Review of Newer Antifungal and Immunomodulatory Strategies ... · pered in antifungals because of...
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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
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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-
Antifungal and Immunomodulatory Therapy • CID 2003:37 (Suppl 3) • S159
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.
S160 • CID 2003:37 (Suppl 3) • Steinbach and Stevens
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-
Antifungal and Immunomodulatory Therapy • CID 2003:37 (Suppl 3) • S161
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-
S162 • CID 2003:37 (Suppl 3) • Steinbach and Stevens
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-
Antifungal and Immunomodulatory Therapy • CID 2003:37 (Suppl 3) • S163
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
S164 • CID 2003:37 (Suppl 3) • Steinbach and Stevens
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-
Antifungal and Immunomodulatory Therapy • CID 2003:37 (Suppl 3) • S165
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
S166 • CID 2003:37 (Suppl 3) • Steinbach and Stevens
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-
Antifungal and Immunomodulatory Therapy • CID 2003:37 (Suppl 3) • S167
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-
S168 • CID 2003:37 (Suppl 3) • Steinbach and Stevens
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
Antifungal and Immunomodulatory Therapy • CID 2003:37 (Suppl 3) • S169
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-
S170 • CID 2003:37 (Suppl 3) • Steinbach and Stevens
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
Antifungal and Immunomodulatory Therapy • CID 2003:37 (Suppl 3) • S171
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
S172 • CID 2003:37 (Suppl 3) • Steinbach and Stevens
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
Antifungal and Immunomodulatory Therapy • CID 2003:37 (Suppl 3) • S173
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
S174 • CID 2003:37 (Suppl 3) • Steinbach and Stevens
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
Antifungal and Immunomodulatory Therapy • CID 2003:37 (Suppl 3) • S175
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.
S176 • CID 2003:37 (Suppl 3) • Steinbach and Stevens
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
Antifungal and Immunomodulatory Therapy • CID 2003:37 (Suppl 3) • S177
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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