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Antifungal and Immunomodulatory Therapy • CID 2003:37 (Suppl 3) • S157

S U P P L E M E N T A R T I C L E

Review of Newer Antifungaland Immunomodulatory Strategiesfor Invasive Aspergillosis

William J. Steinbach1 and David A. Stevens2

1Division of Pediatric Infectious Diseases, Department of Pediatrics, and Duke University Mycology Research Unit, Duke University, Durham,North Carolina; 2Division of Infectious Diseases, Department of Medicine, Santa Clara Valley Medical Center, and California Institutefor Medical Research, San Jose, and Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University,Stanford, California

The incidence of invasive aspergillosis is markedly increasing, and mortality remains dismal. Previously there

were only 2 antifungals with activity against Aspergillus, but over the last few years there has been an explosion

of newer agents and reformulations of older antifungals. Exploration has also begun with immunotherapy,

with use of cytokines and granulocyte transfusions alone or in combination with antifungal therapy. This

review will detail the available in vitro, in vivo, and clinical experience with the newer antifungal and im-

munomodulatory therapies in development for treatment of invasive aspergillosis.

The incidence of invasive aspergillosis (IA) [1] and its

financial burden [2] have increased significantly in the

last several decades with a parallel increase in the num-

ber of immunocompromised patients. The substantial

increase in patients at risk for developing IA is due to

the development of new intensive chemotherapy regi-

mens, increased use of high-dose corticosteroids,

worldwide increase in solid organ and bone marrow

transplantation, and increased use of immunosuppres-

sive regimens for autoimmune diseases [3]. The mor-

tality rate associated with untreated IA is nearly 100%

in some patient groups, and the overall survival rate

among patients treated with amphotericin B is ∼34%

[4, 5].

There has been a recent surge in the development of

newer antifungals, including new formulations of older

drugs (i.e., cyclodextrin-itraconazole, liposomal nys-

Financial support: National Institutes of Health (HD-00850 to W.J.S.).

Reprints or correspondence: Dr. William J. Steinbach, Division of PediatricInfectious Diseases, Box 3499, Duke University Medical Center, Durham, NC 27710([email protected]).

Clinical Infectious Diseases 2003; 37(Suppl 3):S157–87� 2003 by the Infectious Diseases Society of America. All rights reserved.1058-4838/2003/3707S3-0002$15.00

tatin, and PEG-amphotericin B) and entirely new clas-

ses of drugs with novel targets. Newer strategies have

also been explored with combination antifungal ther-

apies, as well as cytokine therapy, with or without an-

tifungals, to ameliorate the host response system. The

therapeutic options for IA have so markedly increased

in the last few years that clinicians need to be aware of

both the proven therapies of today and the anticipated

treatments of tomorrow.

We reviewed available reports on the experimental

and clinical investigations of newer IA therapy. Al-

though a discussion of every prospective antifungal

tested in the last few years is beyond the scope of any

single review, we present the agents with the most

promise for clinical use against IA and those under-

going clinical trials. The discussion does not cover em-

pirical therapy for continued fever, the numerous fun-

gal prophylaxis strategies in use, or the early diagnostic

techniques for IA still in their infancy. New combi-

nation therapies are analyzed in a devoted and more

thorough discussion of combination therapy for IA

elsewhere [6] in this issue of Clinical Infectious Diseases.

Our focus is treatment of suspected or proven IA, with-

out reiteratation of the existing 2000 Infectious Diseases

Society of America Aspergillus guidelines [7]; instead

S158 • CID 2003:37 (Suppl 3) • Steinbach and Stevens

Table 1. Selected antifungal agents with activity against Aspergillus.

Drug class, drug name (brand/investigational name) Formulation

Polyene

Amphotericin B deoxycholate (Fungizone)a Intravenous

Amphotericin B lipid complex (Abelcet)a Intravenous

Amphotericin B colloidal dispersion (Amphocil; Amphotec)a Intravenous

Liposomal amphotericin B (AmBisome)a Intravenous

Liposomal nystatin (Nyotran) Intravenous

Triazole

Itraconazole (Sporanox)a Oral, intravenous

Voriconazole (VFend)a Oral, intravenous

Posaconazole (SCH 56592) Oral

Ravuconazole (BMS-207147; ER-30346) Oral

Echinocandin

Caspofungin (Cancidas)a Intravenous

Anidulafungin (VER-002; LY303366) Intravenous

Micafungin (FK463) Intravenous

a Licensed for clinical use in United States.

we consider the many recent advances and future trends in the

treatment of IA.

OVERVIEW OF ANTIFUNGAL CLASSES

Because this recent increase in the development of antifungals

has created new hope to combat disappointing mortality fig-

ures, clinicians need to be updated as to the agents now avail-

able in the expanding armamentarium and those in develop-

ment (table 1). Until recently, there were only 2 agents with

activity against Aspergillus, amphotericin B deoxycholate and

itraconazole, and those compounds were often ineffective and

frequently toxic or unpredictable [4]. A recent survey of cli-

nicians experienced in the treatment of IA found that most

used amphotericin B monotherapy for their most immuno-

suppressed patients, whereas itraconazole alone or amphoter-

icin B followed by itraconazole was used for less-immunosup-

pressed patients [8].

Polyenes. The oldest antifungal class is the polyene mac-

rolides, amphotericin B and nystatin, which bind to ergosterol,

the major sterol found in fungal cytoplasmic membranes. This

binding creates channels in the cell membrane that increase

permeability and cause cell death through leakage of essential

nutrients. The fungicidal activity is believed to be due to the

lack of a barrier to essential nutrients and ions [9, 10]. These

drugs also have oxidant activity and disrupt cellular metabolism

in the fungal cell [11].

Azoles. The azole antifungals are heterocyclic synthetic

compounds that inhibit the fungal cytochrome P-45014DM (also

known as lanosterol 14a-demethylase), which catalyzes a late

step in ergosterol biosynthesis. The drugs bind through a ni-

trogen group in their 5-membered azole ring to the heme group

in the target protein and block demethylation of the 14C of

lanosterol, leading to substitution of methylated sterols in the

membrane and depletion of ergosterol. The result is an accu-

mulation of precursors with abnormalities in fungal membrane

permeability, membrane-bound enzyme activity, and coordi-

nation of chitin synthesis [12, 13].

The azoles are subdivided into imidazoles and triazoles on

the basis of the number of nitrogens in the azole ring [9], with

the structural differences resulting in different binding affinities

of the azole pharmacophore for the cytochrome P-450 enzyme

system. With the exception of ketoconazole, the imidazoles are

limited to superficial mycoses, and none, including ketocon-

azole, have activity against Aspergillus. Of the older first-gen-

eration triazoles, fluconazole is ineffective against Aspergillus

and itraconazole has unpredictable bioavailability in some pa-

tients. Newer second-generation triazoles (voriconazole, po-

saconazole, and ravuconazole) are modifications of itraconazole

or fluconazole with activity against Aspergillus, and they gen-

erally have lower MICs than do the older compounds [14].

Echinocandins. For years, most development of new sys-

temic antifungals focused on chemically modifying existing

classes [15]. An entirely new class of antifungals, the echino-

candins and the amino-containing pneumocandin analogues,

are cyclic hexapeptide agents that interfere with cell wall bio-

synthesis by noncompetitive inhibition of 1,3-b-d-glucan syn-

thase, an enzyme present in fungi but absent in mammalian

cells [12, 14]. This 1,3-b-glucan, an essential cell wall polysac-

charide, forms a fibril of 3 helically entwined linear polysac-


charides and provides structural integrity for the fungal cell

wall [16, 17].

Echinocandins inhibit hyphal tip and branch point growth,

converting the mycelium to small clumps of cells, but the older

septated cells with little glucan synthesis are not killed [15].

Therefore, the echinocandin in vitro activity end point is mor-

phological change, not clearing of the medium. Echinocandins

are generally fungicidal in vitro, although not as rapidly as

amphotericin B [14, 18], but appear to be more fungistatic

against Aspergillus [19]. As a class, these agents are not metab-

olized through the cytochrome P-450 enzyme system but

through a presumed O-methyltransferase, lessening some of

the drug interactions and side effects seen with the azole class.

Although echinocandin B was first described in 1974 as a

natural product of Aspergillus nidulans [20], drug development

in this class has only recently expanded. Cilofungin, the first

echinocandin B, had activity in a murine model of IA [21], but

clinical investigation was discontinued because of toxicity of

the vehicle, polyethylene glycol [22]. Three new compounds in

this class (caspofungin, micafungin, and anidulafungin) are in

the final stages of clinical trials, including February 2001 US

Food and Drug Administration (FDA) approval of caspofungin

for refractory IA.

Allylamines. The allylamine class of antifungals [23],

which includes terbinafine, inhibits the enzyme squalene epox-

idase in the fungal biosynthesis of ergosterol [24] and is cur-

rently indicated for the treatment of superficial dermatophyte

and yeast infections. In vitro studies have shown terbinafine to

have fungicidal action [25] against Aspergillus similar to that

of amphotericin B or itraconazole [26]. Limited reports show

some efficacy for IA, and combination therapy remains a

possibility.

Nikkomycins. The naturally occurring nikkomycins are

nucleoside-peptide antibiotics that inhibit the synthesis of cell

wall chitin, a polysaccharide found in most fungi but not in

mammalian cells, as competitive analogues of the substrate

UDP-N-acetylglucosamine for the enzyme chitin synthase [14,

27]. The effect is analogous to the action of the b-lactam an-

tibiotics on bacteria cell walls, leading to osmotic lysis [28].

Nikkomycins were first described as a class of drugs in 1976,

isolated as a result of a program to discover fungicides for

agricultural use [29]. The antifungal activity of nikkomycin Z

against opportunistic fungi has been reportedly low in many

experimental fungal models, including ineffective growth in-

hibition against Aspergillus fumigatus [28, 30–32], leaving com-

bination therapy as a potential option.

Other investigational antifungal classes. Protein synthesis

is an attractive target for antibacterials, but that action is ham-

pered in antifungals because of the eukaryotic nature of fungi

and thus similarity between fungal and mammalian protein

synthesis. Most previous targets used for antifungals exploit

differences in the 2 cells, for example ergosterol, 1,3-b-glucan,

or chitin [33]. The new sordarin class affects the translocation

step of the elongation cycle of protein synthesis [34] by inhib-

iting elongation factor 2, an essential factor for fungal protein

synthesis, and halting the addition of amino acid residues to

the growing peptide chain [33].

The sordarins have minimal in vitro activity against Asper-

gillus [35] and in a disseminated murine model have shown

an irregular response and poor survival efficacy [36]. With such

a limited protective effect against IA, this class of antifungals

was pursued for clinical use for other fungal infections, but IA

combination therapy studies remain to be done. Further in-

vestigation of this class for antifungal use is presently suspended

(F. Gomez de las Heras, personal communication).

NC1175 is an a,b-unsaturated ketone and is fungicidal, al-

though only at concentrations much higher than those of am-

photericin B or itraconazole. In vitro data show that overall,

NC1175 is less effective than amphotericin B or itraconazole

against susceptible A. fumigatus isolates. However, there was

demonstrated activity against both amphotericin B–resistant

isolates and itraconazole-resistant isolates. When tested with a

murine model of IA, the survival rate of animals treated with

NC1175 was not significantly better than that of control sub-

jects; however, the fungal load was reduced significantly at a

rate ∼15% less than that of amphotericin B [37]. Toxicity is a

concern, because NC1175 can act as an alkylating agent, but

it may serve as a prototypical molecule for further development

to obtain greater potency and selectivity.

Partricins are semisynthetic polyenes with in vitro and in

vivo activity against other fungi. SPA-S-753 (Societa Prodotti

Antibiotici) is a derivative of partricin A and, unlike ampho-

tericin B, is water soluble. There have been in vivo reports of

lower animal toxicity when SPA-S-753 was administered in-

traperitoneally (ip) [38–40] and substantially less when given

intravenously (iv) [41]. Additionally, in vivo studies have noted

increased [39] or equivalent efficacy [41] of SPA-S-753 com-

pared with that of amphotericin B in other fungal models.

Another partricin, SPA-S-843, showed generally 2-fold–lower

MICs compared with those of amphotericin B for 13 Aspergillus

isolates but similar fungicidal activity [42]. Although no work

has yet examined IA models, these in vitro observations warrant

further development.

The pradimicins and structurally similar benanomycins dis-

rupt the fungal cell membrane by binding to the terminal D-

mannosides. They have shown some in vitro [43] and in vivo

[44] activity against Aspergillus organisms, but further study is

needed to investigate potential use as monotherapy or com-

bination therapy for IA.


ANTIFUNGAL SUSCEPTIBILITY TESTING ANDRESISTANCE

Preceding any discussion of the in vitro results with newer an-

tifungals has to be analysis of antifungal testing. However, before

reviewing antifungal testing, one must evaluate the methodology

of the available data. Studies have used diverging and imprecise

definitions, parameters for estimation of growth inhibition or

measurement, and in vitro conditions. This nonstandardized

methodology plagued early antifungal susceptibility testing, and

these inconsistencies led to conflicting reports. In vivo testing is

therefore preferred, because of a more predictive nature of human

pharmacokinetic effects and toxicities.

The increasing frequency of fungal infections has prompted

better testing in clinical practice and research as well as cor-

relation between in vitro data and in vivo results [45, 46]. In

vitro antifungal susceptibility testing itself has limited utility if

it cannot predict patient outcome. However, clinical relevance

might best be related to patient factors (e.g., recovery of neu-

tropenia, cessation of glucocorticoid therapy) and not intrin-

sically related to the susceptibility of the fungus itself. Never-

theless, isolates with varying MICs have been found, and there

has been a correlation with in vivo outcome, suggesting clinical

significance.

Amphotericin B susceptibility testing can be of limited clin-

ical value; one study was unable to correlate mouse model

outcomes with MICs, despite testing 32 different formats with

varying media, inoculum sizes, incubation temperatures, and

incubation times [47]. However, another study retrospectively

examined Aspergillus isolates from 29 patients, and all 6 infec-

tions with fungi susceptible to amphotericin B were treated

successfully, whereas 22 of 23 patients with isolates resistant to

amphotericin B (MIC 12 mg/mL) died despite standard treat-

ment. The generally amphotericin B–resistant Aspergillus terreus

isolates required considerably higher MICs, and all of those

patients died; MICs for A. fumigatus and Aspergillus flavus were

lower, but strains from patients who died required MICs of 12

mg/mL [48]. Another study, with 6 A. fumigatus patient isolates

and itraconazole [49], was readily able to differentiate the sus-

ceptible and resistant strains by use of both in vitro conditions

and in vivo models, highlighting a direct clinical correlation

between laboratory results and patient outcome.

A number of factors contribute to clinical efficacy, including

the complex interaction between fungal virulence, pharmaco-

kinetics, and availability of antifungals at the site of infection;

intrinsic or acquired fungal resistance; and the host immune

condition. Resistance to antifungals is becoming more common

among Aspergillus species, and the clinician needs to be alert

to resistance as a possible reason for clinical failure. Several

years ago, the first isolates of A. fumigatus resistant to itracon-

azole were reported from 2 patients; MICs correlated with poor

clinical outcome and were consistent with those determined in

a murine model. Two proposed possibilities for resistance in-

cluded changes in the target enzyme, sterol 14a-demethylase,

or alteration in a membrane transporter [50].

Additional studies found the frequency of itraconazole re-

sistance to be low (!2%) in an in vitro analysis of 230 Aspergillus

isolates [51]. In vitro susceptibility of respiratory Aspergillus

samples revealed 3 patients with acquired itraconazole resis-

tance after long-term therapy [52]. However, a recent review

of 593 Aspergillus isolates from a major medical center found

a steady increase in isolation of Aspergillus over a 5-year period

but no increase in antifungal resistance [53]. Cross-resistance

among the azole class, including the 3 newer triazoles, has

emerged in vitro [54]. An in vitro study of 17 A. fumigatus

isolates noted 11 itraconazole-resistant isolates, with a concur-

rent elevation of posaconazole MICs but not elevation of vor-

iconazole or ravuconazole MICs. The susceptibility patterns to

voriconazole and ravuconazole suggest similar mechanisms of

action and resistance, and the susceptibility patterns to posa-

conazole and itraconazole do the same. Interestingly, the lowest

voriconazole and ravuconazole MICs were seen for highly itra-

conazole-resistant isolates [55].

Standardization and testing of filamentous fungi such as As-

pergillus are still in the preliminary stages [56, 57]. Only recently

did the NCCLS publish accepted standard conditions for molds

(M38-A), specifying conidial suspension and MIC end point

criteria [58]. Previous studies used an extrapolation of the M27-

A criteria for yeast testing [59]. Additional studies have com-

pared visual versus spectrophotometric MIC determination

with use of the broth microdilution standards [60] or E-test

(AB Biodisk) as an alternative to broth microdilution [61].

Even standardized antifungal testing is not without its pit-

falls. For instance, the NCCLS in vitro susceptibility testing

method [57] for filamentous fungi cannot clearly identify am-

photericin B–resistant A. fumigatus isolates but can identify

azole-resistant Aspergillus isolates [62]. Echinocandin activity

on Aspergillus does not give classic MICs in vitro by use of

broth dilution techniques, but echinocandins do demonstrate

clear morphological inhibition in vitro [63].

One potentially important distinction between antifungals is

whether their action is fungicidal or fungistatic. The difficulty

lies in testing and interpretation; a drug may be fungicidal or

fungistatic depending on laboratory variables, such as media

used, duration of incubation, drug concentration, or inoculum

size. Drugs determined to be fungistatic in vitro may be fun-

gicidal in vivo in cooperation with host cells. Conversely, drugs

fungicidal in vitro may be fungistatic in vivo because of tissue

penetration, binding, or pH.

Because one of the primary host defenses, neutrophils, is

absent in neutropenic patients, an antifungal with fungicidal

activity may prove to be crucial for patient survival, analogous

to the need for bactericidal agents in neutropenia [64]. Fun-


gistatic activity is reversible and does not clear the organism;

once the drug is removed, the organism recovers rapidly and

functions normally [10]. With a fungistatic agent, a patient

with an impaired immune system will be unable to clear non-

growing organisms, so relapse can occur after cessation of

treatment.

SPECIFIC ANTIFUNGALS

Polyenes

Amphotericin B deoxycholate. Amphotericin B is clearly not

a newer antifungal—since its initial approval for use in 1958,

it has remained the reference standard for treatment of IA [7]

as well as the standard of comparison for all newer antifungal

agents. However, the fact that amphotericin B remains at such

a post is not by virtue of its effectiveness but rather because

alternatives were lacking, until recently [65]. Amphotericins A

and B are natural fermentation products of a soil actinomycete

collected in Venezuela in 1953, but although each has antifungal

properties, amphotericin A was not developed [66]. Ampho-

tericin B is so named because it is amphoteric, forming soluble

salts in both acidic and basic environments [67]. However,

because of its insolubility in water, amphotericin B for clinical

use is actually amphotericin B mixed with the detergent deoxy-

cholate in a 3:7 mixture [67, 68]. The lipophilic amphotericin

B acts by preferential binding to fungal membrane ergosterols,

creating transmembrane channels that cause an increased per-

meability to monovalent cations. It also inhibits proton ATPase

pumps, depletes cellular energy reserves, and promotes lipid

peroxidation, to result in an increase in membrane fragility and

ionized calcium leakage [68].

The drug is released from its carrier and distributes very ef-

ficiently (190% of the drug is distributed) with lipoproteins,

taken up preferentially by organs of the mononuclear phagocytic

system. Amphotericin B has an apparent volume of distribution

of 4 L/kg; distribution follows a 3-compartment model, with

high concentrations reaching the liver, spleen, and lungs. CSF

values are only 2%–4% of serum concentrations [69]. There is

an initial 24- to 48-h half-life, reflecting uptake by host lipids,

very slow release and excretion into urine and bile, and then

metabolism and a subsequent terminal half-life of up to 15 days

[70]. Experimental in vitro and in vivo studies support concen-

tration-dependent killing with a prolonged post-antifungal effect,

suggesting that large daily doses will be most effective and that

achieving optimal peak concentrations is important [18]. Serum

levels of amphotericin B are proportional to doses administered

but reach a plateau at the third consecutive day of a constant

dose [67]. Peak levels are achieved 1 h after the end of a 4-h

infusion, and there is a relationship between total dose admin-

istered and tissue concentrations, suggesting a progressive ac-

cumulation with continued drug administration [71]. Other au-

thors point out the scant support for higher doses of

amphotericin B [72], emphasizing the varying experimental con-

ditions in published studies and no evidence of a clinical dose

effect [73] to support such higher doses.

The recommended dosage of amphotericin B for IA is 1.0–

1.5 mg/kg/day, and optimal duration of therapy is unknown but

largely dependent on underlying disease, extent of the patient’s

IA, resolution of neutropenia, lessening immunosuppression,and

the return of graft function following bone marrow or organ

transplantation [7, 19]. There is no total dose of amphotericin

B recommended, and the suggested key to success is to give a

high dose in the first 2 weeks of therapy and to change to another

treatment if amphotericin B therapy fails [3].

Tolerance of amphotericin B is limited by its acute and

chronic toxicities. In addition to fungal ergosterol, the drug

also interacts with cholesterol in human cell membranes, which

likely accounts for its toxicity [74]. Up to 80% of patients

receiving amphotericin B develop either infusion-related tox-

icity or nephrotoxicity [67], especially with concomitant ther-

apy with nephrotoxic drugs such as aminoglycosides, vanco-

mycin, cyclosporine, or tacrolimus [75–77]. Renal function

usually returns to normal after cessation of amphotericin B,

although permanent renal impairment is common after larger

doses [65]. Risk factors associated with amphotericin B–related

nephrotoxicity include a total dose of 14 g, sodium depletion,

age 130 years, and concomitant therapy with nephrotoxic drugs

[77]. Sodium supplementation has decreased nephrotoxicity,

and it has been recommended to infuse 500 mL of 0.9% saline

before administration of amphotericin B [65, 78].

Amphotericin B lipid formulations. In addition to con-

ventional amphotericin B, 3 fundamentally different lipid-as-

sociated formulations have been developed that offer the ad-

vantage of an increased daily dose of the parent drug, better

delivery to the primary reticuloendothelial organs (the lungs,

liver, and spleen) [79, 80], and reduced toxicity. The US FDA

approved amphotericin B lipid complex (Abelcet; Enzon) in

December 1995, amphotericin B colloidal dispersion (Ampho-

cil; AstraZeneca or Amphotec; Intermune Pharmaceuticals) in

December 1996, and liposomal amphotericin B (AmBisome;

Fujisawa Healthcare) in August 1997 [81]. The different phar-

macokinetics and toxicities of the lipid formulations are re-

flected in the dosing recommendations: amphotericin B lipid

complex is recommended at 5 mg/kg/day, amphotericin B col-

loidal dispersion at 3–5 mg/kg/day, and liposomal amphotericin

B at 1–5 mg/kg/day. Most clinical data have been obtained with

the use of these preparations at 5 mg/kg/day. Animal studies

clearly indicate that on a similar dosing schedule, the lipid

products are almost always not as potent as amphotericin B,

but the ability to safely administer higher daily doses of the

parent drug improves their efficacy [69].

Amphotericin B lipid complex is a tightly packed ribbon-


like structure of a bilayered membrane formed by combining

dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylgly-

cerol, and amphotericin B in a ratio of 7:3:3. Amphotericin B

colloidal dispersion is composed of disk-like structures of cho-

lesteryl sulfate complexed with amphotericin B in an equimolar

ratio. Liposomal amphotericin B, the only true liposomal prod-

uct, consists of small uniformly sized unilamellar vesicles of a

lipid bilayer of hydrogenated soy phosphatidylcholine–distcaryl

phosphatidylglycerol–cholesterol–amphotericin B in the ratio

2:0.8:1:0.4 [82, 83].

According to the US FDA, all 3 lipid formulations are currently

indicated for patients with systemic mycoses, primarily IA, who

are intolerant of or have infections refractory to conventional

amphotericin B, which could be defined as follows: development

of renal dysfunction (serum creatinine level 12.5 mg/dL) during

antifungal therapy; severe or persistent infusion-related adverse

events despite premedication or comedication regimens; and dis-

ease progression after �500 mg total dose of amphotericin B.

In addition, liposomal amphotericin B is approved as empirical

therapy for the neutropenic patient with persistent fever despite

broad-spectrum antibiotic therapy [79]. Guidelines also suggest

possible initial therapy for patients with marginal renal function

or those receiving nephrotoxic drugs [7].

The lipid formulations have unique pharmacokinetics. Lipid

formulations of amphotericin B generally have a slower onset

of action and are less active than amphotericin B in time-kill

studies, presumably because of the required disassociation of

free amphotericin B from the lipid vehicle [84]. It is postulated

that activated monocytes/macrophages take up drug-laden lipid

formulations and transport them to the site of infection, where

phospholipases release the free drug [81, 85]. Lipid formula-

tions have the added benefit of increased tissue concentration

compared with conventional amphotericin B, specifically in the

liver, lungs, and spleen. In animal models, a 5-mg/kg dose of

liposomal amphotericin B can result in a 10-fold–greater con-

centration in reticuloendothelial tissues and a 4-fold rise in

brain tissues compared with a 1-mg/kg dose of conventional

amphotericin B [80]. It is not entirely clear whether these higher

concentrations in tissue are truly available to the microfoci of

infection. Liposomal amphotericin B has comparatively higher

peak plasma levels and prolonged circulation in plasma [83],

whereas amphotericin B colloidal dispersion has a lower plasma

level than amphotericin B after injection but a longer half-life

and larger volume of distribution [86]. A multicenter maxi-

mum-tolerated dose study of liposomal amphotericin B, in-

cluding 39 patients with IA and with doses from 7.5 to 15 mg/

kg/day, found no demonstrable dose-limiting nephrotoxicity or

infusion-related toxicity. There was a nonlinear plasma phar-

macokinetic profile, with a maximal concentration at 10 mg/

kg/day, but the study was not statistically powered to determine

dose-dependent efficacy [87]. In another study, liposomal am-

photericin B at 10 mg/kg for 4 days followed by 3 mg/kg/day

significantly increased survival (76% vs. 45% survival) and re-

duced chitin content in rat lungs compared with results with

amphotericin B at 1 mg/kg/day [88]. However, a randomized

trial of 87 patients found no clinical difference between lipo-

somal amphotericin B at 1 or 4 mg/kg/day [89].

Complexation with lipids appears to stabilize amphotericin

B in a self-associated state, so that it is not available to interact

with the cholesterol of human cellular membranes, the pre-

sumed major site of toxicity [83, 90]. Another theory for the

decreased nephrotoxicity of lipid formulations is the prefer-

ential binding of its amphotericin B to serum high-density

lipoproteins, unlike conventional amphotericin B’s binding to

low-density lipoproteins [91]. The high-density lipoprotein–

bound amphotericin B appears to be released to the kidney

more slowly or to a lesser degree. Amphotericin B lipid complex

was 10-fold less nephrotoxic in mice than was amphotericin B

[92], and lipid formulations have resulted in renal improvement

in patients with amphotericin B–induced or pre-existing renal

insufficiency [77, 93, 94]. For infusion-related toxicity, there is

a general agreement that liposomal amphotericin B has less

toxicity than amphotericin B lipid complex, whereas ampho-

tericin B colloidal dispersion appears closer in toxicity to con-

ventional amphotericin B [95, 96].

There are no data or consensus opinions among authorities

indicating improved efficacy of any new amphotericin B lipid

formulation over conventional amphotericin B [77, 79, 81, 95,

97]. One exception is that liposomal amphotericin B has shown

less frequent infusion-related adverse events than have the other

lipid formulations and conventional amphotericin B [81, 98].

This leaves the clearest indication for a lipid formulation over

conventional amphotericin B to be reducing glomerular tox-

icity. However, mortality was slightly lower in patients treated

with liposomal amphotericin B than in those treated with con-

ventional amphotericin B in a study of a small number of

patients with documented IA [99]. Another retrospective study

with amphotericin B colloidal dispersion showed response rates

and survival rates higher than with conventional amphotericin

B; however, baseline differences in neutropenia in the 2 groups

could confound survival rate findings [100].

Newer amphotericin B formulations. A new formulation

of liposomal amphotericin B is complexed to a hydrophilic

phospholipid derivative of polyethylene glycol 1900 (PEG). This

creates a hydrophilic coating on the surface of the liposomes,

which reduces blood protein binding and uptake by the mono-

nuclear phagocyte system, achieving a long blood residence

time of the liposomes [101]. Superior efficacy of the PEG for-

mulation of amphotericin B over liposomal amphotericin B

was demonstrated in mice with IA [102]. A single dose of PEG-

liposomal amphotericin B gave results similar to those with a

10-day course of amphotericin B, although it showed no sig-


nificant improvement in mortality over amphotericin B, and

increased doses of PEG-liposomal amphotericin B offered no

increased efficacy [101].

Another new formulation of amphotericin B in cholesterol

hemisuccinate vesicles uses cholesterol, linked to a stabilizing

hydrophilic anchor such as succinate, to form bilayer vesicles

similar to liposomes with phospholipid vesicles. One murine

study saw higher therapeutic efficacy than that of amphotericin

B and efficacy and toxicity similar to those of the lipid for-

mulations [103]. Amphotericin B Hydrosomes (Access Phar-

maceuticals) are a novel formulation of hydrophilic, heparin-

surfaced nanoparticles containing amphotericin B designed to

target infected sites by local adhesion. The beef-lung heparin

coats the core amphotericin B and converts the amphoteric

amphotericin B into a completely hydrophilic formulation

[104]. Amphotericin B Hydrosomes are estimated to be 7-fold

less toxic than amphotericin B in a murine model [105], but

there are no published clinical reports.

Aerosolized amphotericin B. Aerosolized amphotericin B

was first mentioned in 1959 [106] as a solution to amphotericin

B’s known distribution to the liver, spleen, and kidneys [71],

leading to both toxicity and potential clinical failure for pul-

monary IA. Aerosolization of amphotericin B is possible with

various nebulizers [107], and in animals, pharmacokinetics

show much higher concentrations of the drug in the lungs

compared with that after ip injection, with undetectable

amounts in the kidneys, spleen, liver, and brain [108]. Con-

ventional amphotericin B is less efficiently nebulized than li-

posomal amphotericin B, but similar measured aerosol con-

centrations reached the lungs, although the lipid remained

associated with the drug after nebulization [109]. The physi-

ological side effects of aerosolized amphotericin B have been

bronchospasm, especially in asthmatic patients, and nausea and

vomiting; however, in general the aerosol is well tolerated [110].

Most studies have focused on prophylaxis [111], but aero-

solized amphotericin B has been shown to prolong survival and

decrease fungal burden in the treatment of murine IA [112].

Aerosolized treatment with conventional or liposomal ampho-

tericin B showed efficacy compared with control rats but did

not reduce dissemination of disease, unlike reports with sys-

temic amphotericin B. However, systemic amphotericin B with

and without aerosolized therapy were not compared [113].

Aerosolized liposomal amphotericin B was well tolerated in 56

patients after lung transplantation, including 6 patients with

IA, and infections resolved [114]. In a similar approach to

aerosolized amphotericin B, CT–guided percutaneous instilla-

tion of amphotericin B into IA lung lesions was recently de-

scribed with some success in patients for whom systemic ther-

apy failed [115].

Liposomal nystatin. Nystatin, a tetraene diene macrolide,

was the first polyene antifungal. It was discovered in 1949 in

a soil sample from Virginia [116] and named after the state of

New York [117]. Nystatin was licensed for use in 1951 against

superficial Candida infections [117]; however, problems with

solubility and toxicity with parenteral use limited nystatin to

topical use [118]. Recent liposomal reformulation has reduced

toxicity and preserved antifungal activity in vitro [119, 120].

The new liposomal nystatin (Nyotran; Antigenics) is a multi-

lamellar liposomal formulation of dimyristoyl phosphatidyl

choline, dimyristol phosphatidyl glycerol, and nystatin (7:3:1)

[121]. Nystatin has plasma pharmacokinetics markedly differ-

ent from those of all 4 amphotericin B formulations, achieving

comparatively high peak plasma concentrations with dose-de-

pendent antifungal efficacy [21], an elimination half-life of �6

h [18], and markedly less toxicity to mammalian cells [122],

with renal toxicity similar to that of conventional amphotericin

B [123].

In vitro studies have been recently reviewed [118] and yield

mixed results. In one in vitro study, liposomal nystatin was more

active against 10 A. flavus isolates than were all 3 lipid formu-

lations of amphotericin B but less active than conventional am-

photericin B. Against 30 A. fumigatus isolates, liposomal nystatin

was more active than amphotericin B colloidal dispersion and

liposomal amphotericin B but was not as active as amphotericin

B lipid complex or amphotericin B deoxycholate [124]. Another

in vitro evaluation of 40 Aspergillus isolates demonstrated MICs

equal to or slightly higher than those of amphotericin B, am-

photericin B lipid complex, amphotericin B colloidal dispersion,

liposomal amphotericin B, and itraconazole [125]. An additional

in vitro study revealed liposomal nystatin MICs higher than those

of all 4 amphotericin B preparations and itraconazole for A.

fumigatus and Aspergillus niger but lower than those of ampho-

tericin B lipid complex and liposomal amphotericin B and equiv-

alent to those of amphotericin B colloidal dispersion and am-

photericin B against A. flavus. However, liposomal nystatin was

markedly better than any amphotericin B preparation against A.

terreus, although still inferior to itraconazole [126]. MICs of

liposomal nystatin have been considerably lower than those of

nystatin [127, 128], suggesting a complex interaction with lipid-

complexed polyenes.

Despite liposomal nystatin having in vitro activity better than

that of amphotericin B against clinical isolates in some studies,

a murine model of IA showed that survival rates for ampho-

tericin B were significantly better than those for liposomal nys-

tatin at 4 mg/kg/day (93% vs. 74% survival). This study also

found the absence of Aspergillus in the liver in all but 1 mouse

treated with liposomal nystatin and absence in all mice treated

with amphotericin B [129]. Liposomal nystatin had activity in

another mouse model with an isolate with reduced suscepti-

bility to amphotericin B, implying that although the mecha-

nisms of action of these 2 polyenes are similar, mechanisms of

resistance are not identical. In this study, liposomal nystatin at


5 mg/kg was the most successful in terms of survival. Liposomal

amphotericin B at 5 mg/kg was second best, followed by equal

results with amphotericin B lipid complex at 5 mg/kg, am-

photericin B at 1 or 5 mg/kg, and liposomal nystatin at 1 mg/

kg. Liposomal nystatin at 5 mg/kg resulted in fewer positive

results of culture of liver samples than did any other treatment

but was inferior to liposomal amphotericin B at 5 mg/kg. Li-

posomal nystatin at 5 mg/kg was superior to all regimens with

respect to positive renal culture results [130].

In a neutropenic rabbit model, the survival rate of animals

treated with liposomal nystatin at 1 mg/kg/day did not differ

from that of control subjects. Treatment with 2 mg/kg/day in-

creased survival to 70% (7 of 10 animals), compared with 40%

(4 of 10) survival in the group treated with 1 mg/kg/day am-

photericin B. However, treatment with standard-dose ampho-

tericin B was significantly more effective at reducing fungal

burden than was treatment with liposomal nystatin, a decrease

to a 7% fungal burden versus 31%–47% on the basis of various

liposomal nystatin doses [123]. No significant difference in

number of pulmonary hemorrhagic infarcts was seen in those

animals treated with amphotericin B versus liposomal nystatin.

In 1 case report, 5 patients with IA were treated with lipo-

somal nystatin after therapy with amphotericin B failed because

of disease progression or toxicity. Liposomal nystatin cured 4

patients, and all 5 had previously elevated serum creatinine

levels return to normal [131]. A phase II trial noted that 7 of

16 evaluable patients with amphotericin B–refractory IA were

alive on day 30 after treatment with 4 mg/kg/day liposomal

nystatin [132]. Two large trials for treatment of IA have been

closed because of lack of enrollment, and the pharmaceutical

company is not pursuing further research with liposomal nys-

tatin at present (E. Hawkins, Antigenics, personal communi-

cation, 2002).

Azoles

Itraconazole. First publicly described in 1983 [133, 134] and

available for treatment of Aspergillus infection in 1990, itra-

conazole (Sporanox; Ortho-Biotech) adopted a triazole nucleus

with higher specificity for the fungal cytochrome P-450 enzyme

system over the older imidazoles [12]. Itraconazole’s fungicidal

activity is not as efficient as that of amphotericin B, because

inhibition of sterol synthesis takes longer than directly creating

channels in the cell membrane [10]. Historically there have

been several constraints with itraconazole: no parenteral for-

mulation, erratic oral absorption in high-risk patients, and sig-

nificant drug interactions.

Itraconazole has a high volume of distribution and accu-

mulates in tissues, and the tissue-bound levels are more clin-

ically relevant than serum levels [13]. Elimination of itracon-

azole is primarily hepatic, so there is no need for dosage

adjustment in renal failure [13]. Itraconazole is poorly water-

soluble, is not reliably absorbed from the gastrointestinal tract

in its capsule formulation, and has high protein binding [18].

It has a relatively long half-life of 25–50 h, which allows for

once-daily dosing [135]. Dosing is dependent on patient pop-

ulation, with children, especially infants, requiring higher doses

[136]. The metabolism of itraconazole is also varied in specific

ethnic populations because of genetic polymorphisms in the

cytochrome P-450 2C19 enzyme [137].

Dissolution and absorption of itraconazole are affected by gas-

tric pH. Patients with achlorhydria or who are using histamine2

receptor antagonists may demonstrate impaired absorption,

whereas coadministration of the capsule with acidic beverages,

such as cola or cranberry juice, may enhance absorption [138].

Administering a dose with food significantly increases the ab-

sorption of the capsule formulation, but the new oral suspension

is better absorbed on an empty stomach [69]. Side effects are

relatively few and include nausea and vomiting (in 10% of pa-

tients) and elevated transaminase levels (5%) [139]. There has

also been a report of 58 cases suggestive of congestive heart failure

associated with itraconazole therapy [140].

As a potent inhibitor of the fungal cytochrome P-450 3A4

enzyme, itraconazole also has some affinity for the human en-

zyme and therefore has important drug interactions. Prior or

concurrent use of rifampin, phenytoin, carbamazepime, and

phenobarbital should be avoided. Any drug handled by this

cytochrome pathway with normally low bioavailability, exten-

sive first-pass metabolism, or a narrow therapeutic window may

be especially vulnerable [24]. Patients with IA often also have

hypochlorhydria and/or enteropathy as a result of a variety of

conditions, so when oral itraconazole is used, serum concen-

trations must be assessed to ensure adequate absorption [7].

Patients receiving cyclosporine who are treated with a triazole

should have an immediate dose reduction of cyclosporine fol-

lowed by frequent monitoring of serum levels. Furthermore,

the azoles may increase serum, and thus intracellular, levels of

cytotoxic drugs such as vincristine and anthracyclines [74] or

protease inhibitors [141]. Azole-drug interactions may lead to

decreased plasma concentration of the azole, related to either

decreased absorption or increased metabolism, or increased

concentration of any coadministered drugs [79]. In an open-

label comparative pharmacokinetic study of 10 patients, there

was an increase in tacrolimus or cyclosporine levels 48 h after

completion of an iv itraconazole loading dose, so a 50% tac-

rolimus or cyclosporine dose reduction is recommended after

an iv itraconazole loading dose [142].

Itraconazole’s insolubility has led to hampered oral absorp-

tion when administered in capsules. To overcome problems

with variable absorption, itraconazole has now been solubilized

in cyclodextrin, with substantial improvement as an oral so-

lution [13, 143]. Cyclodextrins are naturally occurring dough-

nut-shaped glucose oligomers produced by enzymatic degra-


dation of starch that have been chemically modified for medical

use. The external face of the molecule is hydrophilic to facilitate

solubilization of the complex in water and shield a lipophilic

guest molecule [144]. Once itraconazole is released from the

host cyclodextrin, it follows its standard pharmacokinetics, with

steady-state concentration reached in the second week of daily

dosing. However, a murine model showed a 1100-fold increase

in the area under the serum curve (AUC) by cyclodextrin com-

plexation [145], and bioavailability increased from 30%–33%

to 55% in healthy volunteers when given in oral solution com-

pared with capsule formulation [143]. Because elimination of

cyclodextrin is dependent on glomerular filtration, the vehicle

may accumulate in patients with significantly impaired renal

function, but clinical significance has yet to be fully determined

[12, 146].

A new iv formulation of itraconazole was recently approved

by the US FDA for treatment of pulmonary and extrapulmon-

ary aspergillosis in patients who are intolerant of or have in-

fections refractory to amphotericin B [147]. The iv formulation

can rapidly achieve high and steady-state plasma concentrations

[148, 149], as opposed to the 7- to 10-day period needed for

the capsule or oral formulation [150]. Generally, with the oral

formulations of itraconazole, loading doses of 800 mg for 3

days or 600 mg for 4 days are required. Because of this new

formulation, some centers are modifying their protocols to in-

clude use of iv itraconazole during periods of neutropenia and

mucositis and then a switch to oral solution [151]. The iv

formulation was recently shown to be effective in experimental

IA [152, 153]. In a neutropenic rabbit model, excellent toler-

ance and dose-dependent survival were seen with dosages of 8

and 12 mg/kg/day, but seizures were seen at doasges of 16 mg/

kg/day [153].

In a multicenter open-label study, 31 patients with pulmonary

IA received iv itraconazole for 14 days, followed by capsules for

12 weeks. The iv form was well tolerated, and target therapeutic

concentrations (10.25 mg/mL) were obtained within 2 days in

91% of patients and in all patients within 1 week after iv treat-

ment. These levels were also maintained after switching to oral

therapy. A complete or partial response was seen in 48% of

patients (15 of 31 patients) [149]. These 2 new itraconazole

formulations will allow better pharmacokinetics, especially in

high-risk patients who may have difficulty using capsules because

of mucositis, vomiting, or graft-versus-host disease.

The recommended dosage of itraconazole is 400 mg/day, and

recent guidelines dictate a logical approach to include itracon-

azole as oral therapy after disease progression is arrested with

parenteral amphotericin B [7]. Although there is a theoretical

concern of polyene-azole antagonism due to ergosterol mech-

anisms of action, this regimen appeared safe in a recent survey

of clinical practice of 93 patients treated with this sequential

therapy [8]. This reserves itraconazole for maintenance therapy

once the patient’s condition improves or as initial therapy for

less-immunosuppressed patients. In some studies, itraconazole

is a useful alternative therapy to amphotericin B, with com-

parable response rates [154, 155]. The introduction of iv itra-

conazole now offers a new option for patients intolerant of

amphotericin B.

Voriconazole (UK-109,496). Voriconazole (VFend; Pfizer

Pharmaceuticals) is a new second-generation triazole synthetic

derivative of fluconazole. First described in 1995, it was de-

veloped as part of a program to enhance the potency and spec-

trum of activity of fluconazole. The addition of a methyl group

to fluconazole’s propyl backbone and the substitution of a tri-

azole moiety with a fluoropyrimidine group increased the af-

finity for the target enzyme in A. fumigatus by 1 order of

magnitude [156]. Both fungicidal [10, 156, 157] and fungistatic

activity [157, 158] against Aspergillus have been demonstrated.

In addition to its actions on the 14-a-demethylase, like other

azoles, voriconazole also inhibits 24-methylene dehydrolanos-

terol demethylation, explaining why it is active against a mold

such as Aspergillus when fluconazole is not [156]. Independent

of its activity on the cytochrome P-450 enzymes, voriconazole

is also the only antifungal under investigation that inhibits

Aspergillus conidiation and pigmentation [159].

Voriconazole is extensively hepatically metabolized and

shows ∼90% bioavailability. It appears that cytochrome P-450

2C19 plays a major role in the metabolism of voriconazole,

and this enzyme exhibits genetic polymorphism, dividing the

population into poor and extensive metabolizers as a result of

a point mutation in the gene encoding the protein of cyto-

chrome P-450 2C19 [160]. Approximately 5%–7% of the white

population has a deficiency in expressing this enzyme, so ge-

notype plays a key role in the pharmacokinetics of voriconazole

[161].

Voriconazole is 44%–67% plasma-bound, with nonlinear

pharmacokinetics; has a variable half-life of ∼6 h [162], with

large interpatient variation in blood levels [163]; and has good

CSF penetration [12, 14, 164–167]. Its main side effects include

reversible visual disturbances (increased brightness, blurred vi-

sion) in as many as 33% of treated patients, occasional elevated

hepatic transaminases with increasing doses [167], and occa-

sional skin reactions likely due to photosensitization [12, 156].

The skin manifestations are probably consistent with an indirect

retinoid effect of voriconazole associated with a facial photo-

toxic reaction, because some patients showed improvement

with application of sunscreen [168]. A multicenter randomized

dose-escalation study of oral voriconazole treatment for pa-

tients at risk for fungal infection showed a dose-dependent

reporting of visual side effects (mild blurred vision and pho-

tophobia), which were all reversible and transient and required

no intervention [169]. These visual effects can be associated


with changes in electroretinographic tracings, but no perma-

nent retinal damage has been described [170].

Oral absorption was nonlinear and rapid, with an ∼5-fold

accumulation over the 14 days tested in 1 study of patients

with hematologic malignancy, consistent with reports in healthy

volunteers [167]. In a study of 41 healthy men, nonlinear phar-

macokinetics were again proven, and most subjects achieved

steady-state levels by day 4 after switching from iv to oral drug

administration. Maximum plasma voriconazole levels occurred

at the end of the 1-h iv infusion and between 1.4 and 1.8 h

after oral administration. Mean voriconazole levels did fall after

oral administration to 63%–90% of levels achieved after iv

administration, but most subjects achieved steady state at day

4, and trough levels remained above voriconazole MICs for

Aspergillus species [161]. A pharmacokinetic study of 6 hepatic

cirrhosis patients and age-matched control subjects demon-

strated that patients with hepatic impairment should receive

the same oral loading dose but half of the maintenance dose

[171]. In contrast to adults, children show linear elimination

of voriconazole, and they have a higher elimination capacity

and therefore require a larger maintenance dose than adults do

(4 mg/kg twice daily vs. 3 mg/kg twice daily) [172].

As with some other azoles, the potential exists to modify the

metabolism of other drugs, including cyclosporine and phen-

ytoin [14]. Voriconazole does inhibit metabolism of the com-

monly used immunosuppressive drug tacrolimus. In 1 study,

coadministration of voriconazole and tacrolimus elevated

trough tacrolimus levels in 1 patient after liver transplantation

by nearly 10-fold, which was substantiated in an in vitro liver

microsome model that showed that clinically relevant voricon-

azole levels inhibited the metabolism of tacrolimus [173]. In a

study of patients who underwent renal transplantation, con-

comitant administration of voriconazole with cyclosporine also

resulted in a 1.7-fold increase in the geometric mean AUC for

cyclosporine, so it is recommended that the cyclosporine dose

be halved and levels monitored frequently [174].

One study compared in vitro antifungal activity against 62

clinical isolates of A. fumigatus and demonstrated slightly more

in vitro activity of voriconazole than amphotericin B and itra-

conazole [175]. Another study of 29 clinical Aspergillus isolates

reported statistically significantly better in vitro activity of vor-

iconazole compared with that of amphotericin B. The vori-

conazole MICs for all isolates were !4 mg/mL, suggesting that

voriconazole is effective at clinically achievable concentrations,

whereas only 55% of isolates (16 of 29 isolates) had ampho-

tericin B MICs !1 mg/mL, suggesting a possible predisposition

to clinical amphotericin B treatment failure [165]. Another in

vitro report of 23 isolates of A. fumigatus and A. flavus showed

voriconazole MICs less than those of itraconazole and ampho-

tericin B for A. fumigatus but greater than that of itraconazole

for A. flavus [176]. One large review of in vitro studies showed

that voriconazole had activity superior to itraconazole and am-

photericin B against A. fumigatus, although itraconazole was

superior for A. nidulans and A. terreus, and all had equivalent

activity against A. niger [177].

One study compared data for 205 Aspergillus isolates from

patients in clinical trials and showed that voriconazole and

itraconazole were more potent in vitro against Aspergillus than

was amphotericin B, with itraconazole having the lowest MICs

[178]. In another in vitro study of 216 clinical and laboratory

isolates, voriconazole had excellent activity against Aspergillus

species at significantly lower concentrations than required of

amphotericin B, but itraconazole had significantly better activ-

ity compared with voriconazole against A. fumigatus. Against

itraconazole-resistant isolates, a modest rise in voriconazole

MIC was noted, but the drug remained active and indeed was

more active against amphotericin B–resistant isolates than was

itraconazole [179]. Another study of 3 A. fumigatus isolates

that were in vitro and clinically resistant to itraconazole showed

that voriconazole had good in vitro activity [180]. In vitro

testing of 130 clinical and 20 environmental isolates of A. fu-

migatus revealed no confirmed voriconazole resistance and sim-

ilar voriconazole and amphotericin B MICs, although both were

about double that of itraconazole. Voriconazole was also active

against an itraconazole-resistant control isolate [181].

A. terreus infection is known to be frequently refractory to

amphotericin B [182], and in vitro testing of 101 clinical isolates

with amphotericin B compared with voriconazole showed only

2% of isolates were susceptible to amphotericin B (MIC �1

mg/mL) and 100% were susceptibile to voriconazole (mean 48-

h MIC, 0.22 mg/mL) [183]. The minimum lethal concentrations

(MLCs) were elevated to 13.4 mg/mL for amphotericin B and

17.4 mg/mL for voriconazole, suggesting a lack of fungicidal

activity. Voriconazole, usually fungicidal, also demonstrated

fungistatic properties for this Aspergillus species. In an analysis

of 413 clinical isolates of Aspergillus from phase III clinical trials

of voriconazole, in vitro voriconazole was generally equivalent

to itraconazole and superior to amphotericin B; however, itra-

conazole had lower MICs than did voriconazole for 42 A. terreus

isolates [184].

In vivo studies of IA have mirrored the excellent in vitro

efficacy against Aspergillus. In a rat model, voriconazole was

better absorbed than itraconazole after oral administration,

with much higher peak levels. Survival rates were equal with

4 mg/kg ip amphotericin B compared with 30 mg/kg voricon-

azole, but voriconazole was less effective at lower dosages. In

vivo comparison of voriconazole versus itraconazole (both at

30 mg/kg) demonstrated that survival was 100% in the vori-

conazole-treated group and 75% in the itraconazole-treated

group [185]. However, these results were not statistically sig-

nificant, and no report was made of efforts to maximize oral

absorption or monitor for continued therapeutic levels of itra-


conazole. These survival differences may represent the variable

absorption of itraconazole versus the superior bioavailability of

voriconazole. Interestingly, there is a problem with sustaining

measurable concentrations of voriconazole in rodents, attrib-

uted to rapid first-pass metabolism. Oral administration of

grapefruit juice, a known cytochrome P-450 enzyme inhibitor,

does allow measurable concentrations for at least 10 days for

experimentation during in vivo experimentation [186, 187].

In a guinea pig model of disseminated aspergillosis [188],

voriconazole approximated the ability of amphotericin B to re-

duce tissue burden and was more effective in reducing the num-

ber of positive results of culture of liver samples. The same study

also showed increased survival rates with voriconazole at either

5 or 10 mg/kg/day orally or itraconazole cyclodextrin solution

at 10 mg/kg/day orally, compared with itraconazole at 5 mg/kg/

day orally or amphotericin B at 1.25 mg/kg/day ip. However,

this is a low dose of ip amphotericin B and may therefore not

be a fair comparison. In vitro fungicidal activity was documented

for voriconazole and amphotericin B against all isolates and

against most for itraconazole. According to NCCLS criteria, at

48 h amphotericin B lacked fungicidal activity against 4 of 24

isolates (MLC range, 1 to 116 mg/mL) and itraconazole lacked

fungicidal activity against 8 of 28 isolates (MLC range, 2 to 18

mg/mL), but voriconazole maintained fungicidal activity against

all 28 isolates. However, the authors used the upper-limit MLC

values for fluconazole, because none existed for voriconazole

[188]. In another guinea pig IA model, 80% of guinea pigs (12

of 15 animals) treated with voriconazole at 10 mg/kg/day orally

survived, whereas only 20% (3 of 15) treated with amphotericin

B at 1 mg/kg/day ip survived. Fungal burden was also less at

autopsy in voriconazole-treated animals [189].

In a rabbit model, significant reduction in tissue burden in

the liver only was seen with voriconazole at dosages of 30 or

45 mg/kg/day and in the kidney at 45 mg/kg/day. Amphotericin

B at 1.5 mg/kg/day was significantly more effective at decreasing

tissue burdens. The tissue burden in the lungs in the voricon-

azole group was equal to that in untreated, sublethally chal-

lenged control subjects. Voriconazole at both doses and am-

photericin B eliminated mortality in the lethally and sublethally

challenged animals [190]. In a guinea pig model, voriconazole

performed markedly better than itraconazole in cyclodextrin

in prophylaxis and treatment of Aspergillus endocarditis. All of

the animals given oral voriconazole at 10 mg/kg twice daily

survived, compared with none of those treated with oral itra-

conazole at 10 mg/kg twice daily. In addition, the higher-dose

voriconazole sterilized the heart valves, with a dose-dependent

recovery of Aspergillus with the lower doses of voriconazole.

The mitral valves of the itraconazole-treated animals all grew

hyphae, but the mean serum itraconazole level was subthera-

peutic (0.35 mg/mL) [191]. Voriconazole has been shown to

penetrate the aqueous humor of healthy rabbits, but because

of significant variability in levels, more study is required to

determine actual ocular treatment levels [192].

Clinical reports of patients treated with voriconazole are now

appearing. A child with relapsed leukemia who had failure of

therapy with itraconazole, amphotericin B, and liposomal am-

photericin B showed improvement with voriconazole therapy

[193], and 1 patient with cerebral aspergillosis improved after

voriconazole treatment after failure with amphotericin B [194].

Another patient with cerebral aspergillosis had therapy failure

with amphotericin B, liposomal amphotericin B, and then itra-

conazole with intrathecal amphotericin B. Evidence of infection

resolved with voriconazole, but the patient died of leukemia

[195]. A patient with sinusitis worsened and developed osteitis

of the skull base despite 2 courses of itraconazole, but after their

course of therapy was changed to voriconazole, there was marked

clinical improvement and no evidence of disease recurrence 5

years later [196]. There have also been successful responses in

patients with chronic granulomatous disease (CGD) whose treat-

ment included voriconazole [197, 198]. However, it is somewhat

difficult to evaluate all reports of sequential failure with 1 agent

followed by success with another, because the contribution of

the initial therapy to the eventual good outcome can sometimes

be questioned. A review of 42 children with IA treated with

voriconazole showed that the drug was well tolerated and had

an overall response rate of 43% [172].

The largest prospective clinical trial of voriconazole involved

392 patients at 92 centers in 19 countries over 3 years and

compared initial randomized therapy with voriconazole (2

doses of 6 mg/kg on day 1, followed by 4 mg/kg twice daily

for at least 7 days, followed by 200 mg orally twice daily) versus

amphotericin B (1–1.5 mg/kg/day) followed by other licensed

antifungal therapy. Patients who initially received voriconazole

had statistically significantly better complete or partial response

(53% of patients), compared with those initially receiving am-

photericin B (32%). Survival rates also improved to 71% for

voriconazole, versus 58% for those initially receiving ampho-

tericin B [199]. Analysis in an open, noncomparative multi-

center study of 116 patients treated with voriconazole as pri-

mary therapy (60 patients) or salvage therapy (56 patients) also

yielded encouraging results: 14% of patients had a complete

response, 34% had a partial response, and 21% had a stable

response to voriconazole, whereas 31% failed to respond to

therapy [163]. These data forged the way for US FDA approval

of voriconazole for initial therapy for IA in May 2002.

Although the comparator in the pivotal trial was primary

therapy with amphotericin B, extrapolation to the amphotericin

B lipid formulations may be considered. As noted above, no

comparative large clinical trial has documented a difference in

outcome or survival comparing conventional amphotericin B

with any of the lipid products, although there are clear and

relevant differences in toxicity. Therefore, although not specif-


ically studied, it has been argued that the clear superiority of

voriconazole over amphotericin B should extend to the lipid

formulations. However, in that large voriconazole–amphoter-

icin B study, the high dropout rate in the amphotericin B arm

and consequent switch to other licensed antifungal therapy led

to a discontinuation of the comparator and interruption of

therapy in that arm. This raises the question whether better-

tolerated therapy, given continuously, might have produced a

better result. Perhaps it is more accurate to conclude that vor-

iconazole was more successful than other licensed antifungal

therapy for IA.

Although voriconazole therapy has not been compared with

other treatment modalities, such as itraconazole or echinocan-

dins or combination therapy, in a randomized trial, the su-

periority of voriconazole demonstrated over the reference stan-

dard, initial therapy with amphotericin B, makes it the current

first choice for primary therapy for IA, with a few caveats.

Although voriconazole has a broad spectrum of activity, it has

no activity against the Zygomycetes [200], which often produce

a clinically and radiologically indistinguishable picture and

which may also require surgical intervention for improved out-

come [201]. The additional role of voriconazole in combination

with newer agents such as caspofungin remains hopeful yet

unproven [202].

Posaconazole (SCH 56592). Posaconazole (Schering-

Plough Research Institute) is a second-generation triazole and

is closely related to itraconazole. It is fungicidal in vitro, and

its activity is slightly superior at 48 h to that of itraconazole

and voriconazole yet inferior to that of amphotericin B [203].

Posaconazole has a long half-life of at least 18–24 h in humans

[12, 14] and likely time-dependent killing [18]. Presently only

an oral formulation is available, but a new carrier system is in

development.

In vitro testing showed that posaconazole is 4- to 8-fold more

active than itraconazole and 4- to 16-fold more active than

amphotericin B against 22 Aspergillus isolates [204] and overall

more active than itraconazole against 39 other Aspergillus iso-

lates [205]. Another in vitro study showed lower MICs for 4

Aspergillus species compared with itraconazole and amphoter-

icin B [206]. In vitro comparison of the 3 second-generation

triazoles and amphotericin B against 106 A. fumigatus isolates

showed posaconazole with the best activity, followed by vori-

conazole, itraconazole, and then amphotericin B. Of note, the

itraconazole-resistant strain did not show cross-resistance to

posaconazole or voriconazole [207]. Other in vitro studies of

Aspergillus isolates showed posaconazole with generally lower

MICs than voriconazole, itraconazole, and amphotericin B for

all Aspergillus species tested [208], as well as consistently sig-

nificantly lower MICs than all 3 newer triazoles [209].

An in vitro study comparing posaconazole against 2 echin-

ocandins, caspofungin and anidulafungin, demonstrated fun-

gicidal activity for posaconazole and very little fungicidal ac-

tivity for the other antifungals. The same study reviewed

published MIC data demonstrating better in vitro activity of

posaconazole against Aspergillus species than that of ampho-

tericin B, itraconazole, voriconazole, caspofungin, and anidu-

lafungin [210]. A review of 593 isolates showed susceptibility

for all antifungals tested yet lower posaconazole MICs than

amphotericin B, itraconazole, and voriconazole MICs [53].

In 1 study, 60 clinical isolates of Aspergillus were tested to

compare in vitro activity of posaconazole with that of ampho-

tericin B and itraconazole [64]. The study demonstrated that

geometric mean posaconazole MICs were ∼3-fold lower than

itraconazole MICs and 20-fold lower than amphotericin B

MICs. In addition, A. terreus, frequently resistant to ampho-

tericin B, was the Aspergillus species most susceptible to po-

saconazole among those tested, offering another treatment op-

tion for this historically lethal species. Posaconazole was also

active against itraconazole-resistant isolates tested. In another

study, posaconazole had activity similar to that of itraconazole

against an amphotericin B–resistant isolate of A. fumigatus but

superior activity against a voriconazole-resistant isolate [203].

Cross-resistance of posaconazole to itraconazole- and voricon-

azole-resistant isolates, as might be expected because of struc-

tural similarities [64], is low in several studies [203, 211]. One

study evaluating 12 itraconazole-resistant A. fumigatus isolates

found increased posaconazole MICs for 6 isolates, whereas vor-

iconazole and ravuconazole MICs remained similar to those of

susceptible isolates. These strains had a point mutation in the

cyp51A gene. The other 6 isolates required increased MICs of

all antifungals tested, thereby suggesting at least 2 mechanisms

for itraconazole resistance in A. fumigatus [212]. Another study

confirmed the point mutation leading to a single amino acid

substitution and posaconazole resistance [213].

Another in vitro study showed superior activity of posacon-

azole compared with amphotericin B, itraconazole, and vori-

conazole [203]. For 284 clinical isolates of A. fumigatus, po-

saconazole had a lower geometric mean MIC than

amphotericin B, itraconazole, or voriconazole. Additionally, in

a laboratory-derived voriconazole-resistant strain of A. fumi-

gatus, posaconazole maintained the best activity compared with

the same antifungals [211]. A recent in vitro study of 198

Aspergillus isolates showed posaconazole with clearly better ac-

tivity than either amphotericin B or itraconazole and activity

equivalent to or superior to that of both voriconazole and

ravuconazole [214]. An in vitro study of 15 Aspergillus isolates

showed lower MICs and minimum fungicidal concentrations

(MFCs) of posaconazole than of voriconazole, ravuconazole,

and itraconazole by use of various testing media [215]. In vitro

analysis of 353 Aspergillus isolates found posaconazole to be

the most effective triazole, followed by voriconazole, ravucon-

azole, and itraconazole, and all 3 triazoles and caspofungin were


superior to amphotericin B for A. terreus [216]. Another in

vitro study showed that posaconazole and voriconazole had

superior activity against 2 A. fumigatus strains compared with

that of amphotericin B, itraconazole, and terbinafine [217].

A murine pulmonary IA model showed in vivo efficacy of

posaconazole against A. fumigatus and A. flavus strains [205]. In

a rabbit model of pulmonary aspergillosis, survival rates and

antifungal efficacy of posaconazole in clearing tissue burden were

equal to those of amphotericin B at 1 mg/kg/day and superior

to those of itraconazole, given in cyclodextrin, at equal dosages,

with MICs and MFCs consistently 4 times lower [218]. In another

rabbit model, posaconazole significantly prolonged survival com-

pared with itraconazole or amphotericin B; however, fungal tissue

burden was not reduced compared with amphotericin B treat-

ment [219]. In a murine model, treatment with posaconazole,

or itraconazole in cyclodextrin, for infection with an itracona-

zole-susceptible strain showed a significant reduction in mor-

tality, compared with amphotericin B (5 mg/kg/day ip). Posa-

conazole treatment for infection with an itraconazole-resistant

strain achieved 100% survival, compared with 50% survival in

the amphotericin B treatment arm. Posaconazole was also sig-

nificantly better than amphotericin B and itraconazole at reduc-

ing fungal burden in assays of residual fungal tissue burden [220].

In another murine model of IA, posaconazole achieved signifi-

cantly prolonged survival, greater than that seen with high-dose

amphotericin B (6 mg/kg/dose) [221].

Phase I clinical trials demonstrated good oral absorption in

31 patients with cancer and neutropenia [222]. Posaconazole

should not be coadministered with phenytoin because of inter-

actions causing a large variability in antiepileptic concentration

[223]. In a multicenter study, which included 25 patients with

IA, to evaluate salvage therapy for patients who have refractory

invasive fungal infections, posaconazole was well tolerated and

effective in 53% of patients (8 of 15 patients) at week 4 and in

85% (6 of 7) at week 8; however, there was no mention of the

patients who did not have complete follow-up [224].

Ravuconazole (BMS-207147, ER-30346). Ravuconazole

(Bristol-Myers Squibb) is structurally similar to fluconazole and

voriconazole, containing a thiazole instead of a second triazole.

It is often fungicidal [225, 226] and has 47%–74% bioavaila-

bility, with linear pharmacokinetics [12] and a long terminal

half-life of ∼100 h [227]. The drug is well tolerated, with head-

ache a main side effect, and urine studies suggest no cytochrome

P-450 isoenzyme induction [14]. The drug is well absorbed

following oral administration, and its absorption is enhanced

by food [226]. Ravuconazole was well tolerated in healthy hu-

man subjects in single [227] and multiple doses [228]. Ravu-

conazole alone or coadministered with simvastatin was well

tolerated in 20 healthy subjects, and ravuconazole is a less

potent inhibitor of the cytochrome P-450 3A4 enzyme than are

other triazole antifungals [229]. Ravuconazole did not affect

nelfinavir levels in 14 healthy volunteers [230].

One in vitro study of 16 Aspergillus isolates demonstrated

comparable fungicidal activity and MICs of ravuconazole, itra-

conazole, and amphotericin B [231]. In vitro comparisons

against the 2 other new triazoles and amphotericin B and itra-

conazole demonstrated similar MICs among the 4 azole com-

pounds and general superiority over amphotericin B against

various Aspergillus species [232]. Against 177 clinical isolates

of Aspergillus, all 3 new triazoles showed similar and excellent

activity, and all were superior to itraconazole. Overall, posa-

conazole was the most active agent, inhibiting 94% of isolates

at a MIC of �1 mg/mL, followed by ravuconazole (93%) and

voriconazole (89%) [214]. Another study found ravuconazole

MICs to be slightly higher than itraconazole or amphotericin

B MICs, but no ravuconazole-resistant isolates were detected

[225]. One study found good in vitro activity of ravuconazole

against 141 Aspergillus isolates, including A. terreus [233].

One murine study revealed that both ravuconazole and itra-

conazole led to decreased lung fungal burden in a dose-de-

pendent fashion [234]. Penetration of ravuconazole into

healthy rat tissue showed that the concentration of drug in the

lungs was 2–6 times higher than the corresponding blood con-

centration [235]. Another murine model revealed similar sur-

vival efficacy with itraconazole when both were given at 40 mg/

kg, but ravuconazole was better when both were compared at

10 mg/kg [236]. In a guinea pig model of disseminated asper-

gillosis, ravuconazole as well as itraconazole (10 mg/kg/day)

displayed 100% survival (8 of 8 animals), compared with 87%

survival with amphotericin B at 1.25 mg/kg/day ip. Ravucon-

azole was also more effective in reducing positive results of

culture of organ samples than were amphotericin B or itra-

conazole [237].

Another study showed survival efficacy of ravuconazole in a

rabbit model of IA as well as a decrease in tissue fungal burden

comparable to the effect of amphotericin B, and Aspergillus

infection was cleared in 90% of rabbits challenged [238]. This

group of researchers, with 15 years of experience with rabbit

models of IA, report that in studies of the efficacy of ampho-

tericin B, liposomal amphotericin B, fluconazole, sapercona-

zole, itraconazole, voriconazole, and posaconazole, only am-

photericin B and ravuconazole consistently eliminated A.

fumigatus from organ tissues of challenged rabbits [238]. These

findings were reproduced by another group, who showed dose-

dependent reduction in fungal burden and increased survival

with ravuconazole at 5 mg/kg compared with amphotericin B

at 1 mg/kg (95% vs. 50% survival), with significantly less el-

evation in serum creatinine levels [239].

EchinocandinsCaspofungin (MK-0991, L-743,872). Caspofungin (Canci-

das; Merck) is a fungicidal water-soluble semisynthetic deriv-


ative of the natural product pneumocandin B0 [240]. It has

linear pharmacokinetics [241], is hepatically excreted with a b-

phase half-life of 9–10 h [242], and has few adverse effects [164,

243–245]. Parenteral administration must be used because of

the low bioavailability when administered orally [243, 244]. It

is not metabolized by the cytochrome P-450 isoenzyme system

[246]; however, it does have some interaction with cyclosporine

[14]. Moderate hepatic failure does increase serum concentra-

tions of caspofungin, but renal failure does not alter the kinetics.

There appears to be minimal, if any, myelotoxicity or nephro-

toxicity [247].

Caspofungin is also nonhemolytic for human and mouse red

blood cells, and because 1,3-b-glucan is a selective target present

only in fungal cell walls and not in mammalian cells, this elim-

inates drug mechanism–based toxicity [16]. For caspofungin,

there is an ∼1-h lag period before antifungal action, indicating

that fungal growth is required for killing to occur. The rate of

killing for caspofungin is significantly longer than for ampho-

tericin B, which does not require cell growth for activity [16].

Caspofungin was approved by the US FDA in February 2001

and is indicated for patients with refractory aspergillosis or

intolerance to other therapies.

At present there is no maximal tolerated dose and no tox-

icity-determined maximal duration of therapy. The usual

course is to begin with a load followed by a lesser daily dose

[15]. Pharmacokinetics in healthy men revealed linear phar-

macokinetics and dose-proportional AUC concentration data,

with modest nonlinearity following multiple doses [248]. Two

pharmacokinetic studies of mild-to-moderate hepatic insuffi-

ciency showed clinically insignificant elevations of caspofungin

plasma concentrations in patients with mild hepatic insuffi-

ciency, but a dose reduction from 50 mg to 35 mg daily fol-

lowing the standard 70-mg loading dose was recommended for

such patients [248]. Pharmacokinetics appear slightly different

in children, with lower caspofungin levels in smaller children

and a reduced half-life. Pharmacokinetic projections suggest

that dosing at 50 mg/m2appears to be more appropriate than

at 1 mg/kg/day [249]. Plasma concentrations of tacrolimus were

modestly reduced by ∼20% with coadministration of caspo-

fungin, necessitating the close monitoring of tacrolimus levels.

Cyclosporine increased the AUC of caspofungin by ∼35%, al-

though plasma concentrations of cyclosporine were not altered

by coadministration of caspofungin [250]. Mycophenolate and

caspofungin have no relevant interactions [247].

The echinocandin and pneumocandin classes of antifungals

do not give classic MICs for Aspergillus species when testing.

It is generally established that Aspergillus species are only par-

tially susceptible to echinocandins in standard broth dilution

susceptibility assays [251], producing morphological changes

in Aspergillus hyphae [63]. Caspofungin has shown inhibition

of Aspergillus growth in vitro both microscopically and by assay

for both ungerminated and germinated conidia. In vitro data

do not show complete inhibition, but disk diffusion revealed

comparable zones of inhibition to amphotericin B at similar

concentrations [16].

One in vitro study analyzed 61 isolates of A. niger, 35 isolates

of A. nidulans, and 50 isolates of A. terreus, all considered

refractory to antifungal therapy. Potent activity was seen for A.

niger and marginal activity for A. nidulans, but MICs for A.

terreus were generally high [252]. Caspofungin activity against

A. fumigatus was also markedly increased with the in vitro

addition of human serum samples. Most interesting was the

reactivation of inactive caspofungin by the addition of human

sera, a phenomenon never before described with any other

compound [240]. Caspofungin also showed an additive effect

with monocytes and monocyte-derived macrophages, but not

neutrophils, on Aspergillus hyphal growth. The mechanism was

postulated to be effector cells damaging hyphae, allowing better

activity of the antifungal [253].

Another in vitro study of 78 Aspergillus isolates showed good

activity with caspofungin and correlation between the micro-

dilution technique and disk diffusion measuring an inhibition

zone for calculating antifungal activity [254]. One study used

fluorescent dyes to assess various aspects of cell viability and

demonstrated the focus of caspofungin killing on the apical

and subapical branching cells. The aberrant structures that were

formed changed their staining pattern to one consistent with

cell death [255]. Although there was no reduction in the num-

ber of colony-forming units from standard killing curve mea-

surements, there was a change in staining patterns of dyes that

stain viable and nonviable cells, analogous to the fungicidal

activity of caspofungin against Candida albicans.

In a neutropenic rabbit model, improved survival rates over

those of control subjects and linear pharmacokinetics, with

plasma drug levels above the MIC for the entire dosing interval,

were shown. However, despite dose-dependent hyphal damage,

there was no reduction in residual fungal burden or galacto-

mannan antigenemia, unlike results with amphotericin B [256].

Another in vivo study demonstrated equivalent efficacy of cas-

pofungin and amphotericin B for treatment of IA in both tran-

siently and chronically immunosuppressed murine models. In

the transiently immunosuppressed model, treatment with cas-

pofungin at 0.5 and 1.0 mg/kg/day resulted in 70% and 90%

survival rates, respectively, compared with survival rates of 90%

and 50%, respectively, in animals treated with amphotericin B

at the same dosages. In the chronic-suppression model, survival

at day 28 after challenge was nearly identical for caspofungin

and amphotericin B (both 1 mg/kg/day ip) [257].

In a murine disseminated aspergillosis model, caspofungin

showed 50% effective doses (ED50), with daily dosing, com-

parable to those of amphotericin B [244]. Another study dem-

onstrated equivalent efficacy of caspofungin and amphotericin


B in a 5-day course of therapy [258]; quantitative PCR was

used to determine residual fungal burden instead of analysis

of colony-forming units to alleviate the possibility of error in

counting a filamentous hyphal mass.

In a pivotal clinical study leading to US FDA approval, 56

patients with acute IA underwent salvage therapy after expe-

riencing failure of primary therapy (45 patients) for 11 week

or developing significant nephrotoxicity. These patients were

compared with 210 historical control patients evaluated after

1 week of primary therapy. Recipients generally tolerated cas-

pofungin well and had better outcome than did the historical

control patients; 41% of patients (22 of 54) had a favorable

response with caspofungin [259]. A recent update on all 90

patients enrolled in that trial revealed that 45% had a complete

or partial response, and the drug was generally well tolerated

[260]. A Spanish study done before licensure revealed a 67%

(8 of 12 patients) favorable response rate among patients with

proven or probable IA [261].

Micafungin (FK463). Micafungin (Fujisawa Healthcare)

is an echinocandin lipopeptide compound [12, 262, 263] with

a half-life of ∼12 h and, like all echinocandins, is fungistatic

in vitro versus Aspergillus [264]. Compartmental pharmaco-

kinetics in healthy rabbits in doses of 0.5 and 2 mg/kg revealed

a rapid initial distributive phase followed by a slower elimi-

nation phase, with an estimated elimination half-life of ∼3 h

in single-dose studies [265]. There were dose-independent lin-

ear plasma pharmacokinetics and dose-proportional increases

in AUC with increasing dosage after single and multiple dosing.

Plasma drug concentrations fit best to a 2-compartment open

pharmacokinetic model. The highest drug concentrations were

detected in the lung, followed by the liver, spleen, and kidney

at potentially therapeutic concentrations. Drug concentrations

increased proportionally to dosage. There were no abnormal

elevations in serum creatinine or hepatic transaminase levels

after 8 days of treatment. In plasma, drug concentrations were

maintained in a dose-dependent manner severalfold in excess

of the MIC90 for Aspergillus isolates. Micafungin was unde-

tectable in CSF; however, low levels were detected in brain tissue

and the choroidal layer and vitreous humor of the eye but not

in the aqueous humor [241].

In vitro, micafungin was shown to be more efficacious than

either amphotericin B or itraconazole in several studies [264,

266, 267]. In vitro evaluation against 17 clinical isolates of A.

fumigatus showed MICs of micafungin to be lowest compared

with those of itraconazole, amphotericin B, and 5-fluorocy-

tosine [266]. However, MICs reported were determined by the

Japanese Society of Medical Mycology method, which does not

refer to absence of visible growth as the MIC [268]. Against

64 clinical isolates of 4 Aspergillus species, micafungin was more

active than itraconazole and amphotericin B. The MFCs were

much higher than the MICs, indicating fungistatic activity

[264]. Another in vitro study of 44 clinical isolates of A. fu-

migatus and A. niger revealed micafungin MICs lower than

those of both amphotericin B and itraconazole [267]. An in

vitro study of 6 Aspergillus isolates showed activity of mica-

fungin far superior to that of amphotericin B and itraconazole

[269]. In vitro testing showed activity against A. fumigatus, A.

flavus, and A. niger by MIC as well as disk diffusion methods

[270]. In vitro testing of 45 Aspergillus isolates showed mica-

fungin with lower minimum effective concentrations (MECs)

than amphotericin B, including activity against amphotericin

B–resistant isolates [271].

In a murine pulmonary aspergillosis model, mice were

treated with micafungin at 10 mg/kg iv. Under scanning elec-

tron microscopy, the hyphal surfaces were rough and irregularly

deposited, with a fibril-like structure and markedly flattened

hyphae. Transmission electron microscopy revealed swollen

cells, cells with thinned walls resulting in lysis, and a thick and

short hyphal cell with cytoplasmic vacuolation [272]. In a neu-

tropenic rabbit model of pulmonary aspergillosis, rabbits

treated with micafungin dosages of 0.25–2 mg/kg/day had no

quantitative reduction in concentration of A. fumigatus in tissue

compared with that in untreated control subjects, whereas am-

photericin B and liposomal amphotericin B reduced tissue bur-

dens [273]. Additionally, there was a persistence of galacto-

mannan antigenemia, contrasted with the significant reduction

of galactomannan levels in amphotericin B– and liposomal am-

photericin B–treated animals. Micafungin caused dose-depen-

dent damage of hyphal structures in the lung tissues of animals

but did not clear histologically detectable Aspergillus from lung

tissues. However, there was a significant reduction in the level

of organism-mediated pulmonary tissue injury and a significant

improvement in animal survival rates comparable to those

treated with liposomal amphotericin B (5 mg/kg/day). The au-

thors postulated that the survival effect was due to favorable

safety profile and ability to damage hyphae, thereby reducing

levels of angioinvasion.

A murine model of disseminated aspergillosis showed com-

plete survival at 22 days of all mice treated with micafungin at

1 mg/kg/day. However, the ED50 calculated on the basis of

survival rate at 15 days after infection showed that micafungin

had an efficacy 1.7–2.3 times inferior to that of amphotericin

B, although it did have a partial inhibitory effect on fungal

growth [274]. A murine study of pulmonary aspergillosis con-

firmed 100% survival of mice treated with 1 mg/kg/day at 22

days and an ED50 similar to that of amphotericin B [275].

Another murine systemic IA model demonstrated that mica-

fungin at 10 mg/kg/dose was effective at prolonging survival

and reducing fungal burden in the brain and kidney compared

with control animals [276, 277].

In a study of 20 patients, micafungin was well tolerated, with

no severe adverse events, and a maximum tolerated dose was


not reached at 4 mg/kg/day [278]. In an open-label, multicenter

study of micafungin monotherapy that included 10 patients

with IA, overall clinical response was an improvement in 60%

of patients, with no safety-related issues [279]. A recent study

of micafungin combined with an existing antifungal agent in

pediatric and adult bone marrow transplant recipients with IA

revealed an overall complete or partial response by 39% of

patients, including improvement in 40% of allogeneic trans-

plant recipients [280].

Anidulafungin (VER-002, LY-303366). Anidulafungin

(Vicuron) is a semisynthetic terphenyl-substituted antifungal

derived from echinocandin B, a lipopeptide fungal product

[281]. It has linear pharmacokinetics [14], with the longest half-

life of all of the echinocandins (∼18 h) [164, 282] and has

shown fungistatic or fungicidal activity in different settings

[283]. In vitro data on 22 Aspergillus isolates show that it is 2-

to 4-fold more active, measured by a 75% reduction in growth

for the echinocandins, than caspofungin against A. fumigatus

and A. flavus. Both drugs were considerably more active than

amphotericin B or itraconazole, as determined by use of the

same criteria for the echinocandins and itraconazole and a

classic MIC for amphotericin B [284]. An in vitro study with

20 Aspergillus isolates demonstrated lower MECs, but higher

MICs, of anidulafungin than of amphotericin B [281]. In vitro

evaluation of an additional 26 Aspergillus isolates revealed an-

idulafungin MICs lower than caspofungin MICs; however, both

were generally less active than posaconazole [210]. Favorable

antifungal interactions with anidulafungin and neutrophils or

monocytes have been demonstrated [285], similar to findings

with caspofungin [253].

Pharmacokinetic analysis in healthy rabbits revealed linear

pharmacokinetics, with dose-proportional increases in AUC

[286]. Anidulafungin fit a 3-compartment open pharmacoki-

netic model, with a terminal elimination half-life of up to 30

h. Tissue concentrations after multiple dosing were potentially

therapeutic and highest in lung and liver, followed by spleen

and kidney, with measurable concentrations in the brain tissue

at dosages of �0.5 mg/kg/day. No concentration-dependent

effect was seen in IA. The pharmacokinetics showed ∼6-fold

lower mean peak concentrations in plasma and 2-fold lower

AUCs compared with values with similar doses of caspofungin

and micafungin. Anidulafungin had no effect on residual fungal

burden of A. fumigatus in rabbits with IA, despite dose esca-

lation up to 20 mg/kg, and led only to dose-dependent mor-

phological damage of hyphal structures. However, there was a

significant improvement in survival and reduction in pulmo-

nary tissue injury.

Survival in a rabbit model of IA treated with anidulafungin

was comparable to that seen with amphotericin B (1 mg/kg/

day); however, tissue fungal burden was not reduced and was

actually significantly higher than in untreated control subjects.

The serum Aspergillus antigen level was lowered but not elim-

inated, and higher doses produced mottled livers and necrotic

lungs in a rabbit model [238]. Another rabbit model study

confirmed survival efficacy but noted an upper threshold of

toxicity at 20 mg/kg/day. However, although amphotericin B

yielded a significant reduction in A. fumigatus growth in lung

tissues and bronchoalveolar lavage fluid, no difference in

growth was observed in animals treated with either anidula-

fungin or placebo. Analysis of that lung tissue showed dose-

dependent damage of hyphal elements, with a progressive re-

duction in length and increased swelling. By comparison, tissues

from amphotericin B–treated rabbits seldom revealed any hy-

phal elements [283].

In another mouse model, amphotericin B was superior to

anidulafungin in reducing fungal counts in renal tissue, but

anidulafungin was effective against an amphotericin B–resistant

isolate [287]. Of concern is a report of lethal toxicity in a mouse

model, through an unclear mechanism, with anidulafungin and

cortisone, hydrocortisone, or triamcinolone but not dexame-

thasone [288]. Recent studies show that this phenomenon also

occurs with micafungin, so the action might be a property of

the echinocandin class [277]. This animal model finding is of

concern, because most patients with progressive IA are also

taking glucocorticoid therapy for their underlying disease.

A phase I study has shown anidulafungin to be safe and well

tolerated in 29 healthy volunteers, with the highest-dose cohort

experiencing transient elevations in liver function test results

that exceeded twice the upper limit of normal [289]. In a sep-

arate study, 12 subjects with mild or moderate hepatic im-

pairment did not show clinically significant changes in the

pharmacokinetic parameters of anidulafungin [290]. A phase

III open-label, noncomparative multinational study of anidu-

lafungin with a lipid formulation of amphotericin B for IA

scheduled to enroll 60 patients was recently discontinued (Vi-

curon, press release, 2001).

Allylamines

Terbinafine. Since its introduction into clinical practice in

1991, oral terbinafine (Lamisil; Novartis Research Institute) has

been used by clinicians mainly for dermatophyte infections of

the skin and nails [25]. Terbinafine is well tolerated [291], has

a bioavailability of 70%–80% after oral administration, and is

highly lipophilic, with a terminal half-life of up to 3 weeks

[292]. The metabolism of terbinafine is not dependent on the

cytochrome P-450 system, but metabolism takes place exten-

sively in the liver [69], with subsequent excretion in the urine

[25]. Terbinafine is rapidly absorbed following oral adminis-

tration in humans [293], with a maximal plasma concentration

of ∼0.8–1.5 g/mL after a single 250-mg oral dose [294]. There

have been reports of side effects with terbinafine, including


hepatitis [295, 296], pancytopenia [295, 297], hair loss [298],

and drug interaction with tricyclic antidepressants [299, 300].

The lipophilic terbinafine concentrates highest in the sebum

and hair, with quantifiable concentrations 56–90 days after a

final oral dose and a slow redistribution from the peripheral

sites back to the central plasma compartment. Pharmacokinetic

modeling indicated that at steady state, almost all (94%) of the

terbinafine in the human body resides in adipose and skin

tissues, with only 0.4% in the lung [301], which might theo-

retically lead to difficulty in treating systemic infection [302].

Tissue distribution in rats, as determined by high-performance

liquid chromatography after a 6 mg/kg iv dose, revealed a slow

uptake and efflux of terbinafine in the skin, with an estimated

redistribution half-life from the skin of 1.6 days. Approximately

60% and 28% of the apparent volume of distribution of ter-

binafine was to the skin and adipose tissue, respectively [303].

Further analysis of terbinafine distribution in human blood

shows a higher affinity for plasma proteins than blood cells in

a concentration-independent manner [304].

Ryder [305] reviewed several in vitro studies showing good

terbinafine activity against 5 different Aspergillus species. One

study showed that terbinafine was active against A. flavus and

A. niger at one-fourth the concentration of amphotericin B;

however, amphotericin B was fungicidal at one-fourth the con-

centration against A. fumigatus [306]. In vitro testing of 28

Aspergillus species, including 1 strain of A. terreus, showed low

terbinafine MICs (0.3–1.0 mg/mL). Terbinafine serum levels in

rabbits after a 100-mg/kg loading dose and 50 mg/kg twice-

daily dosing have been shown to be considerably above these

MICs for Aspergillus species [307]. In vitro data on 100 clinical

isolates show superior activity, compared with amphotericin B

and itraconazole, against A. flavus, A. niger, and A. terreus but

inferior activity against A. fumigatus. This presents a potential

clinical problem, because A. fumigatus accounts for ∼90% of

human infections in some studies [3]. Interestingly, the ter-

binafine MIC was slightly lower for the itraconazole-resistant

A. fumigatus isolates than for the itraconazole-susceptible iso-

lates. Terbinafine was also more fungicidal for non-A. fumigatus

isolates (88% vs. 35% isolates) [308]. A case report of a patient

who died of Aspergillus ustus infection showed that, compared

with amphotericin B, itraconazole, and voriconazole, terbinaf-

ine had the best in vitro activity and was the most fungicidal

antifungal against that isolate [309].

A rat pulmonary IA model revealed no improvement in sur-

vival over control subjects at doses of 20–80 mg/kg for 1 week,

and the lungs of the few survivors were grossly enlarged and

filled with multiple abscesses at necropsy. Terbinafine concen-

trations determined by bioassay were adequate, and the authors

concluded that terbinafine was inactive in their model despite

adequate lung concentrations. Later in vitro testing revealed that

MICs were 6-fold higher in serum than in laboratory media

[310]. Others speculate that this decreased activity in the presence

of serum is due to a reduced bioavailability resulting from non-

specific binding of the drug to major serum components. This

could account for the low efficacy in experimental animal models

and the high efficacy in dermatophyte infections [311]. After

administration of 14C-labeled terbinafine in a rat model, the high-

est concentrations were found in the liver and pancreas but

declined rapidly, whereas the concentrations in fat tissues de-

clined more slowly, representing the drug’s high lipophilicity

[294].

There are 2 poorly defined case reports of successful treat-

ment of IA with terbinafine, each published as a personal com-

munication. A 15-year-old boy with acute lymphocytic leu-

kemia, graft-versus-host disease, and meningocerebral

aspergillosis was treated with amphotericin B, itraconazole, flu-

conazole, and terbinafine for 169 days. At that point only ter-

binafine treatment continued until day 400, when the patient’s

leukemia was in remission, and he fully recovered from IA [25].

A second patient with pulmonary IA was treated successfully

with amphotericin B plus terbinafine and then continued to

receive terbinafine monotherapy after the patient became in-

tolerant of amphotericin B [312].

IMMUNOMODULATORY THERAPY

Host defense is paramount, because IA generally develops only

in certain subsets of severely immunocompromised patients

[313]. Two lines of host defense exist against Aspergillus. The

first is formed by alveolar macrophages, and in vitro mouse

studies have suggested that resident pulmonary macrophages

are responsible for clearing inhaled Aspergillus conidia from the

lung [314]. Phagocytes appear to kill the inhaled conidia better

once the conidia are metabolically activated and swell, which

takes place in a suitable moist environment such as the terminal

airways of the lung [315]. After phagocytosis, germination of

conidia into the invasive hyphal form is inhibited inside the

phagolysosome. After 24 h, 90% of engulfed conidia are killed,

and, 6–12 h later, they are completely digested [316].

If conidia escape this defense system, then, after transfor-

mation into the invasive Aspergillus hyphae, they become sus-

ceptible to neutrophil killing through the release of toxic oxygen

radicals [315]. It appears that both mechanisms of host defense

are important for Aspergillus to cause invasive disease. The host

may be overwhelmed in the presence of neutropenia, and mac-

rophages may be overcome with high challenge doses, activated

conidia, or corticosteroid suppression of conidiacidal activity

[317]. The ability of polymorphonuclear neutrophils (PMNLs)

to kill hyphae was up-regulated in mice that survived infection

and severely depressed in mice that died of infection [318].

Few patients with persistent neutropenia and IA survive, and

indeed resolution of IA has followed recovery of neutrophil


levels in most cases. In bone marrow transplant recipients, the

risk for IA remains even after engraftment, highlighting the fact

that although the number of phagocytes is important, their

ability to kill must be adequate [319]. Immunotherapy offers

many therapeutic advantages through the availability of a wide

range of recombinant cytokines that exert their effects indirectly

through leukocyte activation rather than directly on the fungus.

Immunotherapy is designed to increase the number of phag-

ocytic cells and shorten the duration of neutropenia, modulate

the kinetics or actions of those cells at the site of infection,

and/or activate the fungicidal activity of phagocytes to kill fun-

gal cells more efficiently [68, 320].

Corticosteroids are a well-known major risk factor for the

development of IA [321] and can suppress the ability of mono-

cytes/macrophages to kill conidia through inhibition of non-

oxidative processes and impairment of lysosomal activity. Cor-

ticosteroids also inhibit PMNLs in their chemotaxis, oxidative

bursts, and activity against hyphae [322–325]. This may be im-

portant in IA, because A. fumigatus has an quickened doubling

time of 48 min in the presence of hydrocortisone in vitro [326].

Generally, corticosteroids suppress macrophages, whereas cy-

totoxic chemotherapy decreases neutrophil number and func-

tion. For instance, granulocytopenic rabbits had more hyphae

in tissue and greater mortality through angioinvasion than did

cyclosporine- and corticosteroid-immunosuppressed animals,

which had more conidia [327]. Dexamethasone has also been

shown to decrease the amount of granulocyte-macrophage col-

ony-stimulating factor (GM-CSF) secreted by monocytes [328].

Interleukins. Antigen-specific immune responses result in

selective stimulation of two CD4+ T helper (Th) cell subsets,

leading to unique patterns of cytokine secretion and a differ-

ential regulation in mice that are resistant or susceptible to IA.

Generally, Th1 cells confer protection against fungal diseases,

and Th2 responses are associated with disease progression [318,

329]. Protective immunity is associated with CD4+ Th1 cells

producing IFN-g, IL-2 , and IL-15, macrophages producing IL-

12, or those mice treated with IL-4 or IL-10 antagonists. Disease

progression is seen in mice producing Th2 cytokines IL-4, IL-

10, and IL-13 or mice treated with neutralizing antibody to

IFN-g or IL-12 [95, 329, 330].

Bronchoscopic findings in a murine model also indicate re-

sistance to disease progression associated with elevated produc-

tion of TNF-a, IL-6, IL-12, IFN-g, and IL-2 [331]. IL-4 pro-

duction is decreased in mice surviving IA infections [318]. IL-4–

deficient mice were found to be more resistant than wild type

mice to infections. When treated with cyclophosphamide, the

wild-type mice died of infection, but the cyclophosphamide-

treated IL-4–deficient mice were resistant to infection and sur-

vived. Lung T helper cells from IL-4–deficient mice produced

50%–60% elevated levels of IFN-g and IL-12 but 50% reduced

IL-5 and IL-10 levels, compared with those in non–IL-4–deficient

mice. This indicates that IL-4 down-regulates IL-12 production

and, consequently, Th1 cell development [331].

Th1 resistance was impaired on IL-12 neutralization and in

IL-12–deficient mice [331]. However, administration of recom-

binant IL-12 failed to increase protective effects in mice [318].

IL-12 immunotherapy remains to be fully studied because of

potential deleterious inflammatory responses [332]. A dissem-

inated aspergillosis murine model demonstrated decreased fun-

gal burden and increased survival of IL-10 knockout mice com-

pared with wild type [333]. One in vitro study found that IL-10

suppressed oxygen radical production and hyphal damage of

A. fumigatus. Anti–IL-10 antibody, IFN-g, and GM-CSF ad-

ministration counteracted the suppressive host defense effects

of IL-10 on phagocyte hyphal damage and oxygen radical pro-

duction [334].

Granulocyte colony-stimulating factor (G-CSF). Human

recombinant G-CSF has been approved for clinical use since

1991 [335]. The potential of exogenously administered G-CSF

therapy seems to be in maintaining the innate signal for longer

production of PMNLs or initiating that signal earlier if en-

dogenous production is decreased or insufficient during a spe-

cific time, such as during neutropenia after bone marrow trans-

plantation [336]. One fear has been the unwanted side effect

of increased inflammatory products, such as the untoward re-

lease of reactive oxygen species and lysosomal contents, with

G-CSF use [337]. However, in vivo and human studies have

also shown that G-CSF reduces production of inflammatory

mediators such as IL-1, TNF-a, and IFN-g [336].

In addition to increasing the number of mature circulating

PMNLs, G-CSF enhances phagocytic activity and oxidative

burst metabolism. Human G-CSF affects function of granu-

locytes only, not macrophages, and has been shown to have a

protective effect in murine models of IA. Prophylaxis with hu-

man G-CSF and amphotericin B or itraconazole showed some

additive effect in neutropenic animal models of IA but not in

those immunosuppressed with cortisone, which has a greater

effect against macrophages. In a neutropenic (cyclophospham-

ide-induced) murine model, human G-CSF alone was ineffec-

tive but with amphotericin B showed synergy in survival greater

than with itraconazole and G-CSF [338].

Pretreatment of neutrophils with G-CSF and/or IFN-g can

attenuate the inhibitory effect of corticosteroids on PMNL-in-

duced hyphal damage [339]. G-CSF administered to human vol-

unteers increased the fungicidal activity through enhanced res-

piratory bursts of their PMNLs against Aspergillus conidia of their

PMNLs by 4-fold [340]. However, there is no clear evidence that

G-CSF benefits patients with aspergillosis. One review found no

significant reduction in fungal infections in patients with acute

myelocytic leukemia treated with G-CSF [341].

GM-CSF and macrophage colony-stimulating factor (M-

CSF). GM-CSF– and M-CSF–treated human macrophages


exhibit enhanced conidial phagocytosis, oxygen radical pro-

duction, and hyphal damage [342, 343]. GM-CSF promotes

differentiation, proliferation, and activation of cells in the mac-

rophage/monocyte system, prevents the defective in vitro an-

tifungal activity of corticosteroid-treated monocytes [322], and

also enhances phagocytic activity of PMNLs [319]. GM-CSF

also doubles the life span of neutrophils, exerts a stimulatory

effect, and enhances their attachment to endothelial cells [344].

GM-CSF has increased spleen cellularity in mice, indicating

potent stimulation of hematopoietic cells, and increased IFN-

a production by concanavalin A compared with control cells

[345]. In another murine model, the antifungal activity of bron-

choalveolar macrophages treated with dexamethasone was sig-

nificantly less than that of macrophages from dexamethasone-

plus GM-CSF–treated mice. This study also showed that

GM-CSF administered before dexamethasone blocked the del-

eterious effects, but if given after dexamethasone, GM-CSF

could not reverse the effect on macrophages [346]. This con-

firmed earlier in vitro work demonstrating that coincubation

of bronchoalveolar macrophages with GM-CSF followed by

dexamethasone significantly prevented the conidiacidal sup-

pression. Also, coincubation of bronchoalveolar macrophages

with dexamethasone and subsequent GM-CSF removed the

deleterious effect if the dexamethasone was discontinued, but

if the dexamethasone pretreatment continued despite GM-CSF

use, the anticonidiacidal effects persisted [347]. In another

study, both murine and human GM-CSF can counteract dex-

amethasone suppression of murine macrophage function [348].

GM-CSF can act synergistically with TNF-a [349], and there

are case reports of success with GM-CSF as part of a treatment

regimen [350, 351]. GM-CSF has been shown to offer some

protection against IA in a clinical trial of patients with acute

myelogenous leukemia, decreasing the fungal infection–related

mortality from 19% to 2% [352]. A small pilot study of GM-

CSF in combination with amphotericin B (1 mg/kg/day) for

treatment of proven fungal infection included 2 patients with

refractory aspergillosis. One patient with pulmonary IA who

underwent bone marrow transplantation because of breast can-

cer showed a partial response, whereas another patient with

acute myelogenous leukemia and sinopulmonary IA had treat-

ment failure [353]. However, use of GM-CSF is not without

its concerns. In 1 case, a child with IA received GM-CSF to

overcome neutropenia and, after bone marrow recovery, de-

veloped pulmonary cavitation and fatal hemoptysis [354]. Bone

marrow recovery may lead to liquefaction of pulmonary foci

and to potential erosive bleeding resulting from an increased

inflammatory response, especially in the first week following

cavitation [355].

M-CSF modulates mononuclear phagocyte functions such

as H2O2 production and phagocytosis and enhances production

of IL-1, IFN-g, and TNF-a [356, 357]. M-CSF was found to

enhance the nonoxidative mechanism of macrophages for in-

hibiting germination, but, although it recruited more macro-

phages to ingest conidia, it did not significantly effect ingestion

of more conidia [357]. A neutropenic rabbit model demon-

strated that prophylactic administration 3 days before inocu-

lation and then throughout neutropenia augmented pulmonary

host defenses against IA. Rabbits receiving M-CSF had in-

creased survival rates and greater numbers of activated pul-

monary alveolar macrophages than did control animals [358].

A phase I trial of M-CSF suggested some benefit in patients

with Aspergillus infections, but an insufficient number of pa-

tients were treated to show a statistical benefit [343, 359].

TNF-a. TNF-a is a proinflammatory cytokine secreted by

various macrophage populations and is shown to be a critical

initiator in innate immunity against respiratory pathogens

[360], including A. fumigatus [361]. In vitro, TNF-a appears

to enhance early host defense against Aspergillus invasion, with

slight increases in oxygen radical production by macrophages,

up-regulation and activation of alveolar macrophage phago-

cytosis, and augmented production of other cytokines such as

GM-CSF. It also augments a late defense with increased PMNL

hyphal damage by production of oxygen radicals [356, 362].

In vitro, GM-CSF and TNF-a administration have been

shown to counteract dexamethasone-induced immunodefi-

ciency [322]. Animal model depletion of TNF-a results in in-

creased fungal burden and mortality [361], and resistance is

further impaired in IFN-g–deficient mice [331]. Treatment of

mice with neutralizing antibodies to TNF-a and GM-CSF re-

duces the influx of PMNLs into the lungs and delays fungal

clearance [363]. Intratracheal administration of a TNF-a ag-

onist resulted in survival benefits when given 3 days before A.

fumigatus inoculation but not when given concomitantly with

conidia, suggesting that pretreatment may provide macrophage

priming [361]. However, excessive toxicities in doses required

to have a biologically useful effect preclude safe administration

to humans [319, 362].

IFN-g. IFN-g promotes TNF-a production [329] and en-

hances PMNL- and mononuclear cell–induced damage by in-

creasing the oxidative burst of PMNLs in response to stimuli

such as nonopsonized hyphae of A. fumigatus [356]. IFN-g and

G-CSF can each enhance the oxidative bursts and fungicidal

activity in vitro of human PMNLs against A. fumigatus hyphae,

with the combination of the 2 cytokines showing an additive

effect [364]. IFN-g can also restore the corticosteroid-sup-

pressed fungicidal activity of human PMNL and elutriated

monocytes [322, 342, 365], and IFN-g–treated human mono-

cytes show enhanced oxygen radical production and damage

to A. fumigatus hyphae [342].

Exogenous administration of IFN-g and TNF-a has resulted

in protective effects in a murine model of IA [366] by decreasing

mortality and the number of organs affected by Aspergillus.


Conversely, IFN-g and TNF-a neutralization resulted in in-

creased disease and increased expression of IL-10. Although

IFN-g is better than G-CSF or GM-CSF at enhancing PMNL

hyphal damage, and both IFN-g and GM-CSF result in en-

hanced hyphal damage by PMNLs in vitro [367], combination

treatment does not increase damage [342, 368]. In vitro, IFN-

g augments PMNLs of patients with CGD by an undetermined

mechanism [369], although previous work demonstrated a

myeloperoxidase-dependent oxidative process [370]. IFN-g has

been proven to help prevent IA in patients with CGD [371],

and there are case reports of the successful use of antifungals

and IFN-g for treatment of patients with CGD [372, 373]. One

recent case report details use of liposomal amphotericin B and

both GM-CSF and IFN-g in successful treatment of sinocer-

ebral aspergillosis, with the addition of the IFN-g temporally

related to clinical resolution [374].

Combination antifungal and immune therapy. An ad-

ditive effect with monocytes and monocyte-derived macro-

phages, but not PMNLs, and caspofungin was seen on Asper-

gillus hyphal growth [253]. An additive antifungal effect of

hyphae cocultured with neutrophils and anidulafungin was also

seen, and monocytes cocultured with hyphae preexposed to

anidulafungin were synergistic [285]. Theoretically, effector cell,

antifungal, and cytokine mechanisms may interact synergisti-

cally, with possible antifungal cell wall damage increased by a

cytokine or cytokine stimulation of effector cells enhancing

penetration of antifungals into cells [375]. Amphotericin B in-

teracting with host cells can also have a positive effect by ac-

tivation of macrophages through an oxidation-dependent pro-

cess [11]. Neutrophils and monocytes with voriconazole have

been shown to act additively in inhibiting fungal hyphal growth.

GM-CSF treatment of neutrophils with voriconazole increases

activity against hyphae compared with that of control neutro-

phils and voriconazole, but no comparable effect was seen on

monocytes [365, 376]. An in vivo additive effect was also found

with G-CSF and posaconazole in 1 study [325], and no an-

tagonism was seen in another in vivo study [377].

Granulocyte transfusions. Granulocyte transfusions were

first used to treat bacterial infections in neutropenic patients

in 1934 [378], but the discovery that functionally intact trans-

fused PMNLs are found in bronchoalveolar lavage fluid [379]

has increased interest in the therapy for IA. However there are

concerns with granulocyte transfusion: alloimmunization

[380], which could complicate subsequent transplantation, or

amphotericin B and neutrophil aggregation leading to pul-

monary leukostasis and respiratory failure [381]. A review of

125 granulocyte transfusions given with concurrent ampho-

tericin B to 31 granulocytopenic patients revealed pulmonary

deterioration temporally related to therapy with amphotericin

B, granulocyte transfusions, or both. However, they failed to

show a specific detrimental interaction between the granulo-

cytes and amphotericin B, beyond the reactions associated with

each modality.

There are anecdotal reports that granulocyte transfusions are

helpful in treating patients with IA [382–385]. Impressive clin-

ical improvement was noted in a patient with aplastic anemia

and IA after 9 transfusions; however, improvement did not

always parallel an increase in neutrophils in the peripheral

blood [385]. A patient with CGD who had failure of antifungal

therapy for pulmonary aspergillosis and osteomyelitis received

transfusions before and during the aplastic period of a periph-

eral blood stem cell transplantation and showed marked clinical

and radiographic improvement [384].

G-CSF–primed donor granulocyte transfusion was used to

treat 15 patients with neutropenia-related fungal infections re-

fractory to amphotericin B, including 7 patients with IA. A

favorable response in 11 of 15 patients, including 3 of 4 with

IA, appeared to be mainly due to the granulocyte transfusion

[386]. Another study showed infections cleared in 5 of 9 pa-

tients with IA [387]. A review of granulocyte transfusions in

treating fungal infections in neutropenic patients following

bone marrow transplantation showed no improvement in can-

didal or noncandidal infections; however, this was before the

use of G-CSF to prime donors, and the mean number of gran-

ulocytes transfused in that study was 1010 per transfusion [388].

By contrast, 1 study revealed that contemporary granulocyte

transfusions, in which donors generally are primed with 600

mg of G-CSF, yield neutrophils, and the addition of10∼ 5 � 10

dexamethasone can yield neutrophils. That same10∼ 8 � 10

study showed a statistically significant difference in bacterial

infections but no difference in mold infections in transfused

versus control patients [389]. Although there has been some

anecdotal success with granulocyte transfusions for treatment

of IA and proven improvement in the duration of neutropenia,

the conclusive answer would lie in a clinical trial comparing

standard-of-care antifungal therapy for IA with and without

adjunctive granulocyte transfusions.

CONCLUSION

For more than 40 years, the treatment for IA has been relatively

toxic amphotericin B administered to patients often in the late

stages of disease. Unfortunately, amphotericin B is at best only

moderately effective against IA [4], and therapy may be inef-

fective in the absence of bone marrow recovery. Although con-

ventional amphotericin B was the drug of choice for IA, its

clinical utility was thwarted by nephrotoxicity and infusion-

related toxicity. Lipid formulations of amphotericin B have

somewhat reduced side effects but retain similar dismal efficacy.

Itraconazole was added in 1990 and, although less toxic, has

uncertain bioavailability in some patients and numerous drug

interactions. Newer formulations may solve the problem of


solubility, but resistance has been described for both ampho-

tericin B and itraconazole. Most experts agree that the present

new antifungal for primary therapy against IA is voriconazole,

with continued mounting evidence for its success.

To date no clinical study has convincingly answered the ques-

tion of combination therapy or sequential therapy. Studies with

amphotericin B and itraconazole, for instance, have produced

a range of effects from synergy to antagonism [390], but guide-

lines [7] and experience [8] dictate clinical safety with am-

photericin B followed by itraconazole. Therapy has most re-

cently been viewed as a sequential approach, in which a patient

with IA first is treated with conventional amphotericin B as a

first-line drug, which is replaced with a lipid formulation if

renal dysfunction occurs. If the patient survives and is able to

take oral therapy, has good intestinal function, and is not taking

drugs that induce the metabolism of the cytochrome P-450

enzymes, itraconazole can be used as a maintenance drug [68].

Combination antifungal therapy for IA is reviewed in detail

elsewhere [202].

Presently we are able to use less-toxic lipid formulations of

amphotericin B, but the future holds the promise of newer

antifungals, such as second-generation triazoles and the echin-

ocandins with novel targets, as well as modification of the host

immune response. Attacking the host immune response instead

of the fungus itself is attractive, yet, to date, only data derived

from vitro animal model studies and isolated or anecdotal clin-

ical reports suggest improvement with immunomodulators.

Data are lacking because of the lack of statistical power of many

clinical studies to demonstrate a conclusive difference with im-

munomodulatory therapy [95, 391]. Newer antifungals and the

possible synergistic effect of antifungal combinations, possibly

with cytokine therapy, are a welcome, optimistic light in the

dark history of treatment of IA.

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