Ibrexafungerp: A Novel Oral Triterpenoid Antifungal in Development for the Treatment of Candida auris Infections

Candida auris is an emerging multidrug-resistant fungal pathogen reported worldwide. Infections due to C. auris are usually nosocomial and associated with high rates of fluconazole resistance and mortality. Echinocandins are utilized as the first-line treatment. However, echinocandins are only available intravenously and are associated with increasingly higher rates of resistance by C. auris. Thus, a need exists for novel treatments that demonstrate potent activity against C. auris. Ibrexafungerp is a first-in-class triterpenoid antifungal agent. Similar to echinocandins, ibrexafungerp inhibits (1→3)-β-D-glucan synthase, a key component of the fungal cell wall, resulting in fungicidal activity against Candida spp. Ibrexafungerp demonstrates broad in vitro activity against various Candida spp. including C. auris and C. auris isolates with fks mutations. Minimum inhibitory concentration (MIC50 and MIC90) values in >400 C. auris isolates were 0.5 μg/mL and 1.0 μg/mL, respectively. Clinical results were reported for two patients with invasive candidiasis or candidemia due to C. auris treated during the CARES (Candidiasis Caused by Candida Auris) trial, an ongoing open-label study. These patients experienced a complete response after treatment with ibrexafungerp. Thus, ibrexafungerp represents a promising new antifungal agent for treating C. auris infections.


Introduction
Candida auris is an emerging fungal pathogen reported on all continents except Antarctica, in at least 39 countries worldwide [1], as well as in 20 states of the United States [2][3][4][5]. Five distinct clades of C. auris were identified with well-defined geographic distributions (South America, Africa, South Asia, East Asia, and West Asia), as well as antifungal resistance patterns and mechanisms that are both

Mechanism of Action
Ibrexafungerp (formerly SCY-078) is the first compound of the enfumafungin-derived triterpenoid class of (1→3)-β-D-glucan synthase inhibitors (GSIs) (Figure 1). Glucan synthase inhibitors were first introduced for the treatment of invasive Candida infections in 2001, with caspofungin the first echinocandin to be approved [19]. This mechanism of action, i.e., blockade of the biosynthesis of ß-(1,3)-D-glucan in the fungal cell wall, was associated with potent and broad-spectrum antifungal activity and clinical efficacy for the treatment of fungal infections. Two additional echinocandins were later introduced, micafungin and anidulafungin. However, echinocandins lack clinically meaningful oral bioavailability, triggering the search for new molecules that shared the glucan synthase inhibition mechanism of action with echinocandins and could also be administered orally. Natural screening efforts led to the identification of enfumafungin derivatives as candidates, and subsequent synthetic modifications to these molecules resulted in increased oral bioavailability, potency, and stability, thereby leading to the discovery of ibrexafungerp. Ibrexafungerp is being developed as the first oral and IV GSI (Intravenous glucan synthase inhibitor) for the treatment and prevention of fungal infections, including serious and life-threatening infections due to Candida spp., Aspergillus spp., and Pneumocystis jirovecii, with the potential to provide the therapeutic advantages of both IV and oral formulations [20]. Ibrexafungerp causes a decrease in (1→3)-β-D-glucan polymers and a weakening of the fungal cell wall [21]. Ibrexafungerp is structurally distinct from echinocandins and interacts differently with the target enzyme ( Figure 2) [22]. Although the binding site on (1→3)-β-D-glucan synthase for ibrexafungerp partially overlaps with a binding site for echinocandins, it appears to be nonidentical, resulting in a lower rate of resistance to ibrexafungerp [22]. In in vitro studies, ibrexafungerp activity against wild-type and echinocandin-resistant strains of Candida spp. in the presence of fks mutations was minimally affected [23]. Thus, ibrexafungerp has limited potential for cross-resistance with echinocandins.

Pharmacokinetics
After oral administration in dog, mouse, and rat, ibrexafungerp is absorbed rapidly from the gastrointestinal (GI) tract, with bioavailability of approximately 35-50% [40]. In animals, ibrexafungerp is widely distributed in tissues with a steady-state volume of distribution (V dss ) of >5 L/kg [41], which is several-fold greater than fluconazole and echinocandins [38]. Higher V dss results in higher tissue-to-plasma concentration ratios could be beneficial for specific fungal infections, such as those located in the skin, liver, spleen, mucosa, bone, and lung, to mention some. Ibrexafungerp undergoes extensive metabolism by cytochrome P450 3A4 isoenzymes in the liver, and <2% of a dose is recovered unchanged in urine [42]. After single oral doses of 10 mg to 1600 mg in healthy subjects, peak ibrexafungerp plasma concentrations were reached after four to six hours, with a mean terminal half-life of approximately 20 to 30 h [43]. Mean area under the concentration-time curve (AUC 0-∞ ) and peak concentration (C max ) were dose-proportional across this dosage range. The proarrhythmic potential of ibrexafungerp was evaluated in animal models and healthy volunteers using cardiodynamic assessments [44]. Ibrexafungerp exhibited no clinically relevant effects on heart rate or PR and QRS intervals. No clinically meaningful effect of ibrexafungerp on the QTcF interval was observed at plasma concentrations up to 4000 ng/mL after IV administration in healthy subjects.
Results from in vitro studies indicated that ibrexafungerp is a substrate of CYP3A4 and a potential inhibitor of cytochrome (CYP) 2C8 [42,45]. In healthy subjects, the interaction potentials of ketoconazole and diltiazem (CYP3A4 inhibitors), rosiglitazone (CYP2C8 substrate), and tacrolimus (CYP3A4 substrate) were evaluated after single or multiple doses co-administered with ibrexafungerp [46]. No clinically relevant effects of ibrexafungerp on CYP2C8 inhibition or CYP3A4 substrates were observed, although a dosage adjustment for ibrexafungerp may be needed when co-administering with potent CYP3A4 inhibitors. Thus, ibrexafungerp shows low potential for CYP-mediated drug interactions at therapeutic exposures.

Clinical Development
Nineteen Phase 1, three Phase 2, and two Phase 3 clinical studies were completed with ibrexafungerp, the latter two (VANISH-303 and VANISH-306), in women with vulvovaginal candidiasis (VVC) (https://clinicaltrials.gov: NCT03734991 and NCT03987620). In both Phase 3 studies, the rate of clinical cure (complete resolution of all vaginal signs and symptoms at Day 10) and mycological eradication were significantly greater with ibrexafungerp than placebo. The most common adverse events reported with ibrexafungerp were mild gastrointestinal disturbances when administered to >1200 patients and healthy subjects.
Currently, ibrexafungerp is undergoing further clinical development in ongoing studies, including CANDLE-304 (clinicaltrials.gov: NCT04029116), a Phase 3 study in women with recurrent VVC, SCYNERGIA, a Phase 2 study of ibrexafungerp combined with voriconazole in patients with invasive aspergillosis (clinicaltrials.gov: NCT03672292), FURI, an open-label study in patients with refractory invasive fungal infections (clinicaltrials.gov: NCT02244606), and CARES (clinicaltrials.gov: NCT03363841), an open-label, emergency protocol study of patients with invasive infections due to C. auris.

In Vitro Activity
The in vitro activity of ibrexafungerp was tested against 16 C. auris clinical isolates obtained from Germany, Japan, India, and South Korea [27]. The MIC 90 for ibrexafungerp was 1 µg/mL. Fluconazole and amphotericin B exhibited less in vitro activity against C. auris with MIC 90 values of >64 and 4 µg/mL, respectively, while the MIC 90 values for anidulafungin, caspofungin, and micafungin were 0.25 µg/mL, 1 µg/mL, and 1 µg/mL, respectively.
The in vitro activity of ibrexafungerp was evaluated against a global collection of 100 isolates of C. auris representing each of the four clades of C. auris known at that time [24]. MICs for ibrexafungerp ranged from 0.0625 to 2 µg/mL, with an MIC 50 of 0.5 µg/mL and MIC 90 1 µg/mL. MIC values for anidulafungin, caspofungin, and micafungin ranged from 0.03 up to >16 µg/mL. Among seven C. auris isolates exhibiting elevated MIC values for echinocandins, the ibrexafungerp MIC ranged from 0.5 to 1.0 µg/mL.
Ibrexafungerp and six comparator antifungal agents were evaluated against 122 C. auris isolates [33]. The MIC range for ibrexafungerp was 0.06 to 2.0 µg/mL. A wide distribution of MIC values was reported for anidulafungin and micafungin, ranging from 0.016 to >32 and 0.03 to >32 µg/mL, respectively ( Table 1). All but one C. auris isolate were resistant to fluconazole. Out of 122 isolates, 8 displayed high MIC values for echinocandins associated with fks mutations (S639F Fks1 alteration). The MIC for ibrexafungerp for these eight resistant isolates ranged from 0.25 to 0.5 µg/mL. Among 102 C. auris isolates with variable resistance to amphotericin B, flucytosine, azoles, and echinocandins, the ibrexafungerp MIC 50 for 97 isolates ranged from 0.06-0.5 µg/mL, and the median and mode MIC were both 0.5 µg/mL [32]. Ibrexafungerp also showed activity against five C. auris isolates considered to be pan-resistant, with a low MIC 50 range of 0.12 to 1 µg/mL. Data were compiled from four studies reporting the in vitro activity of ibrexafungerp against 445 C. auris clinical isolates [47]. Most isolates were obtained from the United States and India and included 32 isolates with increased MIC values to echinocandins. The MIC 50 and MIC 90 for ibrexafungerp across all isolates tested were 0.5 µg/mL and 1.0 µg/mL, respectively (Table 2). Among 32 C. auris isolates with echinocandin resistance, MIC values for ibrexafungerp ranged from 0.5 µg/mL to 1.0 µg/mL. One isolate displayed high MIC values for echinocandins and showed reduced sensitivity (>2 dilutions vs. the mode) to ibrexafungerp, and this isolate exhibited elevated MIC values to anidulafungin, caspofungin, and micafungin (MIC = 1 µg/mL), luconazole (MIC > 256 µg/mL), and amphotericin B (MIC = 1 µg/mL). Thus, ibrexafungerp exhibits in vitro activity against a broad collection of C. auris isolates, including most echinocandin-resistant isolates. The ability of Candida species to form biofilms is associated with catheter and device-related infections and may play a role in C. auris infections considering that many affected individuals are in intensive care units with intravascular lines. In this regard, 97% of patients infected with C. auris had central venous catheters (Sayeed et al., 2019) [48], and a retrospective analysis demonstrated significantly higher use of central venous catheters in patients infected with this multidrug-resistant Candida [49]. The activity of ibrexafungerp against C. auris biofilms was evaluated [27]. Following 48 h of incubation, metabolic activities of biofilms were measured. Images and thicknesses of biofilms growing in the presence or absence of a drug were captured using confocal scanning laser microscopy. Quantitation of the metabolic activity of C. auris biofilms was performed using a biochemical assay, the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay, as described previously [27,50]. Ibrexafungerp demonstrated activity against C. auris biofilms by reducing biofilm thickness and metabolic activity.
The effects of ibrexafungerp and caspofungin on the morphology of C. albicans, C. auris, and C. glabrata were studied using scanning and transmission electron microscopy [51]. When evaluated at respective MIC 50 levels, ibrexafungerp exhibited a profound effect on cellular morphology in caspofungin-resistant organisms, possibly indicative of a difference in target engagement between ibrexafungerp and echinocandins ( Figure 3). Untreated control C. auris cells showed well-defined, oval-shaped yeast morphology, as well as several budding yeasts. In contrast, cells exposed to ibrexafungerp (at a concentration of 1 µL MIC) exhibited a severely distorted yeast cell topography, including cell collapse, deformed cellular appearance, irregular budding, and cells that were fused together and unable to undergo cell division [27].
Antibiotics 2020, 9, x FOR PEER REVIEW 7 of 13 ibrexafungerp and echinocandins ( Figure 3). Untreated control C. auris cells showed well-defined, oval-shaped yeast morphology, as well as several budding yeasts. In contrast, cells exposed to ibrexafungerp (at a concentration of 1 μL MIC) exhibited a severely distorted yeast cell topography, including cell collapse, deformed cellular appearance, irregular budding, and cells that were fused together and unable to undergo cell division [27].

In Vivo Activity
The in vivo efficacy of ibrexafungerp for C. auris was evaluated in a disseminated murine mouse model [37]. Immunocompromised mice were randomized to ibrexafungerp 10, 20, or 30 mg/kg twice daily (BID) vs. a vehicle given by oral gavage. At Day 7, the fungal burden in kidney tissue was reduced by all doses of ibrexafungerp, with a significant difference for the 30 mg/kg dose vs. vehicle. At Day 14, survival rates were 60-70% with ibrexafungerp vs. 20% with vehicle control. Exposures in mice dosed with ibrexafungerp 10, 20, or 30 mg/kg BID were consistent with steady-state plasma exposure (AUC0-24) of 8.4, 24.3, and 40.2 ug*h/mL, respectively. These results demonstrate potent antifungal activity of ibrexafungerp against C. auris.
C. auris colonization is a major problem in hospitals and long-term care facilities. In order to understand the ability of ibrexafungerp to potentially decolonize the skin of C. auris, a study was performed looking at the in vivo efficacy of ibrexafungerp in a cutaneous infection model in Guinea pigs [36]. Animals were treated with ibrexafungerp 10, 20, or 30 mg/kg BID by oral gavage, micafungin 5 mg/kg once daily IP, or vehicle by oral gavage, and prednisone 30 mg/kg SC was given one day before and three days after infection. Tissue burden at Day 7 was lower with all active treatments vs. vehicle. Animals dosed with ibrexafungerp 10, 20, or 30 mg/kg BID showed systemic exposures (AUC0-24) of 2.8, 5.6, and 15 ug*h/mL. Examination of Periodic Acid-Schiff (PAS)-stained

In Vivo Activity
The in vivo efficacy of ibrexafungerp for C. auris was evaluated in a disseminated murine mouse model [37]. Immunocompromised mice were randomized to ibrexafungerp 10, 20, or 30 mg/kg twice daily (BID) vs. a vehicle given by oral gavage. At Day 7, the fungal burden in kidney tissue was reduced by all doses of ibrexafungerp, with a significant difference for the 30 mg/kg dose vs. vehicle. At Day 14, survival rates were 60-70% with ibrexafungerp vs. 20% with vehicle control. Exposures in mice dosed with ibrexafungerp 10, 20, or 30 mg/kg BID were consistent with steady-state plasma exposure (AUC 0-24 ) of 8.4, 24.3, and 40.2 ug*h/mL, respectively. These results demonstrate potent antifungal activity of ibrexafungerp against C. auris.
C. auris colonization is a major problem in hospitals and long-term care facilities. In order to understand the ability of ibrexafungerp to potentially decolonize the skin of C. auris, a study was performed looking at the in vivo efficacy of ibrexafungerp in a cutaneous infection model in Guinea pigs [36]. Animals were treated with ibrexafungerp 10, 20, or 30 mg/kg BID by oral gavage, micafungin 5 mg/kg once daily IP, or vehicle by oral gavage, and prednisone 30 mg/kg SC was given one day before and three days after infection. Tissue burden at Day 7 was lower with all active treatments vs. vehicle. Animals dosed with ibrexafungerp 10, 20, or 30 mg/kg BID showed systemic exposures (AUC 0-24 ) of 2.8, 5.6, and 15 ug*h/mL. Examination of Periodic Acid-Schiff (PAS)-stained skin sections revealed that sections obtained from untreated control animals showed yeast cells, demonstrating that the skin was infected with C. auris. In contrast, examination of multiple skin sections obtained from animals treated with either ibrexafungerp or micafungin did not reveal yeast cells at any of the dose levels tested, indicating that the C. auris infection was cleared. There was no significant difference in clinical scores between the treatment groups [36]. Thus, no fungal elements were observed with ibrexafungerp or micafungin from histological examination.

Clinical Experience
CARES is an open-label study of oral ibrexafungerp in patients with documented candidiasis or candidemia due to C. auris who were treatment naïve or refractory to or intolerant of standard-of-care antifungal agents (clinicaltrials.gov: NCT03363841). Patients were treated with oral ibrexafungerp 750 mg twice daily for two days, then 750 mg once daily for up to 90 days.
In the first two patients from CARES with candidemia due to C. auris, a complete response after 17 and 22 days of treatment was reported with ibrexafungerp [52]. The first patient was a 58-year-old male admitted to the ICU with pneumonia and septic shock. Antibiotics were given together with empiric IV fluconazole. When C. auris was isolated from blood cultures, antifungal therapy was switched to IV micafungin. However, blood cultures remained positive for C. auris after five days, and the patient was switched to ibrexafungerp for 17 days. Subsequent blood cultures at Day 3 of ibrexafungerp therapy were negative for C. auris, and the patient was considered to have a complete response at the end of therapy. Ibrexafungerp-related adverse events were mild loose stools from days two through four of therapy.
The second patient was a 64-year-old female admitted to the hospital with pneumonia, fever, and hypotension. When C. auris was isolated from blood cultures, ibrexafungerp was initiated. A blood culture collected on Day 3 of ibrexafungerp therapy remained positive for C. auris and subsequent cultures at Days 9 and 21 were reported negative. The patient improved clinically, received ibrexafungerp for 22 days and was considered a complete response at the end of therapy. No ibrexafungerp-related adverse events were reported.

Echinocandin Resistance and C. auris
For echinocandins, the primary mechanism of resistance in C. auris species comprises the fks1 and fks2 genes, where mutations of the S639F, S639P, and S639Y amino acid sequences were identified as the cause of elevated MICs to echinocandins [9]. Among 350 C. auris isolates from India, 2% were echinocandin-resistant due to the fks1 mutation expressing the S639F sequence [53]. A similar finding was reported from Kuwait, where 3 (1.0%) of 314 C. auris isolates were echinocandin-resistant due to the fks1 mutation expressing the S639F sequence [54]. Four additional C. auris isolates from a total of 106 isolates were resistant to all tested echinocandins (MIC ≥ 4 µg/mL) and contained an S639F mutation in fks1 [55].
Biagi et al. [56] reported a patient with recurrent candidemia due to C. auris that was echinocandin-resistant but azole-sensitive, who expressed the fks1 mutation for the S639P sequence. A single C. auris isolate was identified in the UK that displayed 5-flucytosine and echinocandin resistance; echinocandin resistance was due to fks1 mutation for the S639Y sequence [57].
Ostrowski and colleagues [18] described three cases of C. auris among 801 patients in New York state with confirmed C. auris that were pan-resistant, i.e., resistant to fluconazole, amphotericin B, and echinocandins. All three patients with pan-resistance were on mechanical ventilators; two died within two to four weeks from isolation of a pan-resistant C. auris, and the third died at 10 months. MICs for fluconazole were >256 µg/mL, 2 µg/mL for amphotericin B, and 2-16 µg/mL for echinocandins. From the same laboratory, an analysis of the susceptibility of five pan-resistant C. auris isolates, defined as in vitro resistance to more than two azoles, all echinocandins, and amphotericin B, reported MIC values to fluconazole (>256 µg/mL), amphotericin B (2 µg/mL), and echinocandins (ranging from 2 to >16 µg/mL) [15]. However, all these pan-resistant isolates exhibited MICs for ibrexafungerp ranging from 0.12 to 1 µg/mL, which were within the wild-type MIC range reported for C. auris.

Summary and Conclusions
In the past decade, C. auris has emerged as a critical public health concern because of its persistence in the environment, contagious nature, and high morbidity and mortality. Unfortunately, rates of resistance to antifungal drugs among C. auris isolates continue to increase with almost universal resistance to fluconazole and growing resistance to other azoles and antifungals, including echinocandins. New antifungal agents in clinical development may provide more effective treatment options to address the growing impact of C. auris. Ibrexafungerp offers the advantages of oral administration, a favorable PK profile, a well-characterized safety/tolerability profile in >1200 patients/subjects, a low risk for cross-resistance to echinocandins, and consistently potent in vitro activity against C. auris, including echinocandin-resistant isolates. Animal studies looking at the tissue distribution of ibrexafungerp achieved high concentrations in the skin, an attribute that may be of importance to limit C. auris skin colonization, with a potential impact of limiting transmission. Ibrexafungerp demonstrates activity across a range of invasive fungal diseases as a monotherapy but also in combination [58,59]. Early clinical evidence from an emergency-use Phase 3 study of ibrexafungerp for invasive candidiasis due to C. auris is promising. Additional preclinical and clinical evidence are awaited to confirm the role of ibrexafungerp in treating infections due to C. auris, as well as other causes of invasive fungal disease.