1. Introduction
There is a critical need for new antifungal agents for the treatment of life-threatening invasive fungal infections which have become more common among immunocompromised patients. These include solid-organ or hematopoietic stem cell transplant recipients, cancer chemotherapy patients, or patients with underlying disease that leads to immunosuppression, as well as patients who are critically ill. Global estimates of life-threatening invasive fungal infections exceed 2 million infections per year that include several invasive mycosis infections: candidiasis (>400,000), pneumocystosis (>400,000), aspergillosis (>200,000), cryptococcal meningitis (>1,000,000) [
1]. Increased use of antibiotics which disrupt normal bacterial colonization, as well as the use of immunosuppressive drugs have contributed to the higher frequency of these infections [
2].
Currently available antifungal agents have one or more liabilities including renal and liver toxicity, treatment spectrum, rising rates of resistance, drug-drug interactions, pharmacokinetic variability and imperfect drug exposure in some infected organs. Amphotericin B (AMB) is only administered intravenously (IV) and demonstrates significant nephrotoxicity [
3]. The widely used azoles demonstrate a broad yeast spectrum, efficacy against
Aspergillus and some Mucorales spp., and are available both IV and orally. However, their use is hampered by liver toxicity, drug-drug interactions, and increased resistance. The azole class is also limited in its spectrum of activity against the rare mold pathogens. According to the Centers for Disease Control and Prevention (CDC), approximately 7% of all
Candida bloodstream isolates are resistant to fluconazole (FLC), most of which are
C. glabrata [
4]. Similarly, >90% of New York
C. auris strains are resistant to FLC [
5,
6]. Among the molds, triazole-resistant
A. fumigatus are a growing concern due to prolonged azole exposure in patients with chronic pulmonary aspergillosis, or due to environmental exposure of isolates to triazoles used in agriculture [
7]. The echinocandins have a more favorable safety profile and are widely used as first-line therapy for
Candida infections; however, the spectrum of activity is variable against molds, they are only available IV, and do not achieve sufficient drug levels in brain tissue, which can be the site of fungal infections [
8]. While echinocandin resistance remains relatively uncommon for most
Candida species, this is not the case for
C. glabrata, with rates exceeding 10% at selected institutions [
9].
Thus, there are limited options for the adequate treatment of many yeast and mold infections, especially rare molds, which often demonstrate high levels of intrinsic resistance to many antifungal agents. High levels of morbidity and mortality are often observed in invasive fungal infections despite antifungal treatment, and mortality ranges from 46–75% for candidiasis, 30–95% for aspergillosis and 30–90% for rare mold infections [
1]. A high percentage of fatalities (>50%) is still observed in invasive pulmonary aspergillosis among severely immunosuppressed patients, such as neutropenic leukemic and transplant patients [
10]. Mortality was >50% in patients with invasive scedosporiosis [
11] and disseminated
Fusarium infections [
12]. Similarly, the overall mortality rate of mucormycosis is >40% and it approaches 100% in patients with disseminated disease, persistent neutropenia, or brain infection [
13,
14,
15]. Clinical trials for the treatment of these invasive fungal infections are difficult due to the relatively small patient populations, compounded by the limited number of patients who are eligible for clinical trials. Consequently, it is more difficult to guide treatment decisions for these infections.
More favorable clinical outcomes are usually associated with improvement in neutrophil counts, rapid diagnosis of the infecting organism, and the early use of the appropriate antifungal therapy. However, diagnosis of fungal disease is often delayed, and thus the need for a safe and effective broad spectrum agent that can be used empirically are important attributes of any new antifungal agent. Fosmanogepix (FMGX, formerly APX001 and E1211) is a first-in-class N-phosphonooxymethylene prodrug that is rapidly and completely metabolized by systemic phosphatases to the active moiety, manogepix (MGX, formerly APX001A and E1210) [
16]. In clinical trials FMGX demonstrated high bioavailability (>90%), and thus both IV and oral formulations are currently being developed.
5. Effects on Virulence Factors and Biofilms
The effect of MGX on
C. albicans virulence factors was examined in a study by investigators at Eisai Co. [
21]. The cell surface expression of the GPI-anchored Als1 adhesin protein was shown to be inhibited by MGX in a concentration-dependent manner by 44%, 58%, and 74% at concentrations of 0.008 (1 × MIC), 0.03 (4 × MIC), and 0.13 µg/mL (16 × MIC), respectively [
21]. This is in contrast to FLC, which showed no effect, and micafungin (MCF), which enhanced Als1p cell surface expression. These data are consistent with the mechanism of action of MGX, which is inhibition of the maturation of GPI-anchored cell surface proteins.
Other virulence factors including adherence and germ tube formation were also examined [
21]. MGX inhibited the adherence of
C. albicans cells to polystyrene with an IC
50 of 0.0039 µg/mL, two-fold below the MIC of 0.008 µg/mL, while the AMB IC
50 was ~ 5-fold higher than its MIC. FLC showed no inhibition and MCF enhanced
C. albicans adherence about 2-fold at concentrations above its MIC value [
21].
C. albicans germ tube formation was inhibited by MGX, MCF, AMB with IC
50 values of 0.0071, 0.015, and 0.24 µg/mL, respectively. MGX, MCF and AMB all inhibited germ tube formation at concentrations near their respective MIC values, whereas FLC did not demonstrate inhibition in this assay at concentrations ≤8 µg/mL [
21]. The effect of MGX on hyphal growth was evaluated by embedding cells within a matrix and following growth. MGX suppressed hyphal growth at 0.002 µg/mL, and inhibited both hyphal and colony growth at 0.008 µg/mL (1 × MIC).
Finally, the inhibition of biofilm formation by MGX was evaluated in an assay that assessed biofilm density after 24 h of growth, using safranin staining of extracellular polymeric materials [
21]. MGX demonstrated an IC
50 of 0.0044 µg/mL and entirely inhibited biofilm formation at a concentration of 0.008 µg/mL, whereas MCF and AMB IC
50 values were 0.014 µg/mL and 0.085 µg/mL, respectively. Although FLC showed some inhibition of biofilm formation, this inhibition was not complete at 16 × MIC. The activity of MGX was also examined against sessile
C. albicans cells. The SMIC
90 of MGX was 8-fold higher for sessile cells than planktonic cells (0.13 µg/mL and 0.016 µg/mL, respectively), similar to MCF and AMB which also demonstrated an 8-fold increase in the MIC
90. FLC was not active against sessile cells (SMIC
90 >16 µg/mL). The activity of MGX was not evaluated against preformed biofilms.
The sum of these data suggests that inhibition of Gwt1 by MGX results in pleiotropic effects on the fungal cell by preventing the maturation of GPI-anchored proteins. MGX has been shown to inhibit a number of important C. albicans virulence factors that include cell surface proteins which affect adherence, hyphal formation and biofilm formation. The inhibition of fungal virulence as well as subsequent cell wall damage are likely contributing to the overall activity of this new antifungal agent.
7. MGX In Vitro Activity against Yeasts
The in vitro activity of MGX against diverse fungal species has been summarized in several publications [
26,
28,
29].
Table 1 summarizes the results of additional studies, including data where >10 isolates per yeast species were examined. The studies include data from the 2017 SENTRY worldwide surveillance study [
30], country specific laboratory surveillance data (Spain, Denmark) [
29,
31] and other collections [
32,
33,
34,
35]. Several studies have been conducted comparing MIC or minimum effective concentration (MEC [defined as the lowest drug concentration resulting in the production of small, rounded, compact hyphal forms]) values using both CLSI and EUCAST methodologies and a high degree of essential agreement has been observed. Where differences are observed, CLSI MIC or MEC values are generally 1–2-fold lower than EUCAST values [
31,
36,
37]. Similar to the echinocandins, endpoints are read at 50% inhibition at 24 h for
Candida and most other yeasts [
38], with MEC values determined at 48 h for most of the molds [
39].
MGX was highly active against most
Candida species tested, with MIC
90 values for key species ranging as follows:
C. albicans (0.008–0.06 µg/mL),
C. auris (0.03 µg/mL),
C. dubliniensis (0.008 µg/mL),
C. glabrata (0.06–0.12 µg/mL),
C. tropicalis (0.03–0.06 µg/mL), and
C. parapsilosis (0.015–0.06 µg/mL) (
Table 1). Among the yeasts, exceptions include
C. krusei and to a lesser extent
C. kefyr [
34] where the MIC
90 values were higher (≥ 0.5 μg/mL) (
Table 1). At least for
C. krusei, the MIC difference is likely not Gwt1 target-based, but rather due to differences in
C. krusei cell permeability or efflux. This is based on the finding that isogenic
S. cerevisiae strains expressing heterologous
C. albicans, S. cerevisiae or
C. krusei proteins demonstrated similar MIC values (within 2–fold), unlike
S. cerevisiae strains expressing the human PIGW protein which, due to target-based differences, resulted in an ~250–fold increase in MIC [
42].
Since
C. auris was not identified in SENTRY surveillance studies until 2017 and less than 10 isolates were observed, a separate review of the activity of MGX, as measured by CLSI methodology, against
C. auris is shown in
Table 2. The CDC collection was comprised of 100 geographically distinct isolates of
C. auris that included the 4 known international clades [
41]. This collection included 6 isolates with elevated MICs to one or more echinocandins, 24 isolates with elevated MICs to AMB, and 2 isolates that were resistant to all three antifungal drug classes. For this collection, the MIC of MGX ranged from < 0.0005–0.015 µg/mL whereas the MICs of the echinocandins ranged between 0.03 to >8 µg/mL for MCF, 0.03 to > 16 µg/mL for caspofungin acetate (CAS), and 0.125 to > 16 µg/mL for anidulafungin (AFG) [
41]. Other studies examined both worldwide and country or state specific isolates [
35]. Arendrup et al. [
37] evaluated 122 strains using both CLSI and EUCAST methodologies and found good essential agreement between the data, although they observed a systematic difference whereby CLSI MICs were generally 1 to 2-fold dilution lower than EUCAST MICs. In addition, the MGX wild-type upper limit (WT-UL)−97.5% and WT-UL-99% were determined using CLSI/EUCAST methodologies: 0.03/0.125 µg/mL and 0.06/0.125 µg/mL, respectively [
37]. A recent study by Zhu et al. [
35] evaluated the activity of MGX against 200 recent
C. auris isolates from a New York outbreak where the MIC
90 was 0.03 µg/mL. All isolates were within the population of wild-type (WT) strains where 0.06 µg/mL defined the WT-UL. MGX was 8- to 32-fold more active than the echinocandins, 16- to 64-fold more active than the azoles, and 64-fold more active than AMB [
35]. Importantly, MGX maintained activity vs. echinocandin-resistant, AMB-resistant, and isolates target-based (
ERG11 [lanosterol 14-α-demethylase]) azole-resistant
C. auris strains. In all studies, MGX demonstrated the lowest
C. auris MIC
50 and MIC
90 values vs. competitor agents (
Table 2).
8. MGX In Vitro Activity against Molds
MGX demonstrates potent activity against
Aspergillus and some species of rare molds. The SENTRY Antimicrobial Surveillance Program evaluated the activity of MGX against 1706 contemporary, world-wide clinical fungal isolates collected in 2017 [
30]. MGX demonstrated potent in vitro activity against recent fungal isolates, including echinocandin- and FLC-resistant strains [
30]. Other laboratories examined more regional organisms, including itraconazole (ITC) resistant strains and cryptic
Aspergillus species. Using EUCAST methodology, a study of contemporary (2016–2017) clinical isolates from Denmark, showed that MEC
50/MEC
90 values for all
Aspergillus spp. ranged between 0.03–06/0.06–0.125 µg/mL and MEC values were unaffected by ITC-resistance [
29].
The data for
Aspergillus spp. is summarized in
Table 3, and MEC
90 values for MGX ranged from 0.015 to 0.06 μg/mL:
A. fumigatus (0.03–0.06 μg/mL),
A. flavus (0.015–0.03 µg/mL),
A. niger (0.015–0.03 µg/mL),
A terreus (0.03–0.06 µg/mL). A study of clinical isolates from patients in Spain (2000–2016) showed that MGX was also active against cryptic
Aspergillus species, with CLSI MEC
90 values ranging between 0.03–0.06 μg/mL for
A. lentulus, A. fumigatiaffinis, A. udagawae, A. calidoustus and
A. alliaceus, whereas posaconazole (POS) MEC
90 values were 1, 1, 0.5, 16 and 2 µg/mL, respectively [
31]. Of note, MGX retained activity against
A. alliaceus and
A. calidoustus, which demonstrated MIC
90 values of 32 µg/mL for AMB, and 16 µg/mL for POS, respectively (
Table 3). The MGX MEC
90 value for
A. thermomutatus was 4-fold higher than other
Aspergillus species (0.25 µg/mL).
MGX also demonstrated significant activity against
Fusarium spp.,
Scedosporium spp. and some fungi from the Mucorales order (
Table 3). The MEC
90 values for MGX against
S. apiospermum, S. prolificans (Lomentospora prolificans), F. solani and
F. oxysporum and
G. fujikuroi were 0.12, 0.12, 0.06, 0.25 and 0.12 μg/mL, respectively in the study conducted by Castanheira et al. [
36], which examined a worldwide collection of isolates. These rare mold species were most often highly refractory to all other comparator agents tested. A study of a collection of isolates from Spain indicated that
Fusarium and
Scedosporium demonstrated a wider range in MEC values, with a corresponding rise in MEC
90 values [
31] (
Table 3). Against
L. prolificans the CLSI MEC
90 of MGX was 0.06 µg/mL, using CLSI methodology, in contrast to MICs of 8 and 16 µg/mL for AMB and POS, respectively [
31] (
Table 3). Although the study of Spanish isolates showed that
Fusarium CLSI MEC
90 values for MGX were > 8 µg/mL, low MEC values (< 0.12 µg/mL) were observed for 4 of 10
F. verticillioides and 8 out of 10
F. oxysporum isolates (MEC
50 = 0.015 µg/mL) suggesting that some of the strains may be clinically treatable. POS MIC
50/MIC
90 values for these strains were >8/ > 8 and 8/ > 8 µg/mL, respectively.
The composite data in
Table 3 shows that
Fusarium spp. MIC
90 value ranges for azoles were variable or high: ITC, >8 µg/mL; VRC, 4 to >8 µg/mL; and POS, 2 to 16 µg/mL. Similarly, azoles MIC
90 values were variable or high for
Scedosporium: ITC, 4 to >8 µg/mL; VRC, 1 to >8 µg/mL; and POS, 2 to 16 µg/mL [
31,
36]. In a recent study of 49
F. oxysporum and 19
F. solani species complexes isolates, MGX MEC values were between <0.015–0.03 µg/mL and <0.015 µg/mL, respectively. However, the AMB MIC values ranged between 1–4 and 0.25–4 µg/mL for
F. oxysporum and
F. solani, respectively [
45]. Thus, the extended spectrum of MGX is notable for its potency against many of the less common but antifungal-resistant fungi
Fusarium spp. and
Scedosporium spp.
Activity against 7 species from the Mucorales order was also investigated [
31]. In general, high MEC
50/MEC
90 values (in µg/mL) were observed:
R. arrhizus >8/ > 8,
R. microsporus 4/ > 8,
R. pusillus > 8/ > 8,
Lichtheimia ramosa 8/ > 8,
L. corymbifera >8/ > 8,
Mucor circinelloides 4/8, and
Cunninghamella bertholletiae > 8/ > 8. However, 3 of 10 strains of
M. circinelloides demonstrated CLSI MEC values ≤ 2 µg/mL. These data are similar to the findings of 41 Mucorales (
Mucor, Rhizomucor, Rhizopus, Lichtheimia, and
Synsephalastrum) isolated between 2017 and 2019 with MEC values ranging between 0.5 to > 8 µg/mL [
28,
30]. Although MGX MECs for Mucorales are variable and generally higher than for the other rare molds, in vivo efficacy has been described in two mouse models of mucormycosis using strains with MEC values of 0.25 µg/mL and 4.0 µg/mL [
46]. These data suggest that some of the Mucorales with lower MEC values may be clinically treatable.
The composite summaries in
Table 1,
Table 2 and
Table 3 show that MGX demonstrates potent broad-spectrum activity against
Candida, Aspergillus and other difficult to treat rare molds. In addition, MGX was highly active against
Coccidioides spp., with an MEC
90 of 0.008 µg/mL [
47].
9. Resistance
The potential for development of resistance to MGX was investigated in
C. albicans,
C. glabrata, and
C. parapsilosis by evaluating spontaneous mutation frequencies [
42]. A large plate format method was utilized to allow assessment of an inoculum of 1 × 10
8 CFU/mL. Median spontaneous mutation frequencies for MGX ranged from 3.25 × 10
−8 (
C. albicans) to <1.88 × 10
−8 (
C. glabrata and
C. parapsilosis). These data are similar to the spontaneous resistance frequency of AFG, CAS and the experimental drug rezafungin (CD101), which were evaluated using a similar methodology [
48].
Serial passage experiments performed using a gradient plate method showed that MIC values increased 8-fold from 0.016 to 0.125 µg/mL for both
C. parapsilosis and
C. albicans, however this occurred after passages 3 and 18, respectively [
42]. No increase in MIC (≤ 2-fold) was observed for
C. glabrata. To further explore the potential for resistance development, the broth macrodilution serial passage method was used for
C. glabrata, C. tropicalis and
C. auris. However, MIC values of MGX did not increase substantially (≤ 2-fold) for these three
Candida isolates [
42].
To understand the underlying resistance mechanisms, the gwt1 target gene was sequenced in strains demonstrating increased MIC values and compared to the sequence of the respective wild-type starting strains. A valine to alanine mutation at position 163 (V163A) in the Gwt1 protein was identified in four C. glabrata mutants and the corresponding valine to alanine mutation at position 162 (V162A) was also identified in a heterozygous C. albicans mutant. A C. glabrata V163A Gwt1 mutant was generated using CRISPR and showed similarly reduced susceptibility. These data suggest the importance of this valine residue to MGX binding to Gwt1 orthologs across different species. MGX MIC values of all of these mutants increased 16–32-fold vs. the isogenic parent strain. However, no changes in MIC values were observed for AMB, CAS, or FLC, demonstrating a lack of cross-resistance.
Two
C. parapsilosis and
C. albicans mutants were identified that demonstrated 4- to 8-fold decreased susceptibility to MGX in which the
gwt1 gene sequence was similar to the wild-type [
42]. These mutants were further investigated and the change in MIC was determined to be efflux-mediated [
49]. The
C. parapsilosis mutant contained a mitochondrial deletion, which activated expression of the major facilitator superfamily transporter gene
MDR1. The
C. albicans mutant demonstrated a gain-of-function mutation in the transcription factor gene
ZCF29, which activated expression of the ATP-binding cassette transporter genes
CDR11 and
SNQ2. These two mutants also showed 2- and 4-fold decreased susceptibility to FLC, but not AMB or CAS [
42,
49]. These data are consistent with the observation that a subset of FLC resistant mutations in several species may lead to small increases in MGX MIC values [
50]. Given the low MGX MIC values of the starting strains and that the two mutant strains demonstrated MIC values ≤ 0.056 µg/mL, these individual mutations may not result in clinically significant resistance and the strains may remain in the clinically treatable range.
10. Activity against Echinocandin-, Azole- and AMB-Resistant Candida spp.
Several studies have examined the activity of MGX against collections of azole-, echinocandin- or AMB-resistant mutants. Pfaller et al. examined 20 FLC-resistant
Candida spp. and 15 CAS-resistant
Candida spp., and no increase in MGX MIC was observed [
32]. Similarly, Miyazaki et al. [
20] reported no cross-resistance to azoles when the MGX MIC
90 values of a collection of 140 FLC-susceptible
Candida spp. was compared to 18 FLC-resistant
Candida spp. (MIC
90 0.06 vs. 0.03 µg/mL, respectively).
The activity of MGX has also been evaluated against specific target-based resistant fungi including those that are echinocandin-resistant (
fks1, fks2) or azole resistant (
erg11). Zhao et al. compared the MGX MIC of 3
C. glabrata strains which were wild-type for Pdr1 and Fks1/2, echinocandin resistant (Fks1-S629P) or multidrug resistant (Pdr1-G1079R, Fks2-S663P) [
51]. The MGX MIC values for the three strains were 0.03, 0.03 and 0.125 µg/mL, respectively, suggesting that the MGX MIC value was elevated 4-fold in the MDR strain in which there is a higher expression of efflux pumps due to the Pdr1-G1079R mutation, but not the echinocandin-resistant strain [
51,
52]
In a study of
C. albicans isolates with known
fks1 mutations or FLC-resistance, MIC values were unchanged vs. the susceptible control strains (≤ 0.03 µg/mL) [
40]. As part of the 2017 SENTRY surveillance study that included 1340
Candida isolates, the MIC values of 10
C. glabrata and 3
C. albicans strains with mutations in
fks1 or
fks2 were also assessed [
30]. MGX showed good activity against both echinocandin-susceptible and echinocandin-resistant
Candida isolates. For the
C. glabrata echinocandin-resistant strains, the MGX MIC values ranged between 0.016–0.12 µg/mL, all below the WT-UL cutoff value of ≤ 0.25 µg/mL. The MGX MICs of the three echinocandin-resistant
C. albicans strains were 0.008 µg/mL, again below the WT-UL of ≤ 0.03 µg/mL. Similarly, a recent study examined 17
Candida isolates with
fks target gene-encoded hotspot alterations and found that MGX activity against these isolates was similar to wild-type strains, with the exception of two
C. albicans with MGX MICs of 0.125 and 0.25 µg/mL which were also pan-azole resistant [
34]. The SENTRY 2018 and 2019 study also evaluated 8
C. glabrata strains with
fks1 and/or
fks2 mutations, and found that MGX MICs ranged between 0.008–0.12 µg/mL, values that are ≤ 0.12 µg/mL, the WT-UL determined from the collection of 460
C. glabrata isolates [
28]. Thus, no cross-resistance with echinocandins was observed.
In some of the studies where MGX was evaluated against resistant clinical isolates, the resistance mechanisms were not defined genetically. These studies included
C. auris isolates that were resistant to azoles, echinocandins and/or AMB [
35,
37,
41], and other FLC or CAS-resistant
Candida spp. [
32]. For the
C. auris isolates, Arendrup et al. [
37] examined the correlation between MGX MICs and other antifungal drugs in a study of 122 isolates from India. No correlation was observed between the MIC values of MGX and AMB, MFG or AFG, consistent with the unique mechanism of action of MGX. However a correlation was observed between MGX and the azoles (CLSI methodology): FLC (
p < 0.001), VRC (
p = 0.017), isavuconazole (ISA) (
p = 0.017) and ITC (
p = 0.016), although the mechanism behind the azole resistance was not characterized in these isolates and the impact on MGX clinical efficacy is unclear.
Despite the fact that FLC and MGX target different fungal enzymes (Erg11 and Gwt1, respectively), a correlation between increased MGX and FLC MIC values was first identified in a study that examined 540 yeast bloodstream isolates from the nationwide Danish surveillance program (2016 and 2017 isolates) along with a collection of 122 clinical isolates of
C. auris from tertiary care hospitals in India (2010 to 2015) [
50]. Single center WT-UL were determined, and 4 of 540 bloodstream isolates (1
C. dubliniensis, 2
C. glabrata, 1
C. tropicalis) were identified with MIC values that exceeded the WT-UL (0.03, 0.25, 0.125 µg/mL, respectively). All four strains were FLC resistant as well (32, > 32, ≥ 16 µg/mL, respectively). However, many FLC-resistant isolates did not show elevated MGX MICs, suggesting that only a subset of FLC-resistant mechanisms may affect MGX susceptibility. In addition, a linear correlation between modal MICs of FLC and MGX was observed for several species including
C. neoformans, C. albicans,
C. dubliniensis,
C. glabrata,
C. tropicalis, but not
C. auris or
C. guilliermondii where MGX modal MICs were low (0.008–0.016 µg/mL) but FLC modal MICs were high (4 µg/mL and 512 µg/mL, respectively) [
50].
A later study by the same investigators examined a collection of 835 yeast isolates received at the reference laboratory in Denmark during 2018 [
34]. The MGX WT-UL was determined for species in this collection, and WT populations of 16/20 yeast species were highly susceptible with modal MICs from 0.004–0.06 µg/mL. Further examination of 5 species with ≥ 10 isolates (
C. albicans, C. dubliniensis, C. glabrata, C. parapsilosis and
C. tropicalis) demonstrated that 3% (11 of 364 isolates) had MGX MIC values above the WT-UL defined for the species. Of the 11 isolates, 9 also demonstrated MIC values above the FLC tentative EUCAST ECOFF values. In contrast, 40 strains with FLC MIC values above the EUCAST ECOFF were within the WT population of MGX MIC values. These data are consistent with the hypothesis of a linkage between MGX and FLC MIC values in a small subset FLC-resistant strains. These data may be explained by the analysis of laboratory generated mutants in which some isolates with efflux related mutations displayed a 4- to 8-fold increase in the MIC of MGX and a concomitant 2- to 4-fold increase in the FLC MIC [
49]. This is in contrast to target-based Gwt1 mutants in which increased MIC values for MGX, but not FLC, were observed [
42]. Further studies are necessary to understand the impact of target-based vs. efflux-based mechanism(s) in clinical isolates, as well as their impact on FMGX efficacy.
13. In Vivo Efficacy
Earlier studies conducted by Eisai Co. evaluated the efficacy after administration of the active moiety, MGX, rather than the prodrug. Reduction in CFU was observed in the oropharyngeal candidiasis model, and increased survival was observed in models of disseminated candidiasis, pulmonary aspergillosis and disseminated fusariosis as summarized in
Table 4 [
53]. Eisai Co. also examined the effect of the co-administration of FMGX and echinocandins in an
A. flavus pulmonary infection model where mice were immunosuppressed with 200 mg/kg of 5-FU given 5 days prior to infection, treatment was administered Day 0–3, and Day 14 survival was evaluated [
53]. The combinations of FMGX plus MFG or FMGX plus CAS significantly increased survival over the administration of a single agent. A caveat to this study was that both FMGX and the echinocandins were administered at doses in mice that give rise to exposures well below what is achieved clinically. Thus, it is not clear whether combination dosing in humans at approved dosing regimens would recapitulate these findings and improve patient outcome over monotherapy.
The prodrug FMGX has been examined in a wide variety of animal models of invasive fungal infections including disseminated models due to
C. albicans, C. glabrata, C. auris, C. immitis, C. neoformans, F. solani infections, and pulmonary models of
A. fumigatus, A. flavus, S. prolificans, S. apiospermum and
R. arrhizus which are summarized in
Table 4. All of the models utilized mice, with the exception of a rabbit model of
Candida endophthalmitis and hematogenous meningoencephalitis [
55]. In addition to demonstration of increased survival, several of the models demonstrated reduction in fungal burden in lung, kidney, spleen, eye, and brain after FMGX administration. This latter is particularly important in some invasive infections given that the echinocandins do not show significant efficacy in brain infections. The results of these models are summarized below.
In addition to the PK/PD study conducted with
C. albicans, C. glabrata and
C. auris [
54], the efficacy of FMGX was evaluated in two other disseminated
C. auris models which examined both survival and reduction in fungal burden. In the first model, 90–100% survival and significant reductions in CFU in kidney, lung, and brain were observed when 78 mg/kg BID or 78 mg/kg thrice daily (TID) was administered intraperitoneally (IP) [
33]. In a second
C. auris model, treatment was delayed for 24 h post-inoculation [
43]. Significant improvements in survival at Day 21 were observed in each group administered FMGX (104 and 130 mg/kg IP twice daily [BID] or 260 mg/kg IP BID) and CAS (10 mg/kg IP once daily [QD]); however FLC (20 mg/kg administered orally [PO], QD) was not effective in this model [
43]. Significant reductions in kidney fungal burden vs. control were observed with 260 mg/kg BID FMGX (3.86 log
10 CFU/g) or with 10 mg/kg CAS (3.41 log
10 CFU/g). Significant reductions were also observed in brain in mice treated with 260 mg/kg BID FMGX (2.99 log
10 CFU/g) compared with that of the vehicle control. In contrast, brain fungal burden was not significantly reduced in mice treated with the lower doses of FMGX, FLC, or CAS [
43].
The efficacy of FMGX was examined in a disseminated rabbit model of
Candida endophthalmitis and meningoencephalitis where antifungal therapy was initiated 48 h after inoculation and continued throughout the course of the experiments for 7 days [
55]. Rabbits treated with FMGX at 25 mg/kg BID, 50 mg/kg BID, and 100 mg/kg BID demonstrated quantitative clearance of
C. albicans from tissues including cerebrum, cerebellum, spinal cord, cerebrospinal fluid (CSF), meninges, and aqueous humor.
The in vitro and in vivo activity of MGX/FMGX was evaluated against
Cryptococcus [
55]. The MIC
90 of 9 strains of
C. neoformans and 9 strains of
C. immitis was 0.5 µg/mL, indicating that MGX was less potent against
Cryptococcus spp. than
Candida spp. The efficacy of administration of FMGX, FLC, or the combination of FMGX and FLC was evaluated in a disseminated mouse model of cryptococcal meningitis using strain H99 (H99 MGX MIC = 0.25 µg/mL). In this model, MGX exposures were similar to those obtained in Phase 1 clinical studies, and the reduction in fungal burden in brain tissue for FMGX and FLC was 0.78 and 1.04 log
10 CFU/g, respectively whereas the combination resulted in a reduction of 3.52 log
10 CFU/g, suggesting possible additivity brain tissue [
56]. However, in lung tissue, no statistically significant differences between the treatment groups was observed.
The PK of MGX after oral administration of FMGX has been evaluated in mice, and the half-life was determined to be 1.4–2.75 h, which is significantly shorter than what has been observed in healthy human volunteers during Phase 1 clinical studies (2 to 2.5 days) [
54,
63,
64]. As a result, BID, TID, or more frequent dosing intervals have been utilized in order to achieve efficacy in mouse models. To counter this rapid metabolism in mice, oral administration of 1-aminobenzotriazole (ABT), a non-specific inhibitor of cytochrome P450 enzymes, 2 h prior to each FMGX dose was shown to increase the MGX half-life in mice to approximately 6–9 h and greatly enhanced exposures. Several studies conducted with different fungi showed that there is no antifungal effect of ABT alone either in vitro or
in vivo, and that there was no synergy between ABT and antifungal agents in vitro or in vivo [
51,
59]. Thus, pretreatment with 50 mg/kg ABT 2 h prior to FMGX was utilized as a standard dosing regimen for several efficacy experiments where 78 mg/kg FMGX plus ABT gives rise to MGX exposures similar to what was observed in healthy human volunteers.
Subsequent efficacy experiments in mice have shown that once daily dosing of FMGX in conjunction with ABT results in improved efficacy as demonstrated by reduction in fungal burden, increased survival, and histological improvement. In invasive candidiasis, FMGX was shown to be an effective antifungal agent for the treatment of susceptible, echinocandin-resistant, or MDR
Candida infections. Administration of 26 mg/kg plus ABT sterilized kidneys in mice infected with
C. albicans, while FMGX alone at the same dose resulted in a modest fungal burden reduction of only 0.2 log
10 CFU/g, relative to the vehicle control [
51]. In the presence of ABT, 2 days of once-daily dosing with FMGX at 26 mg/kg also demonstrated significant in vivo efficacy in the treatment of
C. glabrata infections in mice [
51]. Potent kidney burden reduction was achieved in mice infected with susceptible, echinocandin-resistant, or multidrug resistant strains. In contrast, the standard of care MFC was ineffective in treating infections caused by the resistant
C. glabrata isolates.
In a pulmonary coccidioidomycosis model where treatment was initiated one week after infection, treatment of mice with 26 mg/kg FMGX (QD, with ABT) or FLC (25 mg/kg BID) reduced log
10 CFU in the lung by >2.5 fold (
p < 0.001) versus the ABT control and completely prevented dissemination to the spleen [
47]. The FMGX treated mice also demonstrated significantly longer survival than control or FLC-treated mice.
The efficacy of FMGX in conjunction with ABT was examined in an invasive pulmonary aspergillosis (IPA) model in mice infected with
A. fumigatus [
59]. This model utilized a severely immunocompromised mice in which the cyclophosphamide/cortisone acetate treatment results in pancytopenia for at least 9 days from the first administered dose. Treatment of mice with FMGX at 78 mg/kg QD, 78 mg/kg BID, or 104 mg/kg QD significantly enhanced median survival time and prolonged Day 21 post-infection overall survival when compared to placebo. Furthermore, administration of FMGX resulted in a significant reduction in lung fungal burden [4.2 to 7.6 log
10 conidial equivalents (CE)/gram tissue] vs. the untreated control and resolved the infection as judged by histopathological examination. The observed survival and tissue clearance were comparable to a POS dose that was at least twice as high as the POS dose required in mice to achieve an AUC consistent with efficacy in the clinic [
65].
Galactomannan (GM) detection in biological samples has been shown to predict therapeutic response by azoles and polyenes [
66]. GM was evaluated as a biomarker of FMGX efficacy in the IPA model, as described [
60]. Cohorts of mice were sacrificed at 48 h, 72 h, and 96 h to evaluate changes in CFU/g of lung tissue as well as serum GM and bronchoalveolar lavage (BAL) GM levels from the same mice. FMGX or POS treatment resulted in a ~6–7 log reduction in CE/g lung tissue after 96 h versus placebo. Changes in GM levels in BAL and serum mirrored reductions in lung CE with significant decreases seen after 96 h or 72 h for FMGX or POS, respectively, suggesting the potential use of GM as a biomarker of FMGX efficacy in immunosuppressed mice.
The efficacy of FMGX was also evaluated in highly immunosuppressed murine models of disseminated fusariosis and pulmonary scedosporiosis [
61]. In the pulmonary scedosporiosis model,
S. apiospermum DI16–478, which is susceptible to azoles as well as to MCF, was utilized and treatment was initiated 16 h post infection. Treatment of mice once daily with 78 mg/kg FMGX (plus ABT) or 104 mg/kg FMGX (plus ABT) significantly increased median survival time vs. placebo from 7 days to 13 and 11 days, respectively and enhanced overall survival by Day 21 [
61]. Neither POS (30 mg/kg BID) nor liposomal AMB (L-AMB) (10 mg/kg) prolonged median survival time vs. the placebo. When tissue fungal burdens were examined, all doses of FMGX (104 to 264 mg/kg) plus ABT significantly reduced tissue fungal burden in lung and brain and were comparable to that of L-AMB treatment. In kidney, all doses of 104 mg/kg to 264 mg/kg plus ABT significantly reduced fungal burden, but only the highest dose (264 mg/kg plus ABT) demonstrated statistical significance vs. L-AMB treatment. All FMGX treatments resulted in a 2-log
10 reduction in lung, brain, and kidney CE.
In the disseminated fusariosis model (
F. solani 95–2478), once daily 78 mg/kg and 104 mg/kg FMGX plus ABT significantly enhanced median survival time from 7 days to 12 and 10 days, respectively [
61]. Furthermore, FMGX plus ABT or L-AMB treatments equally enhanced overall survival by Day 21. Administration of a high dose of L-AMB (15 mg/kg) or FMGX (78 mg/ kg, 104 mg/kg, and 130 mg/kg) plus ABT resulted in significant reductions in kidney and brain burdens. Compared to placebo, treatment with 78, 104, or 130 mg/kg FMGX plus ABT reduced kidney counts by 2.10, 2.21, and 3.14 log
10 CE, respectively, while L-AMB treatment resulted in a 3.96-log
10 reduction in kidney counts. Thus, in both a pulmonary scedosporiosis model and a disseminated fusariosis model, administration of FMGX resulted in increased survival and a 2- to 3-log
10 reduction in kidney and brain CE [
61]. Reduction in tissue fungal burden was corroborated with histopathological data, with target organs showing reduced or no abscesses in FMGX-treated mice.
The efficacy of FMGX was evaluated in two pulmonary mucormycosis models using two
Rhizopus arrhizus strains that demonstrated an 8-fold difference in MGX MEC values: 0.25 μg/mL and 4 μg/mL for
R. arrhizus var.
delemar 99–880 and
R. arrhizus var.
arrhizus 99–892, respectively [
62]. The ISA MIC values for the two strains were 2.0 μg/mL and 1.0 μg/mL, respectively. In the
R. arrhizus var.
delemar 99–880 infection model, administration of once daily 78 mg/kg FMGX with ABT and 104 mg/kg FMGX with ABT demonstrated significantly improved survival at Day 21, similar to 110 mg/kg ISA TID, a dose which gives rise to clinically relevant exposures of ISA in mice. Tissue fungal burden was also assessed and the 78 mg/kg and 104 mg/kg FMGX dosing groups resulted in a 1.3 and 1.97 log
10 reduction in CE/gram of lung tissue, which was similar to what was observed for ISA (1.79 log
10 reduction in CE). Reductions in log
10 CE/gram of brain tissue were 0.93, 1.78 and 1.65 for 78 mg/kg FMGX, 104 mg/kg FMGX, and 110 mg/kg TID ISA, respectively.
In the second
R. arrhizus var.
arrhizus 99–892 model (MGX MEC 4 μg/mL) both the 104 mg/kg FMGX (with ABT) or ISA (110 mg/kg TID, PO) treatments demonstrated significant efficacy (30% survival Day 21) vs. placebo control (0% survival), and the two drug treatment survival curves were not significantly different from each other. Assessment of lung and brain tissue at Day + 4 demonstrated that administration of FMGX resulted in significant reductions in lung (log
10 1.15) and brain (log
10 1.14) burden, whereas ISA reduced the fungal burden lung (log
10 1.69) and brain (log
10 1.14). ISA and FMGX CE reductions were not significantly different from each other [
62]. Thus, FMGX showed significant efficacy against two strains of
Rhizopus, with both high (4 µg/mL) and low (0.25 µg/mL) MEC values, that was as protective as ISA in two invasive pulmonary mucormycosis infection models, using highly immunosuppressed mice.