Research Article
25 April 2019

A Novel 1,3-Beta-d-Glucan Inhibitor, Ibrexafungerp (Formerly SCY-078), Shows Potent Activity in the Lower pH Environment of Vulvovaginitis

ABSTRACT

Ibrexafungerp (IBX) (formerly SCY-078) is a novel glucan synthase inhibitor whose oral availability is being evaluated for efficacy against vulvovaginal candidiasis (VVC). Bioavailability and in vitro activity are important efficacy indicators, but accepted susceptibility methods do not always accurately predict activity in an acidic environment, such as the vagina. Studies were 3-fold, as follows: (i) pharmacokinetic study following oral administration in a murine model; (ii) susceptibility testing of isolates from a phase 2 VVC clinical trial by CLSI M27-A4 methodology; and (iii) susceptibility testing of Candida albicans and Candida glabrata isolates obtained from this trial group in RPMI 1640 adjusted to 3 different pH values, 7.0, 5.72, and 4.5, compared to susceptibility testing for micafungin and fluconazole. IBX readily accumulated in vaginal tissues and secretions following oral administration. Potent in vitro activity was demonstrated against Candida strains obtained at baseline and end of study visits. Moreover, the geometric mean (GM) values for IBX at pH 4.5 were dramatically lower than those at pH 7.0 and 5.72. The MIC90 values of micafungin remained the same regardless of pH value, while those of fluconazole tended to increase with lower pH values. IBX is able to reach target tissues following oral administration at pharmacologically meaningful levels. IBX demonstrated potent in vitro activity, with no development of resistance, following repeated exposure over the course of the clinical trial. Importantly, activity of IBX in an acidic medium suggests a therapeutic advantage of this novel antifungal in the treatment of vaginal Candida infections.

INTRODUCTION

Vulvovaginal candidiasis (VVC) accounts for up to one-third of all vaginitis reported in the United States (1, 2) and is recurrent in 5% to 9% of patients (3). Although most cases of VVC are caused by Candida albicans, Candida glabrata and less commonly fluconazole (FLU)-resistant C. albicans are also causes of recurrent cases (4). C. glabrata has shown intrinsic resistance to multiple azoles, including FLU, which is widely used for treatment of this infection. Thus, eradication of FLU-resistant C. albicans and C. glabrata from the vagina has proven difficult (4).
Complicating the task of choosing appropriate therapy is the fact that the accepted antifungal broth dilution susceptibility method, Clinical and Laboratory Standards Institute (CLSI) M27-A4 (5), does not always predict resistance in test isolates from cases of VVC. It is thought that this disconnect may be due to the unique acidic vaginal environment, which may influence antifungal activity. CLSI broth dilution susceptibility testing is conducted in a medium buffered to a pH of 7.0, while the normal pH of the vagina, 4 to 4.5, remains acidic during VVC episodes (6). To that point, Marr et al. have shown that an acidic pH tends to result in higher MICs of FLU for some Candida species (7), which in turn may indicate true FLU resistance.
Ibrexafungerp (IBX) (formerly SCY-078) is a member of a new class of glucan synthase inhibitors that interferes with the synthesis of the fungal cell wall polymer β-(1,3)-d-glucan. IBX is currently in clinical development for use in the treatment of various fungal infections and is available as oral and intravenous formulations. In vitro studies have demonstrated that IBX has fungicidal activity against azole-resistant Candida spp. isolates similar to the echinocandins but, importantly, shows activity against the majority of clinical isolates demonstrating echinocandin resistance due to FKS gene mutations (8).
IBX has also shown broad-spectrum activity against Aspergillus strains (9), and while these in vitro tests would predict therapeutic success in cases such as invasive candidiasis or aspergillosis, the effect of an acidic environment on the activity of IBX is not known.
In order to further characterize the activity of IBX in the treatment of vulvovaginitis, we evaluated the potential for vaginal distribution of orally administered IBX in mice, while simultaneously determining whether changes in test medium pH that mimic the vaginal environment had an effect on the in vitro susceptibility of C. albicans and C. glabrata vaginal isolates as measured by MIC. Additionally, we evaluated the MICs of Candida spp. isolates from patients with VVC before and after IBX therapy to make an initial assessment of the risk of resistance development.

RESULTS

Pharmacokinetic studies.

IBX demonstrated a high potential to accumulate in vaginal tissues and fluids following oral administration, with concentrations in both increasing in a dose-dependent manner. The content of IBX in vaginal tissue was 2- to 5-fold higher than the respective plasma levels across all dose groups (Table 1).
TABLE 1
TABLE 1 Ibrexafungerp plasma: tissue concentration ratio and accumulation potentiala
Dose groupp.o. regimen (mg/kg single dose)Total doses (no.)AUC0–24 (μg · h/ml) mean ± SD for:
PlasmaVaginal secretionsVaginal tissue
11018.4 ± 1.51.3 ± 0.729.4 ± 18.1
220119.4 ± 3.31.8 ± 1.454.1 ± 14.1
340136.1 ± 7.04.2 ± 2.8186.8 ± 107.7
480175.6 ± 12.410.8 ± 8.4168.9 ± 36.3
a
p.o., oral; AUC0–24, area under the concentration-time curve from 0 to 24 h.

In vitro activity of IBX at various pH levels.

MIC ranges for IBX at pH values of 7.0, 5.72, and 4.5 against the C. albicans isolates were 0.125 to 0.5 μg/ml, 0.125 to 0.25 μg/ml, and <0.016 to 0.031 μg/ml, respectively. The MIC50 values (defined as the lowest concentration to inhibit 50% of isolates tested) for IBX at pH values 7.0 and 5.72 were both 0.25 μg/ml, while the MIC90 values (defined as the lowest concentration to inhibit 90% of isolates tested) were 0.5 and 0.25 μg/ml at pH 7.0 and 5.72, respectively. The MIC50 and MIC90 values for IBX at pH 4.5 were both <0.016 μg/ml. The geometric mean (GM) of the MIC values for IBX against the C. albicans isolates tested at pH 4.5 was significantly lower than those at pH 7.0 and 5.72 (P values of <0.0001) (Table 2).
TABLE 2
TABLE 2 MIC results for IBX, MICA, and FLU against 10 C. albicans isolates tested at three pH levels
ParameterResults (μg/ml) for IBX with pH:Results (μg/ml) for MICAa with pH:Results (μg/ml) for FLUb with pH:
7.05.724.57.05.724.57.05.724.5
Range0.125–0.50.125–0.25<0.016–0.0310.250.063–10.25–0.5<0.125–1<0.125–10.25–8
MIC500.250.25<0.0160.2510.50.25<0.1250.25
MIC900.50.25<0.0160.2510.50.250.251
GM0.2500.2180.0170.2500.5750.4670.3150.3970.500
a
MICA, micafungin.
b
FLU, fluconazole.
MIC ranges for IBX at pH values of 7.0, 5.72, and 4.5 against the C. glabrata isolates were 0.5 to 1.0 μg/ml, 0.5 μg/ml, and 0.031 to 0.063 μg/ml, respectively. The MIC50 and MIC90 values for IBX at pH 7.0 were both 1.0 μg/ml, while the MIC50 and MIC90 values for IBX at pH 5.72 were one dilution lower (0.5 μg/ml). The MIC50 and MIC90 values for IBX at pH 4.5 were both 0.063 μg/ml. In this model system, the GM of the MIC values for IBX against the C. glabrata isolates tested at pH 4.5 was also significantly lower than those at pH 7.0 and 5.72 (P values of <0.0001) (Table 3).
TABLE 3
TABLE 3 MIC results for IBX, MICA, and FLU against 10 C. glabrata isolates tested at three pH levels
ParameterResults (μg/ml) for IBX with pH:Results (μg/ml) for MICAa with pH:Results (μg/ml) for FLUb with pH:
7.05.724.57.05.724.57.05.724.5
Range0.5–10.50.031–0.0630.25–0.50.250.250.5–22–161–16
MIC5010.50.0630.250.250.25188
MIC9010.50.0630.50.250.2521616
GM0.7580.5000.0510.3080.2500.2501.2316.9648.000
a
MICA, micafungin.
b
FLU, fluconazole.
MIC ranges for micafungin (MICA) at pH values of 7.0, 5.72, and 4.5 against the C. albicans isolates were 0.25 μg/ml, 0.063 to 1.0 μg/ml, and 0.25 to 0.5 μg/ml, respectively. The MIC50 and MIC90 values of MICA against C. albicans were equal at each pH level (pH 7.0, MIC50 and MIC90 = 0.25 μg/ml; pH 5.72, MIC50 and MIC90 = 1.0 μg/ml; pH 4.5, MIC50 and MIC90 = 0.5 μg/ml) (Table 3). The MIC range for MICA against C. glabrata isolates was 0.25 to 0.5 μg/ml at pH 7.0, while all MIC values were 0.25 μg/ml at pH 5.72 and 4.5. The MIC90 of MICA at pH 7.0 was one dilution higher at 0.5 μg/ml (Table 3).
MIC ranges for FLU against the C. albicans isolates were <0.125 to 1.0 μg/ml at pH values of 7.0 and 5.72 and 0.25 to 8.0 μg/ml at pH 4.5. The FLU MIC50 and MIC90 values were 0.25 μg/ml at pH 7.0; <0.125 and 0.25 μg/ml, respectively, at pH 5.72; and 0.25 and 1.0 μg/ml, respectively, at pH 4.5 (Table 3). MIC ranges for FLU at pH values of 7.0, 5.72, and 4.5 against the C. glabrata isolates were 0.5 to 2.0 μg/ml, 2.0 to 16 μg/ml, and 1.0 to 16 μg/ml, respectively. The MIC50 values of FLU were 1.0 μg/ml at pH 7.0 and 8 μg/ml at pH 5.72 and pH 4.5, while the MIC90 values were each one dilution higher (2 μg/ml at pH 7.0 and 16 μg/ml at pH 5.72 and pH 4.5) (Table 3).

Activity of IBX against baseline versus end of therapy collected clinical trial isolates.

MICs of IBX were determined against all isolates obtained from a phase 2 dose-finding clinical trial involving subjects with acute vulvovaginal candidiasis (ClinicalTrials.gov identifier NCT02679456). The majority of subjects in the study had C. albicans detected at baseline (86%). Other Candida species isolated at baseline included Candida parapsilosis, C. glabrata, Candida krusei, Candida tropicalis, Candida lusitaniae, and Candida nivariensis. The study included 50 subjects with recurrent VVC (RVVC) that received oral IBX and for which vaginal cultures were positive for Candida spp. at baseline with subsequent cultures scheduled 1, 2, 3, and 4 months after treatment. These study results have been previously reported showing high clinical cure rates (i.e., 76%) with the oral IBX regimens tested (S. Helou and D. Angulo, A multicenter randomized, evaluator blinded, active-controlled study to evaluate the safety and efficacy of oral SCY-078 vs. oral fluconazole in 96 subjects with moderate to severe vulvovaginal candidiasis, presented at the Infectious Diseases Society for Obstetrics and Gynecology 44th Annual Meeting, Park City, UT, August 2017).
The IBX MIC range, MIC50, and MIC90 for baseline C. albicans isolates among the IBX-treated groups (43/50; 86%) were 0.06 to 0.5 μg/ml, 0.25 μg/ml, and 0.25 μg/ml, respectively. Thirty-one of the 43 IBX-treated subjects (72%) with C. albicans infection experienced mycological eradication. Overall, there were no changes in MIC values obtained for the C. albicans isolates from the 12 subjects with positive culture at the end of the study; the IBX MIC range, MIC50, and MIC90 were 0.06 to 0.5 μg/ml, 0.25 μg/ml, and 0.25 μg/ml, respectively (Table 4).
TABLE 4
TABLE 4 Distribution of IBX MICs against C. albicans isolates collected at baseline and from per protocol patients on study drug at the last visit
Time of collectionNo. of isolates with IBX MIC:
0.06 μg/ml0.125 μg/ml0.25 μg/ml0.5 μg/ml
Baseline (n = 43)116251
Last visit (n = 9) 27 

DISCUSSION

VVC and recurrent VVC (RVVC) are serious conditions with significant morbidity and with very limited oral treatment options. VVC affects approximately 70% to 75% of women at least once in their lifetime (3), and RVVC, defined as 3 or more episodes of symptomatic acute Candida vaginitis in a 12-month period, is estimated to occur in 6% to 9% of women during their reproductive years (10). There is no product approved for the prevention of RVVC, and current treatment alternatives have significant limitations for the treatment of VVC, particularly in patients with other comorbidities, in patients taking prohibited concomitant medications, during pregnancy, or for episodes caused by an azole-resistant microorganism.
Single dosing of FLU has long been shown to produce efficacious concentrations in vaginal secretions (11, 12), with a reported clinical cure rate of 69%. However, according to the Centers for Disease Control and Prevention, conventional antifungal treatment may not be effective against C. glabrata and other non-albicans Candida species observed in 10% to 20% of women with recurrent VVC (13). Further, antifungals normally prescribed for treatment do not always eradicate the total Candida population, likely due to their fungistatic properties, leaving a reservoir for the establishment of a recurring infection (14).
In vitro susceptibility data showing decreased activity of certain antifungals at lower pH levels may explain the high rates of Candida survival in the acidic vaginal environment. Establishing an alternative susceptibility standard employing a lower pH medium to accurately predict therapeutic outcome would be lengthy and cost prohibitive to develop and standardize; thus, identifying a novel compound that is efficacious against Candida at lower pH would be highly advantageous. In this study, we compared the MICs of IBX, a novel glucan synthase inhibitor, to those of FLU against C. albicans and C. glabrata strains isolated from patients with recurrent vulvovaginitis. Though not a therapeutic agent for vaginitis due to its intravenous application, we also took the opportunity to include an echinocandin, MICA, as a comparator in order to provide additional data for this drug at different pH levels.
Interestingly, IBX showed significantly increased activity against the C. albicans and C. glabrata strains in an acidic medium. IBX MIC values at pH 4.5 were up to 5-fold lower than those at pH values of 7.0 and 5.72 for both C. glabrata and C. albicans strains. The opposite effect was seen with FLU, for which the MICs tended to be higher at the lower pH levels. These data agree with those of Danby et al., who reported elevated MIC values at pH 4.5 for FLU, voriconazole (VOR), and posaconazole (15). Similar data have been published for clotrimazole and miconazole as well as FLU (16). Combined, these data indicate that FLU is less effective at eradicating these yeast strains in acidic environments, such as the vagina, and suggest a therapeutic advantage of IBX in the treatment of vaginal Candida infections.
Further, our data show that pH levels had no effect on the activity of MICA. The MIC50 and MIC90 values of MICA against both Candida species were within 2 dilutions at all pH levels (a 2-dilution variance is considered equivalent). These results suggest that IBX may inhibit glucan synthase in an alternative manner to that of echinocandins, perhaps due to its different molecular structure. In addition to possessing antifungal activity at a wide pH range, IBX has an advantage over MICA and other echinocandins because of its bioavailability in an oral formulation.
Importantly, the levels of IBX accumulation, even at the lowest testing dose of 10 mg of drug/kg of body weight in the murine model, were more than 2.5 times greater in vaginal secretions than at the highest MIC value obtained from clinical trial isolates and 53 times greater in vaginal tissue. These data, coupled with the lack of resistance development following repeated exposure and a previously reported capacity to reduce kidney fungal burden in a murine model (17), suggest that IBX has utility for treatment of VVC.

MATERIALS AND METHODS

Pharmacokinetic studies.

A dispositional pharmacokinetics study with IBX was conducted in female CD-1 mice following oral (gavage) dosing. Mice (n = 27 per group) were dosed with a single dose of IBX at 10, 20, 40, or 80 mg/kg. Whole blood, vaginal lavage fluid, and vulvovaginal tissue samples were collected from 3 mice per group at predose, 1, 2, 3, 6, 8, 12, 18, and 24 h postdose. Plasma (separated from whole blood collected in K2EDTA tubes) and vulvovaginal tissue were stored frozen until analysis. Vaginal lavage fluid was collected with a solution of phosphate-buffered saline (PBS)-0.2% tocopherol polyethylene glycol succinate (TPGS) (to mitigate nonspecific binding to pipette tips and collection vials), centrifuged to produce supernatant, and frozen until analysis.
All samples were extracted via protein precipitation. Vaginal lavage fluid samples were corrected for dilution based on blood urea levels (18). Calculated concentrations for all samples were interpolated from matrix-matched calibration curves using Applied Biosystems Analyst 1.4.2 software. Standard curves were created by generating least-squares fitting plots of peak area ratio (analyte peak area divided by internal standard peak area) versus nominal concentration. Incurred sample concentrations were calculated from results of least-squares fits.

MIC testing of clinical trial isolates.

Susceptibility testing was performed using a broth microdilution method according to CLSI M27-A4 guidelines (5). RPMI 1640 with morpholinepropanesulfonic acid (MOPS) was the medium for all testing, and the inoculum size was 0.5 × 103 to 2.5 × 103 CFU/ml. MIC endpoints for IBX were recorded at 50% inhibition, compared to those of the growth control, after 24 h incubation. MICs of the comparators FLU (Sigma-Aldrich) and MICA (Astellas Pharma) were recorded at 50% inhibition following 24 h of incubation.

MIC testing at various pH levels.

IBX (Scynexis, Inc.) and MICA (Astellas Pharma) were tested in a range of 0.015 to 8 μg/ml, while FLU (Sigma-Aldrich) was tested in a range of 0.125 to 64 μg/ml. Serial dilutions of each antifungal were prepared in RPMI 1640 (USBiological) at three different pH levels; pH 7, pH 5.72, and pH 4.5. Adjustments in pH were made using sodium hydroxide or hydrochloric acid, with buffering by morpholinepropanesulfonic acid (MOPS) (0.165 M) as described by Marr et al. (7).
Ten strains each of C. albicans and C. glabrata were obtained from recent clinical trial patients with vulvovaginal candidiasis (VVC) prior to treatment. Inocula of each strain were prepared in RPMI 1640 at each pH level to a concentration of 0.5 × 103 to 2.5 × 103 blastospores/ml. Inocula and antifungal dilutions were added to the wells of microtiter plates in equal 100 μl amounts and incubated at 35°C for 24 h. Wells containing no antifungal served as the growth control. C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 were tested concurrently to ensure quality control.
Inhibition endpoints were read visually as a 50% reduction in growth compared to the growth control. Differences in the geometric mean of the MIC values were assessed for significance by analysis of variance (ANOVA) using Tukey’s posttest for multiple comparisons.

ACKNOWLEDGMENT

This work was supported by Scynexis, Inc. M.G. received grants from Scynexis, Inc.

REFERENCES

1.
American College of Obstetricians and Gynecologists. 2006. ACOG practice bulletin. Clinical management guidelines for obstetrician-gynecologists, number 72, May 2006: vaginitis. Obstet Gynecol 107:1195–1206.
2.
American College of Obstetricians and Gynecologists. 2009. Vulvar disorders. ACOG clinical updates in women’s health care, vol 8, no. 2, p 36. American College of Obstetricians and Gynecologists, Washington, DC.
3.
Sobel JD. 2007. Vulvovaginal candidosis. Lancet 369:1961–1971.
4.
Sobel JD. 2003. Management of patients with recurrent vulvovaginal candidiasis. Drugs 63:1059–1066.
5.
Clinical and Laboratory Standards Iinstitute. 2018. Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard—3rd ed. CLSI document M27-A4. Clinical and Laboratory Standards Institute, Wayne, PA.
6.
Linhares IM, Summers PR, Larsen B, Giraldo PC, Witkin SS. 2011. Contemporary perspectives on vaginal pH and lactobacilli. Am J Obstet Gynecol 204:120.e1–120.e5.
7.
Marr K, Rustad T, Rex J, White T. 1999. The trailing end point phenotype in antifungal susceptibility testing is PH dependent. Antimicrob Agents Chemother 43:1383–1386.
8.
Scorneaux B, Angulo A, Borroto-Esoda K, Ghannoum M, Peel M, Wring S. 2017. SCY-078 is fungicidal against Candida species in time-kill studies. Antimicrob Agents Chemother 61:e01961-16.
9.
Ghannoum M, Long L, Larkin EL, Isham N, Sherif R, Borroto-Esoda K, Barat S, Angulo D. 2018. Evaluation of the antifungal activity of the novel oral glucan synthase inhibitor SCY-078, singly and in combination, for the treatment of invasive aspergillosis. Antimicrob Agents Chemother 62:00244-18.
10.
Sobel JD. 2016. Recurrent vulvovaginal candidiasis. Am J Obstet Gynecol 214:15–21.
11.
Pfizer, Inc. 2011. Diflucan package insert. Pfizer, Inc. New York, NY.
12.
Debruyne D. 1997. Clinical pharmacokinetics of fluconazole in superficial and systemic mycoses. Clin Pharmacokinet 33:52–77.
13.
Centers for Disease Control and Prevention. 2015. Sexually transmitted diseases treatment guidelines. Centers for Disease Control and Prevention, Atlanta, GA.
14.
De Punzio C, Garutti P, Mollica G, Nappi C, Piccoli R, Genazzani AR. 2003. Fluconazole 150 mg single dose versus itraconazole 200 mg per day for 3 days in the treatment of acute vaginal candidiasis: a double-blind randomized study. Eur J Obstet Gynecol Reprod Biol 106:193–197.
15.
Danby CS, Boikov D, Rautemaa-Richardson R, Sobel J. 2012. Effect of pH on in vitro susceptibility of Candida glabrata and Candida albicans to 11 antifungal agents and implications for clinical use. Antimicrob Agents Chemother 56:1403–1406.
16.
Spitzer M, Wiederhold NP. 2018. Reduced antifungal susceptibility of vulvovaginal Candida species at normal vaginal pH levels: clinical implications. J Low Genit Tract Dis 22:152–158.
17.
Wiederhold NP, Najvar LK, Jaramillo R, Olivo M, Pizzini J, Catano G, Patterson TF. 2018. Oral glucan synthase inhibitor SCY-078 is effective in an experimental murine model of invasive candidiasis caused by WT and echinocandin-resistant Candida glabrata. J Antimicrob Chemother 73:448–451.
18.
Rennard SI, Basset G, Lecossier D, O'Donnell KM, Pinkston P, Martin PG, Crystal RG. 1986. Estimation of volume of epithelial lining fluid recovered by lavage using urea as marker of dilution. J Appl Physiol 60:532–538.

Information & Contributors

Information

Published In

cover image Antimicrobial Agents and Chemotherapy
Antimicrobial Agents and Chemotherapy
Volume 63Number 5May 2019
eLocator: 10.1128/aac.02611-18

History

Received: 14 December 2018
Returned for modification: 9 February 2019
Accepted: 10 March 2019
Published online: 25 April 2019

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Keywords

  1. ibrexafungerp
  2. SCY-078
  3. vulvovaginitis

Contributors

Authors

E. L. Larkin
Center for Medical Mycology, Case Western Reserve University and University Hospitals Cleveland Medical Center, Cleveland, Ohio, USA
L. Long
Center for Medical Mycology, Case Western Reserve University and University Hospitals Cleveland Medical Center, Cleveland, Ohio, USA
N. Isham
Center for Medical Mycology, Case Western Reserve University and University Hospitals Cleveland Medical Center, Cleveland, Ohio, USA
K. Borroto-Esoda
Scynexis, Inc., Jersey City, New Jersey, USA
S. Barat
Scynexis, Inc., Jersey City, New Jersey, USA
D. Angulo
Scynexis, Inc., Jersey City, New Jersey, USA
S. Wring
Scynexis, Inc., Jersey City, New Jersey, USA
M. Ghannoum
Center for Medical Mycology, Case Western Reserve University and University Hospitals Cleveland Medical Center, Cleveland, Ohio, USA

Notes

Address correspondence to M. Ghannoum, [email protected].

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