Ibrexafungerp Demonstrates In Vitro Activity against Fluconazole-Resistant Candida auris and In Vivo Efficacy with Delayed Initiation of Therapy in an Experimental Model of Invasive Candidiasis

ABSTRACT

Candida auris is an emerging pathogen that has rapidly spread to many countries on multiple continents. Invasive infections caused by this species are associated with significant mortality, and treatment options are limited due to antifungal resistance. Ibrexafungerp is the first-in-class member of the triterpenoids, which inhibit the production of (1,3)-β-d-glucan and can be administered orally. We evaluated the in vitro activity and in vivo efficacy of ibrexafungerp against C. auris. Antifungal susceptibility was tested by broth microdilution against 54 C. auris isolates. Neutropenic mice were intravenously infected with a clinical isolate, and a 7-day treatment course was begun 24 h postinoculation with vehicle control, ibrexafungerp (20, 30, and 40 mg/kg orally twice daily), fluconazole (20 mg/kg orally once daily), or caspofungin (10 mg/kg intraperitoneally once daily). Fungal burden was assessed by colony counts in the kidneys on day 8 and on day 21 or as mice became moribund in the survival arm. Ibrexafungerp demonstrated consistent activity, with MICs ranging between 0.25 and 2 μg/ml against all isolates. Marked improvements in survival were observed in mice treated with the higher doses of ibrexafungerp and caspofungin. Similarly, reductions in kidney fungal burden were also observed in these groups. No improvements in survival or reductions in fungal burden were observed with fluconazole, consistent with the in vitro resistance of the isolate used to establish infection to this azole. These results demonstrate that ibrexafungerp is effective in vivo against C. auris even when the start of therapy is delayed.

INTRODUCTION

Invasive candidiasis is a significant challenge to clinicians, as Candida species are a common cause of nosocomial bloodstream infections (1) and are associated with substantial morbidity and mortality (1, 2). Attention has now focused on Candida auris, which was first described in 2009 as an isolate collected from the external ear canal of a patient in Japan (3) but quickly emerged as a pathogen, spread to multiple countries on several continents, and has become a significant clinical problem (4). In one retrospective review of 54 patients, most of whom had multiple risk factors for invasive disease, candidemia was observed in 61% and the mortality rate was 59% (4). Available therapy options against invasive infections caused by C. auris are limited, as up to 90% of isolates are resistant to fluconazole and 50% have reduced susceptibility to voriconazole (4, 5). Currently, the echinocandins are recommended for the treatment of C. auris infections (6). However, elevated MICs secondary to hot spot regions in the FKS genes (FKS1 and FKS2) known to cause resistance to the echinocandins in other Candida species and Aspergillus fumigatus have been documented in some C. auris isolates (5, 7, 8). In addition, a few isolates have been found to be resistant to all clinically available classes of antifungal agents. Thus, there is a clear need for the development of new therapeutic candidates and novel treatment strategies to combat infections caused by this emerging pathogen.

Scynexis, Inc. (Jersey City, NJ), has developed a novel antifungal class, the triterpenoids, that like the echinocandins target the fungal cell wall through inhibition of glucan synthase but unlike the echinocandins can be administered orally (9). Ibrexafungerp (formerly SCY-078), the first representative of this class, has potent in vitro and in vivo activity against Candida species, including isolates with mutations in FKS1 hot spot regions that confer resistance to the echinocandin caspofungin (10–13). Studies have also reported in vitro and in vivo activity against C. auris (9, 14–17). The objective of this study was to further evaluate the in vitro activity and in vivo effectiveness of ibrexafungerp in a murine model of invasive candidiasis caused by C. auris following an initial delay in the start of therapy.

RESULTS

In vitro susceptibility.

Ibrexafungerp demonstrated consistent activity against Candida auris isolates, with MICs ranging from 0.25 to 2 μg/ml, MIC₅₀ and MIC₉₀ values both of 1 μg/ml, and a geometric mean (GM) MIC of 0.764 μg/ml. The MICs of caspofungin and micafungin (range, 0.06 to >8 μg/ml) were generally 1 to 2 dilutions lower than that of ibrexafungerp, with GM MICs of 0.249 and 0.217 μg/ml, respectively. Although no clinical breakpoints have been established against C. auris, based on general guidance provided by the U.S. Centers for Disease Control and Prevention, all but two of these isolates would be considered susceptible to the echinocandins (18). Two isolates had elevated echinocandin MICs (caspofungin MIC, >8 μg/ml for both, and micafungin MICs of 4 and 8 μg/ml), and one is known to harbor an FKS mutation (19). The ibrexafungerp MIC was 0.5 μg/ml against both isolates. Against UTHSCSA DI17-46, which was used in the murine model, the ibrexafungerp, fluconazole, and caspofungin MICs were 1, >64, and 0.25 μg/ml, respectively.

Survival.

Treatment with ibrexafungerp was associated with a survival advantage in this experimental model of invasive candidiasis caused by C. auris. On day 8 postinoculation, 1 day after therapy was stopped, each dose level of ibrexafungerp resulted in a significant survival advantage compared to that of vehicle control (P ≤ 0.02 for all comparisons) (Fig. 1A and B). Similar results were observed with caspofungin (P = 0.001) but not fluconazole, which is consistent with the in vitro susceptibility results for the isolate used to establish infection. When assessed on day 21 postinoculation, 14 days after therapy had stopped, survival was also improved in mice treated with ibrexafungerp 30 mg/kg compared to the vehicle control (P = 0.0371), and there was a trend toward improved survival in mice treated with ibrexafungerp 40 mg/kg (P = 0.0535) (Fig. 1C and D). Survival was also improved with caspofungin (P = 0.0010) but was not enhanced in mice treated with the lowest dose of ibrexafungerp (20 mg/kg).

FIG 1.

Survival curves in mice inoculated intravenously with C. auris and treated with vehicle control, fluconazole 20 mg/kg per os (p.o.) once a day (QD), or caspofungin 10 mg/kg intraperitoneally (i.p.) QD (A and C) or with ibrexafungerp (IBX) at doses of 20 mg/kg, 30 mg/kg, or 40 mg/kg p.o. twice a day (BID) (B and D). Treatment started 1 day postinoculation and continued for 7 days. Mice were then followed off therapy until day 8 postinoculation, 1 day after therapy stopped (A and B), and at day 21, 14 days after therapy stopped (C and D). Black squares, vehicle (untreated) control; white squares, fluconazole 20 mg/kg; gray diamonds, caspofungin 10 mg/kg; black circles, ibrexafungerp 20 mg/kg; gray circles, ibrexafungerp 30 mg/kg; white circles, ibrexafungerp 40 mg/kg. n = 10 mice per group. *, P value < 0.05.

Fungal burden.

Treatment with ibrexafungerp also resulted in a dose-dependent reduction in fungal burden on day 8, 1 day after treatment was stopped (Fig. 2A). Mean CFU counts in each ibrexafungerp group (range in mean log₁₀ CFU/g, 1.83 to 3.85) were significantly lower than those observed in the vehicle control group (5.36 log₁₀ CFU/g). In the ibrexafungerp 30 mg/kg and 40 mg/kg groups, fungal burden was reduced by >1.5 log₁₀ CFU/g and >2.5 log₁₀ CFU/g, respectively, compared to that measured at 24 h postinoculation just prior to the start of therapy (4.62 log₁₀ CFU/g). In contrast, no reductions in fungal burden were observed in mice treated with fluconazole (5.79 log₁₀ CFU/g). Although CFU counts in the caspofungin group (4.50 log₁₀ CFU/g) were numerically lower than those observed in mice that received vehicle control, this difference did not reach significance.

FIG 2.

Kidney fungal burden (CFU/g) in mice with invasive candidiasis secondary to C. auris after 7 days of therapy in the fungal burden arm on day 8 postinoculation, 1 day after therapy stopped (A), and in the survival arm on day 21, 14 days after therapy was stopped or as mice became moribund (B). n = 10 mice in the vehicle control and treatment groups; n = 5 mice in the 24-h control group. Black circles represent mice that were moribund prior to the day 21 endpoint in the survival arm. IBX, ibrexafungerp. *, P value < 0.05.

Fungal burden was also assessed in the survival arm on day 21 postinoculation or as mice became moribund or succumbed to infection. As shown in Fig. 2B, CFU counts in the ibrexafungerp 30 mg/kg and 40 mg/kg dose groups (5.10 and 4.14 log₁₀ CFU/g, respectively) were significantly lower than those observed in the vehicle control group (7.42 log₁₀ CFU/g; P ≤ 0.04). In the ibrexafungerp 40 mg/kg group, fungal burden was also >1.5 log₁₀ CFU/g lower than that measured 24 h postinoculation at the start of therapy (5.92 log₁₀ CFU/g). Although the mean fungal burden was numerically lower in the ibrexafungerp 20 mg/kg group (5.74 log₁₀ CFU/g) than in the vehicle control group, this difference was not significant. Fungal burden was also significantly lower in mice treated with caspofungin (3.90 log₁₀ CFU/g), consistent with the survival results, but not in mice treated with fluconazole (6.11 log₁₀ CFU/g). In mice that were moribund or succumbed to infection prior to day 21, fungal burden in each group was higher in most mice than in those that survived to the study endpoint.

DISCUSSION

Following the initial report of its isolation and description in 2009 (3), Candida auris has rapidly spread to many countries on several continents (4, 5, 20). This emerging pathogen has several characteristics that make its management particularly difficult. These include a high mortality rate in patients with invasive disease (4), its ability to form biofilms, making removal from environmental surfaces or clearance of colonization in patients difficult (21), and the reported resistance to multiple classes of antifungals (4, 20). Although the echinocandins are effective against C. auris infections and are currently recommended as first-line therapy (6), they are limited by only being able to be administered intravenously, and the development of in vitro resistance in C. auris leading to clinical failure following exposure to this class has been documented (19). Several new antifungals that are currently in preclinical or clinical development have demonstrated promising results against C. auris, including the arylamidine T-2307, which causes collapse of fungal mitochondrial membrane potential (22), the echinocandin rezafungin (23, 24), the tetrazole VT-1598, which inhibits the biosynthesis of ergosterol similar to the azoles (25), and fosmanogepix, the prodrug of manogepix, which inhibits glycosylphosphatidylinositol-anchored protein maturation in fungi (26–28).

Ibrexafungerp, a member of the triterpenoid class of antifungals, is structurally different than the echinocandins but also causes a reduction in (1,3)-β-d-glucan synthesis, an important component of the cell wall of many pathogenic fungi, through noncompetitive inhibition of glucan synthase (29). Unlike the echinocandins, ibrexafungerp is absorbed from the gastrointestinal tract and can be administered orally (30). This agent, which is in phase 3 clinical trials, has demonstrated in vitro and in vivo activity against various Candida species, including some isolates harboring point mutations in FKS1 and FKS2 hot spot regions that cause echinocandin resistance (13, 29). In the current study, ibrexafungerp maintained in vitro activity against two C. auris isolates that were resistant to caspofungin and micafungin, including the one known to harbor an FKS mutation (19). Previous in vitro studies have also reported good activity against C. auris isolates (15, 17), similar to what was observed in the current study. One in vitro study also reported good activity for ibrexafungerp against C. auris biofilms (21).

The in vitro activity for ibrexafungerp against C. auris observed in our study also translated into in vivo efficacy in this neutropenic murine model of invasive candidiasis. Following a 24-h delay in the initiation of therapy, ibrexafungerp resulted in both improvements in survival and reductions in fungal burden as measured by CFU counts. The survival advantage with ibrexafungerp was observed with the higher doses of this agent, as were reductions in fungal burden, which were >1.5 log₁₀ than the fungal burden measured just prior to the initiation of therapy. Once therapy was stopped, survival in mice treated with ibrexafungerp did decrease, which may be due to waning exposures over time. However, this was not fully evaluated, as ibrexafungerp concentrations were not measured in this study. Similar improvements in survival and reductions in fungal burden with higher doses of ibrexafungerp in neutropenic mice have also been reported in abstract form (14). Ibrexafungerp has also been reported to reduce the severity of lesions and fungal burden caused by C. auris in a guinea pig cutaneous infection model (16). Although ibrexafungerp maintained activity against the two clinical isolates that were resistant to caspofungin, it is unknown if this would translate into in vivo efficacy against candidiasis caused by strains of C. auris that are echinocandin resistant. This was not evaluated in this study, as echinocandin-resistant C. auris isolates are rare, and the advantage of ibrexafungerp over the echinocandins is that this agent can be administered orally, thus possibly reducing the reliance on azoles when transitioning from intravenous to oral therapy.

Overall, our results are encouraging and demonstrate that the in vitro activity observed for ibrexafungerp against C. auris does translate into in vivo efficacy. These results are further supported by preliminary data from an ongoing phase 3 open-label study in which two cases of C. auris candidemia were successfully treated with oral ibrexafungerp (31). Additional studies are warranted in order to further evaluate the utility of ibrexafungerp for the treatment of infections caused by the emerging pathogen C. auris.

MATERIALS AND METHODS

Antifungals.

Stock solutions and initial dilutions of ibrexafungerp (Scynexis, Inc., Jersey City, NJ), fluconazole and caspofungin (Sigma-Aldrich, St. Louis, MO), and micafungin (Astellas Pharma US, Northbrook, IL) for in vitro testing were prepared in dimethyl sulfoxide (DMSO). Further dilutions were made in RPMI 1640 (0.165 M MOPS [morpholinepropanesulfonic acid], pH 7.0, without bicarbonate). For dosing in the murine model, ibrexafungerp was provided in a methylcellulose formulation for oral administration, and the clinically available intravenous formulations of fluconazole and caspofungin were used.

Isolates.

For in vitro testing, 10 C. auris isolates from the CDC FDA Antibiotic Resistance Isolate Bank (https://www.cdc.gov/drugresistance/resistance-bank/index.html), 14 isolates from the Westerdijk Fungal Biodiversity Institute (https://wi.knaw.nl/), and 30 clinical isolates received and identified as C. auris through DNA sequence analysis of the internal transcribed spacer (ITS) ribosomal DNA (rDNA) region by the Fungus Testing Laboratory at the University of Texas Health Science Center at San Antonio were used (32–34). For the in vivo model, a clinical isolate of C. auris (UTHSCSA DI17-46), originally cultured from the bloodstream of a patient with invasive candidiasis, was used to establish infection. The isolates were subcultured twice at 37°C for 48 h on Sabouraud dextrose agar. Prior to inoculation in the in vivo model, colonies were taken from the second subculture, placed into brain heart infusion broth, and grown overnight at 37°C with shaking at 200 rpm; cells were then collected by centrifugation and washed three times in sterile saline with 0.1% Tween 20. The starting inoculum was determined by counting Candida cells using a hemocytometer and adjusting that number to the target number of cells. Following the preparation of the inocula, viability was assessed by serially diluting an aliquot and plating it on Sabouraud dextrose agar to determine the number of colonies after incubation at 37°C.

Antifungal susceptibility.

MICs were determined by broth microdilution as described in the CLSI M27 standard (35). Following 24 h of incubation at 35°C, MICs were read for ibrexafungerp, caspofungin, and micafungin as the lowest concentration that inhibited 50% of growth compared to drug-free growth control. The fluconazole MIC was also measured against the strain used to establish infection using this same endpoint.

Murine model.

Outbred male ICR mice weighing ∼28 g were used. Mice were housed 5 per cage and had access to food and water ad libitum. A single dose of pharmaceutical grade 5-fluorouracil (5 mg/mouse intravenously 1 day prior to inoculation) was used to induce neutropenia in mice. To prevent bacterial superinfections, mice received antibacterial prophylaxis consisting of enrofloxacin at 50 ppm in their drinking water beginning 1 day prior to inoculation. On day 0, mice were infected intravenously via the lateral tail vein with 0.2 ml of C. auris UTHSCSA DI17-46. This isolate was chosen since it is fluconazole resistant and the majority of C. auris isolates are resistant to this azole (4, 5). In addition, this isolate results in consistent infection in this experimental model (22, 25, 26). The target inoculum was 1 × 10⁷ cells/mouse in the survival arm, and a lower inoculum was used in the fungal burden arm (5 × 10⁶ cells/mouse) in order to prevent morbidity prior to the day 8 fungal burden time point.

To evaluate in vivo efficacy as treatment, therapy began 24 h following inoculation and continued through day 7. Treatment groups consisted of a vehicle control (methylcellulose), ibrexafungerp administered by oral gavage at three different dose levels (20, 30, and 40 mg/kg twice daily), and two positive controls: fluconazole 20 mg/kg administered by oral gavage twice daily and caspofungin administered at a supratherapeutic dose of 10 mg/kg by intraperitoneal injection once daily. The doses of ibrexafungerp used are known to result in exposures that are achievable in humans (11, 12, 36). The doses of fluconazole and caspofungin were chosen as controls since they result in consistent survival and fungal burden results in this model (22, 25, 26).

Both survival and fungal burden were used to measure in vivo efficacy. In the survival arm, mice were monitored at least twice daily until day 21, 14 days after therapy had been discontinued, to allow for adequate washout of the antifungal agents. Tissue fungal burden was measured using enumeration of CFU in the kidneys. In the fungal burden arm, kidneys were collected 1 day after treatment stopped (day 8), and in the survival arm, kidneys were collected at the prespecified endpoint for survival (day 21 postinoculation) or as mice succumbed to infection. The numbers of CFU were also measured at 24 h postinoculation in both arms to establish fungal burden prior to the initiation of antifungal therapy. Tissues from each animal were weighed, placed into sterile saline containing gentamicin and chloramphenicol, and homogenized, and the numbers of CFU/gram of tissue were determined.

Throughout the study, mice were observed multiple times per day and their overall health was determined, and any animal that appeared moribund prior to the scheduled endpoint was euthanized. The animal protocol was approved by the University of Texas Health Science Center at San Antonio Institutional Animal Care and Use Committee.

Data analysis.

Descriptive statistics were used to evaluate the in vitro activity, including MIC range, concentrations that inhibited 50% and 90% of the isolates tested (MIC₅₀ and MIC₉₀, respectively), and geometric mean (GM) MICs. Survival was plotted by Kaplan-Meier analysis, and differences in survival were analyzed by the log rank test. Differences in kidney fungal burden were compared by analysis of variance (ANOVA) with Tukey’s posttest for multiple comparisons. A P value of < 0.05 was considered statistically significant for all comparisons.