. tropicalis

(Barchiesi et al., 2000) and C. neoformans (Spitzer & Spitzer, 1997).

Changes in the interaction of the drug with the target enzyme

In an azole-resistant C. albicans strain (NCPF 3363) isolated from

a patient with chronic mucocutaneous candidosis (Smith et al.,

1986) we observed a diminished afﬁnity of the cytochromes

P450 for azole antifungals and a red shift in the maximum of the

CO absorption spectrum from 448 to 450 nm (Vanden Bossche

et al., 1990). We suggest that these changes resulted from a

mutation in the gene coding for the 14a-demethylase. More

recently, it was found that strain NCPF 3363 contained a

Y132H mutation (replacement of tyrosine with histidine at

amino acid 132) on both of its CYP51 alleles (Marichal et al.,

1999b) (Table 2).

An alteration of tyrosine 132 was found commonly among

ﬂuconazole-resistant C. albicans isolates (in Table 2 examples of

reported amino acid polymorphisms are listed). The importance

of this Y132H mutation for azole susceptibility has been

demonstrated by Sanglard et al. (1998b), Asai et al. (1999)

and Kelly et al. (1999a). The results obtained by Kelly et al.

(1999a) and Asai et al. (1999) show that the Y132H substitu-

tion occurred without drastic perturbation of the 14a-demethy-

lase activity allowing mutants to produce ergosterol and retain

viability, an efﬁcient strategy for resistance. Other mutations not

only reduce substantially the inhibitory effect of ﬂuconazole but

also result in a reduced catalytic activity of the sterol 14a-deme-

thylase. Examples are the T315A (Lamb et al., 1997), G464S

(Kelly et al., 1999b) and the R467K (White, 1997; Lamb et al.,

2000b) amino acid substitutions. In the A. fumigatus CYP51

sequence isoleucine 301(I301) corresponds to C. albicans

T315 (Edlind et al., 2001). Edlind et al. (2001) propose that

the presence of I301 contributes to A. fumigatus ﬂuconazole

resistance.

As shown in Table 2, a number of mutations affect only the

inhibitory effect of ﬂuconazole and not the MICs of itraconazole.

These results suggest that the afﬁnity of itraconazole is much less

affected by these mutations. The latter was further proved by

measuring the effects of these mutations on subcellular ergos-

terol biosynthesis. For example, C. albicans isolate C26 (Table 2)

contained two mutations S405F and Y132H, next to a mutation

also found in some azole-sensitive isolates (D116E). For subcel-

lular fractions of sensitive C. albicans isolates 50% inhibition of

ergosterol synthesis was found at about 40 n

ﬂuconazole,

whereas for the C26 isolate 4880 n

ﬂuconazole was needed

(Marichal et al., 1999b). With itraconazole, 50% inhibition of

ergosterol synthesis by subcellular fractions of azole-sensitive

C. albicans isolates was 33–44 n

, whereas only 20 n

was

required to reach 50% inhibition of ergosterol synthesis by the

subcellular fraction of the C26 isolate (Marichal et al., 1999b).

The smaller, hydrophilic ﬂuconazole molecule has fewer stabil-

ization sites in the active pocket compared with the lipophilic

itraconazole molecule which may explain this difference in

activity (Vanden Bossche & Koymans, 1998).

A common mechanism of resistance on eukaryotic cells is

gene ampliﬁcation (White et al., 1998). In a subcellular fraction

of a ﬂuconazole-resistant clinical isolate of C. glabrata, ergosterol

synthesis was 8.2-fold higher than that from the pretreatment

isolate; the increased synthesis coincided with an increase in

microsomal P450 content (Vanden Bossche et al., 1992). This

increased level of the protein was associated with CYP51

(ERG11) gene ampliﬁcation (Vanden Bossche et al., 1994b;

Marichal et al., 1997). The CYP51 mRNA transcript in the

resistant isolate was eight times greater than it was in the

susceptible isolate (Marichal et al., 1997). This increase in copy

number is due to duplication of the chromosome containing the

CYP51 gene. As expected, the ampliﬁcation of an entire

chromosome has tremendous effects on the protein expression

in the cells: from 1377 proteins which were identiﬁed, 25 were

up-regulated by more than a factor of three and 76 were down-

regulated by the same factor (Marichal et al., 1997). Further

studies are needed to identify the up- and down-regulated

products. For example, it would be of interest to know whether

some of the products are involved in drug efﬂux. In fact, changes

in drug accumulation were observed in this ﬂuconazole-resistant

C. glabrata isolate (Vanden Bossche et al., 1992).

Changes in other enzymes of the ergosterol pathway

Fungi achieve effective resistance to azoles by circumventing or

compensating the toxic consequences of the changes in sterol

composition. As already mentioned azole-induced growth inhi-

bition results from the depletion of ergosterol and the coinci-

dental accumulation of membrane-disturbing 14-methylated

sterols and 3-ketosteroids. However, not all 14-methylated

sterols disturb membranes; for example, 14-methylfecosterol

can fulﬁl some of the functions of ergosterol (Watson et al.,

16 H. Vanden Bossche et al.

2003 Blackwell Publishing Ltd, J. vet. Pharmacol. Therap. 26, 5–29

1989). Accumulation of 14-methylfecosterol is achieved when

C. albicans clinical isolates deﬁcient in D

8)7

desaturase (product

of the ERG2 gene) and D

5)6

desaturase (product of the ERG3

gene) are incubated in the presence of, for example, ﬂuconazole

(Kelly et al., 1997b). Azole antifungal agents induce the

accumulation of the membrane disturbing 14amethyl-ergosta-

8,24(28)-dien-3b,6a-diol (3,6-diol). Alterations in ERG3 prevent

the synthesis of this 3,6-diol and thus protect C. albicans against

the toxic effects of azole antifungals.

Summary of resistance to azole antifungal agents

Resistance seems to evolve over time as the result of several

alterations due to continuous selective pressure from the

antifungal used. The accumulation of alterations has been

documented by White et al. (1998) for a series of 17 clinical

C. albicans isolates. After the initial selection of a substrain of

C. albicans, the CaMDR1 efﬂux pump was overexpressed.

Subsequently, three simultaneous changes occurred at ERG11:

a point mutation, a gene conversion or mitotic recombination of

the ERG11 point mutation converting both alleles to the mutant

version, and overexpression of ERG11. Finally the CDR efﬂux

pumps are overexpressed. Perea et al. (2001b) investigated the

molecular mechanisms of resistance to azoles in C. albicans

strains displaying high-level ﬂuconazole resistance

(MICs‡64 mg ⁄L). Their analysis conﬁrmed the multifactorial

nature of ﬂuconazole resistance and the prevalence of these

mechanisms of resistance in C. albicans isolates, with a predom-

inance of overexpression of genes encoding efﬂux pumps,

detected in 85% of all resistant isolates, being found. Alterations

in the target enzyme, including functional amino acid substitu-

tion and overexpression of the gene that encodes the enzyme,

were detected in 65 and 35% of the isolates, respectively.

Overall, multiple mechanisms were combined in 75% of these

C. albicans isolates.

Selectivity and adverse reactions

Ketoconazole affects, at higher doses than those needed to inhibit

ergosterol synthesis, other cytochrome P450s. For example, at

concentrations >100 n

it inhibits not only the mammalian

14a-demethylase (CYP51) (involved in the biosynthesis of

cholesterol), but also 17-hydroxylase-17,20-lyase (CYP17), the

cholesterol side-chain cleavage (CYP11A1) and the

11b-hydroxylase (CYP11B1) (Vanden Bossche, 1992; Vanden

Bossche et al., 1995).

Enilconazole shows high selectivity; it has 79, 98 and 108

times more afﬁnity for the C. albicans P450s than for those of

piglet testes microsomes, bovine adrenal mitochondria and rat

liver microsomes, respectively. Parconazole is also a selective

inhibitor; this imidazole derivative has 11, 122 and >200 times

more afﬁnity for C. albicans P450 than for those of piglet testes

microsomes, bovine adrenal mitochondria and rat liver micro-

somes (Vanden Bossche, 1991). Itraconazole inhibits at 0.01 m

the aromatase by 18% only (Vanden Bossche et al., 1989),

whereas at this concentration ﬂuconazole inhibits this enzyme

by 70% (Latrille et al., 1989).

Ketoconazole is an inhibitor of CYP3A4, a major drug-

metabolizing P450 isoform. Co-administration of ketoconazole

with CYP3A4 substrates such as cyclosporine, tacrolimus,

lovastatin, terfenadine and astemisole can result in clinically

signiﬁcant drug interactions (Venkatakrishnan et al., 2000).

Itraconazole is almost devoid of effects on P450-dependent

steroid biosynthesis and catabolism (Vanden Bossche et al.,

1986, 1989). However, similar to ketoconazole, itraconazole

inhibits CYP3A4 (Venkatakrishnan et al., 2000). Lamb et al.

(2000a) compared the inhibition by ketoconazole and itracon-

azole of human CYP3A4 and C. albicans CYP51 following

heterologous expression in S. cerevisiae.IC

-values for ketocon-

azole and itraconazole CYP3A4 inhibition were 250 n

and

200 n

, respectively. These values compared with much lower

concentrations required to obtain 50% inhibition of CYP51,

where IC

-values of 8 n

and 7.6 n

were observed for

ketoconazole and itraconazole, respectively. Chronic administra-

tion of itraconazole inﬂuences the metabolism of single dose

astemisole in normal volunteers; the reduction in astemisole

clearance may result in a marked increase in astemisole plasma

concentrations and QTc alterations during chronic combined

intake of astemisole with itraconazole (Lefebvre et al., 1997). A

prolonged QT-interval in ECG and symptomatic torsades de

pointes ventricular tachycardia has been reported as a conse-

quence of the interaction of itraconazole and terfenadine

(Pohjola-Sintonen et al., 1993). Ketoconazole, miconazole, hyd-

roxy-itraconazole, itraconazole and ﬂuconazole inhibit the in vitro

metabolism of cisapride, a prokinetic drug that is predominantly

metabolized by CYP3A4 (Bohets et al., 2000).

Fluconazole and miconazole are potent inhibitors of CYP2C9.

Co-administration of phenytoin, warfarin, sulphamethoxazole

and losartan with ﬂuconazole results in clinically signiﬁcant drug

interactions (Venkatakrishnan et al., 2000). For example, ﬂucon-

azole inhibits the metabolism of losartan to the active metabolite

E-3174, a CYP2C9-dependent reaction (Kaukonen et al., 1998).

In rats, the induction of certain cytochrome P450 isozymes

was observed 24 h after azole administration (Suzuki et al.,

2000). For example, CYP1A, CYP2B and CYP3A2, but not

CYP2E, were observed 24 h after clotrimazole, ketoconazole, or

miconazole treatment. Clotrimazole was the most potent inducer

of CYP3A and miconazole was a more potent inducer of CYP1A

and CYP2B. Ketoconazole induced CYP1A and CYP2B, but the

inducibility of ketoconazole was less than that of clotrimazole or

miconazole. Enhanced expression of CYP1A, CYP2B, CYP2C and

CYP3A has been demonstrated in small intestinal mucosa and

liver of mice treated for 3 days with 10 mg enilconazole ⁄kg ⁄day

(Muto et al., 1997).

In rat and dogs treated for periods of 3–12 months with orally

administered ketoconazole no signs of toxicity occurred with doses

up to 10 mg ⁄kg (Heel, 1982). In dogs treated daily with 40 mg ⁄kg

for 1 year appetite was reduced with emesis and decreased weight

gain. Liver weight was increased. This coincided with increased

levels of serum transaminases and alkaline phosphatase. Ketoc-

onazole is contraindicated in patients with known liver disease.

Gynaecomastia was reported in two patients during treatment

with 200 mg ketoconazole daily and in some patients receiving

Antifungal agents 17

2003 Blackwell Publishing Ltd, J. vet. Pharmacol. Therap. 26, 5–29

high multiple doses (600–1200 mg ⁄day) (reviewed in Vanden

Bossche, 1992). This rare side-effect suggested that in such

patients ketoconazole affected androgen synthesis. Measuring

the testosterone serum levels, a dose related reversible decrease

was observed. This was related to the effect of ketoconazole on

the 17,20-lyase, a key enzyme in the androgen biosynthesis

pathway. This inhibition was observed in subcellular fractions of

rat, pig, dog, bovine and human testes, and bovine adrenals

(Vanden Bossche, 1992).

In laboratory animals the toxicity proﬁle of itraconazole is

qualitatively similar to, but quantitatively clearly different from,

that of ketoconazole (Lampo et al., 1993). At normal doses

itraconazole causes in man only minor side-effects, most of

which are related to the gastrointestinal tract. Prophylactic

administration of itraconazole oral solution to patients with

haematological malignancy and profound neutropenia induced

drug-related adverse events in 5% of the patients (Harousseau

et al., 2000). The main drug-related events were local intoler-

ability (1.8%), nausea (0.7%) and vomiting (1.1%). Diarrhoea

induced by treatment with the oral solution is most likely due to

the hydroxypropyl-b-cyclodextrin carrier; nausea and vomiting

belong to the adverse event proﬁle of both itraconazole capsules

and oral solution (Harousseau et al., 2000).

Fluconazole treatment is associated with very few side-effects.

The most common adverse effects are gastrointestinal, cutaneous

eruption and headache (Gupta & Summerbell, 2000).

Haematological reactions have occurred in seriously ill patients.

In a patient with renal transplant and cryptococcosis, adminis-

tration of ﬂuconazole for 16 weeks may have contributed to a

reversible thrombocytopenia (Osterloh & Pluck, 1993).

ALLYLAMINES AND THIOCARBAMATES

The allylamines (naftiﬁne and terbinaﬁne) (Ryder & Mieth,

1992) and the thiocarbamates (e.g. tolnaftate) (Barrett-Bee et al.,

1986; Ryder et al., 1986) inhibit the fungal squalene epoxidase.

The prototype of the allylamines, naftiﬁne, proved to be an

efﬁcacious antimycotic for topical application in dermatomycoses

(Stu

¨tz, 1988).

The discovery of the antifungal activity of naftiﬁne was the

starting point for studies on structure–activity relationships

resulting in the development of terbinaﬁne (Fig. 3), an allylamine

with considerably enhanced antimycotic properties (Stu

¨tz, 1988).

Terbinaﬁne has oral and topical formulations. It is strongly

lipophilic and is well distributed in the skin, fat and nails. It binds

strongly to plasma proteins and has high afﬁnity for tissues. Oral

terbinaﬁne is well absorbed, with 70–80% of the ingested dose

being absorbed. The peak plasma levels are reached within about

2 h. Terbinaﬁne undergoes extensive hepatic metabolism, with

excretion being primarily (>70%) in the urine. Maximal levels in

plasma after a single oral dose of 250 or 500 mg of terbinaﬁne are

0.9 and 1.7–2.0 mg ⁄L, respectively. Steady-state levels are

attained after 10–14 days of treatment, and low levels of

terbinaﬁne in plasma can be measured for as long as 6 weeks after

cessation of therapy (Elewski, 1998; Gupta & Summerbell, 2000).

Terbinaﬁne is rapidly delivered to the stratum corneum, nails

and hair – both through sebum and by direct diffusion through

dermis–epidermis (Faergemann et al., 1994).

The much older squalene epoxidase inhibitor, tolnaftate

(naphthiomate T; Fig. 3) belongs to the class of the thiocarba-

mates. It is almost insoluble in water, soluble in acetone,

chloroform and polyethylene glycol.

Terbinaﬁne spectrum of activity

Mieth (1993) collected the MIC values obtained at different

laboratories. All dermatophytes tested were signiﬁcantly more

susceptible to terbinaﬁne than to naftiﬁne. The MIC values

against T. rubrum,T mentagrophytes,T. verrucosum,M. canis and

E. ﬂoccosum were 0.001–0.038, 0.001–0.006, 0.001–0.006,

0.006–0.08 and 0.001–0.047 mg ⁄L, respectively. Similar results

were obtained more recently by using the NCCLS broth micro-

dilution (Jessup et al., 2000b; Ferna

´ndez-Torres et al., 2001) and

Fig. 3. Chemical structures of terbinaﬁne,

tolnaftate, thiabendazole and ﬂucytosine.

18 H. Vanden Bossche et al.

2003 Blackwell Publishing Ltd, J. vet. Pharmacol. Therap. 26, 5–29

macrodilution methodologies (Perea et al., 2001a). Jessup et al.

(2000b) found excellent in vitro activity against Rhizopus spp.,

Alternaria spp., Phialophora spp., Chrysosporium spp. and Exophiala

spp. Good antifungal activity is also found against

A. ﬂavus,A. niger,A. terreus, but some A. fumigatus strains are

much less sensitive (MIC range: 0.05–5 mg ⁄L; Mieth, 1993).

Moore et al. (2001) compared the in vitro activities of terbinaﬁne,

itraconazole and AmB against Aspergillus species. The in vitro

activity of terbinaﬁne was inferior against A. fumigatus and

superior against A. ﬂavus,A. terreus and A. niger. However, this

comparison should be made in vivo, as trailing endpoints are

problematic. Fusarium spp. varied greatly in their susceptibility to

terbinaﬁne; MIC range: 0.5 to >64 mg ⁄L (Mieth, 1993).

Excellent in vitro activity was obtained against the dimorphic

fungi B. dermatitidis,C. immitis,H. capsulatum and S. schenckii

(Mieth, 1993).

For yeasts, most susceptibility studies with terbinaﬁne showed

poor activity. Jessup et al. (2000b) found a mean MIC of

10.56 ± 0.133 mg ⁄L (0.06 to >16 mg ⁄L) for C. albicans and

Mieth (1993) found in the literature that, for the yeast form, MIC

ranged from 6.25 to >128 mg ⁄L. A number of non albicans

Candida species exhibited susceptibility to terbinaﬁne with a mean

MIC of 4.91 ± 0.79 mg ⁄L. Candida parapsilosis and C. lusitaniae

had mean MICs of 1.16 ± 0.38 and 0.38 ± 0.4 mg ⁄L, respect-

ively. For C. guilliermondii and C. tropicalis MICs ranged from

0.25 to 2 mg ⁄L and 0.06–16 mg ⁄L. But, for C. glabrata,C. krusei

and C. kefyr the MICs were 2–16, 2–16 and >16 mg ⁄L (Jessup

et al., 2000b).

The in vitro susceptibility of C. neoformans highly depends on

the strains tested, MICs: from 0.06 to >16 mg ⁄L. Trichosporum

beigelii strains are more sensitive with MIC ranges of 0.13–

1mg⁄L (Jessup et al., 2000b).

Minimum inhibitory concentrations of terbinaﬁne against

M. furfur,M. sympodialis,M. sloofﬁae,M. pachydermatis,M. glo-

bosa,M. obtusa and M. restricta ranged from £0.03 to 64 mg ⁄L

(Gupta et al., 2000).

Tolnaftate

The use of tolnaftate is essentially restricted to dermatophyte

infections. When Sabouraud broth was used for testing the

antifungal activity of tolnaftate, the following MICs (mg ⁄L)

were found: M. canis (10–100 mg ⁄L), M. audouini (1000 mg ⁄L),

M. gypseum (1000 mg ⁄L), T. mentagrophytes (10 mg ⁄L), T. rubrum

(0.1 mg ⁄L) and T. verrucosum (0.1 mg ⁄L) (Thienpont et al.,

1975). When cultured on Sabouraud’s dextrose agar medium,

much lower MICs were found for T. mentagrophytes (0.05–

0.2 mg ⁄L), T. rubrum (0.006–0.39 mg ⁄L) and M. canis (0.05–

0.39 mg ⁄L) (Fukushiro et al., 1992).

Adverse reactions

In some patients (108 of 1583) treated with naftiﬁne local side-

effects such as irritation, burning sensation, or dry skin were

observed (Bra¨ utigam & Weidinger, 1993).

A postmarketing surveillance study showed that gastrointest-

inal, skin and taste disturbances are the most common adverse

events related to the use of oral terbinaﬁne in man (Hall et al.,

1997). A rare adverse reaction is hepatobiliary dysfunction

(one of 45 000–54 000 patients); this may be related to

7,7-dimethylhept-2-ene-4-ynal, a reactive metabolite of terbin-

aﬁne (Iverson & Uetrecht, 2001).

Mechanism of action

The membrane-bound squalene epoxidase (product of the ERG1

gene) plays a key role in the biosynthetic pathway from acetate

to sterols (see Ryder, 1988; Mercer, 1991; Ryder et al., 1992;

Vanden Bossche, 1995; Favre & Ryder, 1997). The blockade of

this enzyme results in the accumulation of squalene and

deﬁciency of ergosterol.

Measuring the effects of naftiﬁne, terbinaﬁne and tolnaftate on

fungal and mammalian microsomal squalene epoxidation

revealed a high degree of selectivity for the fungal enzyme

system (Ryder et al., 1992; Nozawa & Morita, 1992). Naftiﬁne

and terbinaﬁne are reversible noncompetitive inhibitors of the

microsomal squalene epoxidase of C. albicans with IC

-values of

1.1 and 0.03 l

, respectively. Tolnaftate is less active with an

-value of 5 l

. Terbinaﬁne is a reversible competitive

inhibitor of the rat liver microsomal enzyme. Fifty per cent

inhibition of rat liver squalene epoxidase was achieved at

0.077 m

terbinaﬁne (Ryder et al., 1992) and 50% inhibition of

the squalene epoxidase of guinea-pig liver microsomes was

achieved at 0.004 mM terbinaﬁne (Ryder et al., 1992).

Favre and Ryder (1996) characterized the squalene epoxidase

activity from T. rubrum and its inhibition by terbinaﬁne. Terbinaﬁne

was a potent inhibitor, with a 50% inhibitory concentration of

15.8 n

. Inhibition was also demonstrated by tolnaftate (IC

51.5 n

). Trichophyton rubrum squaleneepoxidase was only slightly

more sensitive (approximately twofold) to terbinaﬁne inhibition

than was the C. albicans enzyme. Therefore, this difference cannot

fully explain the much higher susceptibility ‡100-fold of dermat-

ophytes than of C. albicans and other yeasts to this drug.

Resistance

Resistance to allylamines has been reported only rarely. However,

the potential to develop resistance by the action of multidrug

efﬂux transporters does exist. Over-expression in S. cerevisiae of

the C. albicans CDR1 and CDR2 genes and of the CaMDR1 gene

confer resistance to terbinaﬁne (Sanglard et al., 1995), suggesting

that terbinaﬁne is a substrate for these transporters (see

discussion on resistance to azole antifungal agents).

BENZIMIDAZOLES

Benzimidazole derivatives were introduced commercially as

anthelmintics and ⁄or antifungals. They are effective at relatively

low doses for the inhibition of a broad range of fungi (literature

reviewed by Delp, 1995).

Mechanism of action

Biochemical, genetic and cytological studies provide evidence for

an effect of benzimidazoles on microtubule assembly (Davidse &

Antifungal agents 19

2003 Blackwell Publishing Ltd, J. vet. Pharmacol. Therap. 26, 5–29

Flach, 1978). Microtubules are found in all eukaryotic cells.

They form a component of the cellular cytoskeleton and the

mitotic spindle. Microtubules are thus important for the

structural integrity of the cell and cell division. They also

function in organelle positioning and movement (Oakley &

Morris, 1980; Burland & Gull, 1984). The major component of

microtubules is tubulin which exists as a heterodimer composed

of the subunits a- and b-tubulin. Microtubules are never static,

and their constant assembly and disassembly involving tubulin

ﬁlaments and various accessory proteins is an integral part of

their function (Hollomon et al., 1998). This essential microtu-

bule property of dynamic instability is based on the binding,

hydrolysis and exchange of GTP in each tubulin dimer (Nogales,

1999). Benzimidazole derivatives interfere with microtubule

assembly by binding to b-tubulin (Davidse & Flach, 1978;

Burland & Gull, 1984; Hollomon et al., 1998). This results not

only in blockage of nuclear division but, for example, in hyphal

tip cells of F. acuminatum, also in displacement of mitochondria

from hyphal apices, disappearance of Spitzenko¨ rpers (vesicle

supply centres) and reduction of linear growth rate (Howard &

Aist, 1977, 1980).

Resistance

In plant pathogenic fungi resistance to the benzimidazole

derivatives is a major problem. Resistance is caused by point

mutations in b-tubulin (for a review see Davidse & Ishii, 1995).

In resistant fungi changes in the b-tubulin genes are restricted

to codons 198 and 200: mutations included replacement of the

glutamine at position 198 with alanine, lysine, valine or

glycine, and of the phenylalanine at position 200 with

tyrosine. The same mutations are also seen in veterinary

medicine where parasitic worms such as Haemonchus contortus

have become resistant to treatment with benzimidazoles (Kwa

et al., 1995).

Thiabendazole

Thiabendazole (Fig. 3), ﬁrst sold as an anthelmintic was then

developed for postharvest fruit treatment and was later used in

veterinary medicine for the treatment of dermatoses. In some

countries thiabendazole is available as a smoke pellet or smoke

generator formulation for application in chicken hatcheries and

poultry houses (Desplenter, 1988). This benzimidazole derivative

is soluble in DMF and dimethyl sulphoxide; maximum solubility

in water at pH 2.2 is 3.84%.

Spectrum of activity

In vitro thiabendazole is active against Trichophyton spp.,

Microsporum spp., less active against A. fumigatus and inactive

against Candida spp. For example, it proved to be fungicidal on

specimens of skin scrapings infected with arthroconidia and

mycelial elements of T. verrucosum. The killing effect was

achieved using dilutions of 1:10 000 and 1:20 000 at 4 and

7 days, respectively (Gabal, 1986).

Thiabendazole inhibited A. fumigatus isolates from avian

species at concentrations between 25 and 50 mg ⁄L (Redig &

Duke, 1985).

Thiabendazole is active against some isolates of Fusarium spp.;

complete inhibition was achieved at 1.5–5 mg ⁄L (Hide et al.,

1992). In vitro tests did not show activity against M. furfur and

M. pachydermatis (Lorenzini et al., 1985). This indicates that

there is no relationship between in vitro activity and clinical

outcome. Indeed, thiabendazole in combination with other drugs

is used in topic otic preparations (de Jaham et al., 2000).

This antimicrotubule benzimidazole is also active against

Pneumocystis carinii at 10 mg ⁄L (Bartlett et al., 1992).

FLUCYTOSINE

5-Fluorocytosine (5-FC) (Fig. 3) is soluble in water

(1.5 g ⁄100 mL at 25 C). 5-FC is well absorbed after oral

administration, penetrates well into body tissues and is excreted

mainly by the kidneys. Drugs that impair glomerular ﬁltration

will decrease the elimination of 5-FC and thus prolong its half-life

(Vermes et al., 2000).

Spectrum of activity

5-Fluorocytosine has a narrow spectrum of activity. Using the

NCCLS M27A broth microdilution method the following MIC

ranges (mg ⁄L) were found: C. albicans (£0.125 to ‡64), C. glabrata

(£0.125 to ‡64), C. tropicalis (0.125–2), C. dubliniensis (0.125–

0.5) and C. parapsilosis (0.125–0.25) (Arthington-Skaggs et al.,

2000). Candida krusei was less sensitive (MIC range: 4–32 mg ⁄L).

Using the same NCCLS method an MIC range of 1 to >64 mg ⁄L

was found for C. neoformans (Arthington-Skaggs et al., 2000;

Barry et al., 2000). Flucytosine showed generally poor activity

against most T. beigelii isolates (Perparim et al., 1996).

A broth microdilution method was also used to determine the

in vitro activities of 5-FC against Cladosporium spp., E. dermatit-

idis,Cladophialophora spp., Paecilomyces spp. and Aspergillus spp.

(A. fumigatus and A. niger) (Llop et al., 2000). The mean MICs

were: 4.59, 0.25, 0.59, 3.17, and 9.75 mg ⁄L, respectively. 5-FC

has no activity against S. chartarum and F. solani (MICsof

256 mg ⁄L). Fusarium moniliforme,F. oxysporum and F. semitec-

tum isolates were also not sensitive to this pyrimidine derivative

(Reuben et al., 1989).

Although 5-FC inhibits growth of many Aspergillus isolates its

clinical efﬁcacy in aspergillosis is equivocal. This difference in

sensitivity may result from the fact that in Aspergillus 5-FC exerts

only fungistatic activity whereas a fungicidal effect is seen in

yeast after prolonged contact (Polak, 1992).

Adverse effects

The severe side-effects of 5-FC include hepatotoxicity and bone

marrow depression. In most patients, these side-effects are

concentration dependent, predictable, and mostly avoidable with

close monitoring to maintain 5-FC concentrations at

20 H. Vanden Bossche et al.

2003 Blackwell Publishing Ltd, J. vet. Pharmacol. Therap. 26, 5–29

<100 mg ⁄L. These side-effects are reversible with drug dis-

continuation or reduction of the dose (Vermes et al., 2000).

Mechanism of action

5-Fluorocytosine is taken up into the cell by a cytosine permease,

an enzyme responsible for the uptake of adenine, guanine,

hypoxanthine and cytosine. Inside the cell 5-FC is rapidly

deaminated into 5-ﬂuorouracil (5-FU) by a cytosine deaminase.

This is a critical enzyme: without conversion into 5-FU no

antifungal activity is observed. As mammalian cells have little or

no cytosine deaminase, 5-FC is fungus-speciﬁc (Polak, 1990).

After the formation of 5-FU two pathways are possible:

5-Fluorouracil can be converted by pyrimidine-processing

enzymes into 5-ﬂuoro-dUMP which is a speciﬁc inhibitor of the

thymidylate synthase, an essential enzyme of DNA synthesis.

This inhibition is at the origin of 5-FC’s fungicidal effect. Via the

pyrimidine salvage pathway, 5-FU can be metabolized into

5-ﬂuoroUTP, which is incorporated into RNA, thus disrupting

protein synthesis. This leads to a cytostatic effect in C. albicans,

Cryptococcus and Aspergilli (Polak, 1990).

Resistance

Monotherapy with 5-FC is limited because of the frequent

development of resistance (Vermes et al., 2000). Indeed the

incidence of primary and the selection of secondary resistance

during monotherapy with 5-FC is a major problem (Polak &

Scholer, 1980). However, in a more recent study on the in vitro

susceptibility of 8803 Candida spp. isolates a very low level of

primary resistance among virtually all Candida spp. was found

(Pfaller et al., 2002). Only for C. krusei a high level of primary

resistance was found: 28% of the 184 isolates tested were

resistant (Pfaller et al., 2002). In C. neoformans 1.8% of strains

tested were found to be naturally resistant, but during treatment

resistant mutants are easily selected (Polak & Scholer, 1980).

The lowest 5-FC resistance frequency is seen in C. albicans,

followed by C. neoformans, and the highest is seen in Aspergillus

(Polak & Hartman, 1991).

Intrinsic resistance to 5-FC is usually the result of a defect in

the cytosine deaminase. The most common enzyme associated

with secondary resistance to 5-FC in clinical isolates of C.

albicans is a decrease in the activity of the uracyl phosphor-

ibosyl transferase (UPRTase). This enzyme is involved in the

synthesis of 5-FUMP and 5-FdUMP (Whelan, 1987; Vanden

Bossche et al., 1987a, 1994a). Resistance in C. neoformans

usually results from the loss of cytosine deaminase or UPRTase

(Whelan, 1987).

ACKNOWLEDGEMENTS

We would like to thank Dr Lieven Meerpoel for providing us with

the chemical structures of the antifungal agents. The assistance

of Christel Herman in preparing this manuscript is gratefully

acknowledged.

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Inhibitors of Brassinosteroid Biosynthesis and Signal Transduction

Article

Full-text available

Nov 2019
MOLECULES

Chemical inhibitors are invaluable tools for investigating protein function in reverse genetic approaches. Their application bears many advantages over mutant generation and characterization. Inhibitors can overcome functional redundancy, their application is not limited to species for which tools of molecular genetics are available and they can be applied to specific tissues or developmental stages, making them highly convenient for addressing biological questions. The use of inhibitors has helped to elucidate hormone biosynthesis and signaling pathways and here we review compounds that were developed for the plant hormones brassinosteroids (BRs). BRs are steroids that have strong growth-promoting capacities, are crucial for all stages of plant development and participate in adaptive growth processes and stress response reactions. In the last two decades, impressive progress has been made in BR inhibitor development and application, which has been instrumental for studying BR modes of activity and identifying and characterizing key players. Both, inhibitors that target biosynthesis, such as brassinazole, and inhibitors that target signaling, such as bikinin, exist and in a comprehensive overview we summarize knowledge and methodology that enabled their design and key findings of their use. In addition, the potential of BR inhibitors for commercial application in plant production is discussed.

Eukaryotic Infections in Dairy Calves: Impacts, Diagnosis, and Strategies for Prevention and Control

Article

Full-text available

Nov 2023

Eukaryotic infections are common among dairy calves and can have significant impacts on their health and growth rates. Fungal infections caused by Aspergillus fumigatus, Trichophyton verrucosum, and Candida albicans can cause respiratory diseases, dermatophytosis, and diarrhea, respectively. Protozoan parasites, including Cryptosporidium parvum, Giardia duodenalis, and Eimeria spp., are also common in dairy calves. C. parvum is highly contagious and can cause severe diarrhea and dehydration, while Giardia duodenalis can lead to poor growth and is transmissible to humans through contaminated food or water. Eimeria spp. can cause coccidiosis and lead to reduced growth rates, poor feed conversion, and death. The common helminthic infections in dairy calves include Ostertagia ostertagi, Cooperia spp., Fasciola hepatica, and Strongyloides papillosus. These parasitic infections significantly impact calf health, growth, and dairy industry productivity. Diagnosis of these infections can be made through fecal samples using microscopy or molecular methods. However, diagnosis of the infections can be challenging and requires a combination of clinical signs and laboratory tests such as culture and PCR. Preventing and controlling eukaryotic infections in dairy calves requires several measures. Good hygiene and sanitation practices, proper management strategies, and timely treatment of affected animals are important. It is also necessary to avoid overcrowding and consider vaccination against ringworm. Further research is needed to better understand the epidemiology and characterization of eukaryotic infections in dairy calves, which will help in the development of more effective prevention and control strategies. In general, good hygiene practices, appropriate management strategies, and timely treatment of affected animals are crucial in preventing and controlling the infections, ensuring the health and well-being of dairy calves.

Antifungal Activity of Disalt of Epipyrone A from Epicoccum nigrum Likely via Disrupted Fatty Acid Elongation and Sphingolipid Biosynthesis

Article

Full-text available

Aug 2024
J. Fungi

Multidrug-resistant fungal pathogens and antifungal drug toxicity have challenged our current ability to fight fungal infections. Therefore, there is a strong global demand for novel antifungal molecules with the distinct mode of action and specificity to service the medical and agricultural sectors. Polyenes are a class of antifungal drugs with the broadest spectrum of activity among the current antifungal drugs. Epipyrone A, a water-soluble antifungal molecule with a unique, linear polyene structure, was isolated from the fungus Epiccocum nigrum. Since small changes in a compound structure can significantly alter its cell target and mode of action, we present here a study on the antifungal mode of action of the disalt of epipyrone A (DEA) using chemical-genetic profiling, fluorescence microscopy, and metabolomics. Our results suggest the disruption of sphingolipid/fatty acid biosynthesis to be the primary mode of action of DEA, followed by the intracellular accumulation of toxic phenolic compounds, in particular p-toluic acid (4-methylbenzoic acid). Although membrane ergosterol is known to be the main cell target for polyene antifungal drugs, we found little evidence to support that is the case for DEA. Sphingolipids, on the other hand, are known for their important roles in fungal cell physiology, and their biosynthesis has been recognized as a potential fungal-specific cell target for the development of new antifungal drugs.

Molecular Structure, Lattice Energy, Hirshfeld Surface and NCI-RDG Analysis, DFT Calculations, and In Silico Molecular Docking of an Imidazole Derivative

Article

Dec 2024

This paper describes a comparative study concerning the molecule 1-methyl-2-[(E)-2-(4-methylphenyl) ethenyl]-4-nitro-1H-imidazole (EMENI). The study involved experimental analysis using high-resolution X-ray diffraction and DFT calculations using the B3LYP functional, along with the 6–311G(d,p) basis set. An accurate depiction of the molecular electron charge density distribution in the compound was achieved using Hansen and Coppens' multipolar model. The study also involved a topological analysis of the total electron density and the location of critical points of the bond, which helps in evaluating the molecular reactivity. Additionally, Hirshfeld surface (HS) and reduced density gradient (RDG) analyses were conducted to explore the specific intermolecular interactions responsible for the stiffness of the molecular arrangement in the compound's packaging. The analyses revealed that H...H interactions significantly influenced the intermolecular contacts, comprising 34.3% of the total interactions as indicated by the 2D fingerprint plots. Furthermore, the study included an exploration of charge transfer and establishment of the electronic characteristics of EMENI through Frontier molecular orbitals (FMO) analysis and HOMO-LUMO energies. Finally, the current study involved an in-silico investigation using molecular docking to evaluate the biological activity of the compound studied. This investigation showed that the title compound exhibits exceptional antiprotozoal activity against Trichomonas

Anticandidal applications of selenium nanoparticles biosynthesized with Limosilactobacillus fermentum (OR553490)

Article

Full-text available

Jul 2024

Candida albicans is one of the most dangerous pathogenic fungi in the world, according to the classification of the World Health Organization, due to the continued development of its resistance to currently available anticandidal agents. To overcome this problem, the current work provided a simple, one-step, cost-effective, and safe technique for the biosynthesis of new functionalized anticandidal selenium nanoparticles (Se NPs) against C. albicans ATCC10231 using the cell-free supernatant of Limosilactobacillus fermentum (OR553490) strain. The bacterial strain was isolated from yogurt samples available in supermarkets, in Damietta, Egypt. The mixing ratio of 1:9 v/v% between cell-free bacterial metabolites and sodium selenite (5 mM) for 72 h at 37 °C were the optimum conditions for Se NPs biosynthesis. Ultraviolet–visible spectroscopy (UV–Vis), Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), X-ray diffraction (XRD), Zeta analyses, and elemental analysis system (EDS) were used to evaluate the optimized Se NPs. The Se NPs absorption peak appeared at 254 nm. Physicochemical analysis of Se NPs revealed the crystalline-shaped and well-dispersed formation of NPs with an average particle size of 17–30 nm. Se NPs have − 11.8 mV, as seen by the zeta potential graph. FT-IR spectrum displayed bands of symmetric and asymmetric amines at 3279.36 cm ⁻¹ and 2928.38 cm ⁻¹ , aromatic and aliphatic (C–N) at 1393.32 cm ⁻¹ and 1237.11.37 cm ⁻¹ confirming the presence of proteins as stabilizing and capping agents. Se NPs acted as a superior inhibitor of C. albicans with an inhibition zone of 26 ± 0.03 mm and MIC value of 15 µg/mL compared to one of the traditional anticandidal agent, miconazole, which revealed 18 ± 0.14 mm and 75 µg/mL. The cytotoxicity test shows that Se NPs have a low toxic effect on the normal keratinocyte (IC 50 ≈ 41.5 μg/mL). The results indicate that this green synthesis of Se NPs may have a promising potential to provide a new strategy for drug therapy.

In vitro effects of omeprazole in combination with antifungal compounds against Malassezia pachydermatis

Article

Full-text available

Oct 2023

Background Fungal and yeast infections, including those caused by Malassezia spp., are becoming increasingly difficult to treat, likely due to the occurrence of drug resistance. Objectives This study aimed to evaluate the antifungal effects of omeprazole (OME), a proton pump inhibitor, against antifungal‐resistant Malassezia pachydermatis and to investigate the potential synergistic effects between OME and other antifungal compounds. Methods In total, 15 samples of M. pachydermatis isolated from the skin of dogs were tested. The susceptibility of M. pachydermatis to itraconazole, ketoconazole, miconazole, terbinafine and OME was assessed using a modified broth microdilution (BM) method. The in vitro efficacy of OME alone and in combination with other antifungal compounds was evaluated for all isolates using the BM chequerboard method. The data obtained were analysed using the fractional inhibitory concentration index (FICI). Results The minimum inhibitory concentration (MIC) values of antifungal compounds and OME against quality control strain (M. pachydermatis CBS1879) were lower than the MIC90 values of same drugs against clinically collected strains. There was no significant difference in MIC values between drugs alone and combination. According to the analysis by the FICI method, no interaction was observed with OME and antifungal compounds. Conclusions Most M. pachydermatis strains were resistant to azole antifungal compounds. OME exerted antifungal effects against Malassezia spp. and even showed good effects on antifungal‐resistant strains. No synergistic effects were observed between the antifungal compounds and OME.

Tubulin targeting agents and their implications in non-cancer disease management

Article

Mar 2025
DRUG DISCOV TODAY

An insight into Synthetic, Structural and Medicinal perspective of imidazole analogs: A review

Article

Mar 2025
EUR J MED CHEM

Imidazole is a versatile heterocyclic moiety that has been found to be widely present in many natural and synthetic molecules. It is a ubiquitous moiety that has been widely explored by the scientific community owing to its broad spectrum of biological activities. To pave the way for future research, a comprehensive study on the imidazole moiety was carried out. This review paper will assist researchers and academics in developing more therapeutically active imidazole-based compounds. Herein, we provide a thorough overview of the current research status of imidazole-based compounds with a wide variety of biological activities, including antiviral, anti-cancer, anti-microbial, analgesic and anti-inflammatory, antidiabetic, antiprotozoal, antipsychotic, anti-aging, antioxidant, and anthelmintic. Through this review article, our focus is to contribute towards the recent advancements in the synthetic methodologies and SAR study of imidazoles that contribute as a useful class of heterocyclic compounds involved in the development of therapeutically active molecules. We sincerely hope that this review will serve as a pertinent guide for imminent research in the development of imidazoles and their derivatives.

A simple green synthesis and characterization of selenium nanoparticles and evaluation of their in vitro anticandidal activity

Article

Dec 2024

Malassezia Yeasts in Animals in the Next-Generation Sequencing Era

Chapter

Nov 2024

The yeasts of the genus Malassezia are part of the normal skin microbiota of a wide range of warm-blooded animals, including humans. Currently, this genus includes 18 species that have been only isolated from different wild and domestic animals and humans. A main characteristic of these yeasts is their lipid dependency, which is caused by the lack of the gene encoding the fatty acid synthase. Thus, Malassezia yeasts have a predilection for sebaceous skin sites. Despite being part of the normal skin microbiota, the population of Malassezia can overgrow under certain circumstances and these yeasts can become opportunistic pathogens causing Malassezia-related diseases such as dermatitis, otitis, and other skin diseases. In dogs, Malassezia pachydermatis is considered a common etiological agent causing otitis and dermatitis. Although azole antifungals are usually effective in resolving these diseases, some cases of treatment failure have recently been reported.

Chemistry and biology of the polyene macrolide antibiotics

Article

Jun 1973

Jeremy Hamilton-Miller

Chemistry and biology of the polyene macrolide antibiotics.

Article

Sep 1973

Jeremy Hamilton-Miller

Rapid, Transient Fluconazole Resistance in Candida albicans Is Associated with Increased mRNA Levels of CDR

Article

Feb 1999

Anti-Candida Drugs — Mechanisms of Action

Chapter

Jan 1991

Hugo Vanden Bossche

During recent years considerable advances have been made in the identification of potential targets for antifungal agents (for a review see Kerridge and Vanden Bossche, in press). A number of them are in the cell wall, others are in the plasma membrane, endoplasmic reticulum, nucleus, mitochondria, cytoskeleton or cytosol (Fig. 1).

Mykosebehandlung mit Imidazolderivaten

Book

Jan 1978

Wolfgang Raab

Mechanisms of resistance to azole antifungal compounds

Article

Jan 1999

Patrick Marichal

Antifungal dermatologic agents: Azoles and allylamines

Article

Jun 2000
COMP CONT EDUC PRACT

A companion article discussed the pharmacology and clinical uses of the more traditional antifungal therapies: polyenes, griseofulvin, and iodides. The availability of newer antifungal drugs, which are often more efficacious with fewer side effects, has led to many safe and effective applications in the management of small animal cutaneous fungal infections. This article describes the pharmacokinetics, modes of action, principal adverse effects, and clinical uses of antifungal agents of the azole (triazoles, imidazoles) and allylamine (terbinafine) classes for treating cutaneous fungal diseases in small animals. Clinical experience gained with the newer antifungals will aid practitioners in choosing appropriate drugs from an expanded armamentarium.

Biosynthesis of the polyene antifungal antibiotic nystatin in Streptomyces noursei ATCC 11455: analysis of the gene cluster and deduction of the biosynthetic pathway (vol 7, pg 395, 2000)

Article

Jul 2000
CHEM BIOL

Fluconazole, but not itraconazole decreases the metabolism of losartan to E-3174.

Article

Feb 1998

Treatment of Aspergillus Keratitis with Imidazoles and Related Compounds

Chapter

Jan 1988

In this study conducted over a period of thirty months, 228 cases of mycotic keratitis were treated with ketoconazole, itraconazole or amphotericin B. This included 69 cases of Aspergillus keratitis. In general, severity of the keratitis influenced response to therapy with all three compounds. A positive correlation was made between age and response of Aspergillus keratitis to itraconazole. Aspergillus keratitis was found to respond more frequently to itraconazole than to ketoconazole or amphotericin B. Itraconazole was significantly superior to ketoconazole and apparently superior to amphotericin B in the treatment of severe Aspergillus keratitis. Although oral azole therapy was not associated with major drawbacks, topical azole therapy was frequently hampered by ocular irritation and inefficacy in treating severe keratitis. Itraconazole may emerge as the drug of choice in treating Aspergillus keratitis.

Antifungal agents of use in animal health - Chemical, biochemical and pharmacological aspects

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