Anti-Aspergillus activities of olorofim at sub-MIC levels during early-stage growth

ABSTRACT Olorofim, the first member of the novel class of antifungal drugs, the orotomides, shows promising anti-Aspergillus activity and is currently in phase III clinical development. Using high-throughput microscopy, we monitored olorofim’s antifungal potential at sub-minimum inhibitory concentration (MIC) levels with a focus on early-stage growth. Unlike voriconazole, olorofim showed significant growth inhibitory activities against three main pathogenic Aspergillus species, Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger, at concentrations >100,000-fold below its MIC. IMPORTANCE Among antifungal compounds in clinical development for systemic disease, the orotomide olorofim is one of only two that target a completely new mechanism of action. Olorofim is highly potent against pathogenic Aspergillus species including cryptic species that frequently show increased resistance to current agents. In this study, our primary focus was on evaluating in detail the inhibitory activity of voriconazole and olorofim against different pathogenic Aspergillus species employing high-throughput microscopy. Compared to standardized, less-sensitive visual assessment-based methods, microscopy-assisted growth monitoring allowed us to detect sub-MIC drug concentration ranges with significant inhibitory activity at early-stage growth. This revealed that olorofim exerts growth inhibition at concentrations that are several magnitudes below those of voriconazole.

In this work, we assessed in detail the antifungal activity of olorofim at early-stage growth and compared it to that of the first-line treatment agent voriconazole (2) with a focus on its inhibitory potential at sub-MIC level.For this, we carried out high-throughput microscopy to detect growth-based confluence values using the IncuCyte S3 Live-Cell Analysis System (Essen Bioscience Inc., Ann Arbor, MI, USA), which proved to be a powerful instrument for the detection of antifungal effects during the early growth (11).The analyses comprised three major pathogenic Aspergillus species A. fumigatus (ATCC 204305), A. flavus (ATCC 204304), and A. niger (ATCC 9029).GraphPad Prism 9 software (Dotmatics, Boston, MA, USA) was used to analyze and display results.All experiments were carried out in triplicate.While cultivation steps were performed following the broth microdilution reference method of the European Committee on Antimicrobial Suscepti bility Testing (12) using an inoculum of 1 × 10 5 conidia/mL, the monitored concentration ranges for voriconazole were 4 to 0.008 mg/L and 0.2 to 3.73 × 10 −10 mg/L for olorofim, as the drug showed activity far below its MIC.For microscopic analysis, strains were incubated for 12 h at 37°C.Growth was monitored in the absence (no drug control) and presence of serial dilutions of each drug, which allowed the determination of the inhibitory effects of each drug concentration.Percent growth reduction was calculated by normalizing the measured confluence values to the respective no-drug control.
Statistically significant (P < 0.05) growth reduction of all species was achieved at a concentration of 0.063 mg/L for voriconazole, whereby the highest growth reduction at this drug level was observed for A. fumigatus (78%) followed by A. flavus (46%) and A. niger (19%) (Table 1 displays growth rates as a % of no drug control; further details are provided in supplemental data 1).For olorofim concentrations, several magnitudes below its detected MIC significantly inhibited growth of each species; i.e., 2.98 × 10 −9 mg/L were required for A. fumigatus (12%) and 1.19 × 10 −8 mg/L and 1.91 × 10 −7 mg/L, respectively, for A. flavus (33%) and A. niger (38%).Due to the small activity range of voriconazole compared to olorofim (up to 4 and 23 serial dilutions below the MIC, respectively), this would not allow an adequate comparison of the two drugs' activities.Therefore, we further determined the smallest concentrations of each compound that led to severe growth reduction (>90%) (Fig. 1).This was achieved with voriconazole at 0.125 mg/L for A. fumigatus (96.0%), 0.5 mg/L for A. flavus (93.3%), and 0.25 mg/L for A. niger (94.3%).The minimal olorofim levels required were 3.05 × 10 −6 mg/L for A. fumigatus (93.3%) and 9.77 × 10 −5 mg/L for both A. flavus and A. niger (91.4% and 91.6%, respectively).Considering similar growth inhibition at these concentrations (<5% difference; 91.4-96.0%)with both drugs for all species, we further determined fold differences (molar ratios) employing the respective voriconazole and olorofim concen trations.The molar concentration ratios (voriconazole vs olorofim) were 58,467 for A. fumigatus (0.358 µM vs 6.12 × 10 −6 µM), 7,308 for A. flavus (1.431 µM vs 1.96 × 10 −4 µM), and 3,654 for A. niger (0.716 µM vs 1.96 × 10 −4 µM), which further emphasizes the large differences in the activities of the two drugs on early-stage growth inhibition.
In previous work, the inhibitory potential of olorofim against planktonic cells of A. fumigatus isolates at sub-MIC levels and different time points (4 h, 12 h, 24 h, and 48 h) has already been demonstrated (13), however at a comparably low concentration range (up to 3 serial dilutions below the MIC).Here, we monitored and demonstrated its potent anti-Aspergillus activity using an extended sub-MIC range (up to 27 serial dilutions below the MIC), which allowed us to determine the smallest concentration that led to significant growth inhibition of each Aspergillus species tested.In contrast to the first-line treatment agent voriconazole, the in vitro activity of which was restricted to a relatively small concentration range for the Aspergillus spp.tested in this work (2-4 serial dilutions below the MIC at 48 h), at 12 h olorofim significantly inhibited the growth of A. fumigatus, A. flavus, and A. niger, respectively, 23 (8,388,608-fold), 21 (2,097,152-fold), and 18 (262,144-fold) serial dilutions below the finally detected MIC at 48 h.The low olorofim concentrations that were required to almost fully inhibit growth of the different Aspergillus species (>90%) at 12 h re-enforce the requirement of hyphae for pyrimidines to facilitate active growth.
The observed sub-MIC effects suggest that olorofim may continue to exert a growth inhibitory effect at levels below established MIC thresholds.This and the previously observed post antifungal effects (14) are helpful properties of olorofim as a new antifungal agent and suggest, like previous work (13), that olorofim might have antifun gal effects also at low doses if drug levels fall below targeted therapeutic concentrations during standard dosing.However, it is important to note that population pharmacoki netic modeling (in-house data) has predicted that, with the standard dosing regimen, ≥94% of patients with invasive fungal infection (IFI) will have plasma concentra tions above the therapeutic threshold for 24 h a day, with ≥98% of patients exceeding the therapeutic threshold for over 20 h a day.Sub-MIC concentrations of antimicrobials are associated with an increased risk of resistance, particularly for antibacterials.Previously, exposure to sub-MIC concentrations of olorofim during serial passage experiments did not give rise to increased MICs after 40 passages (8).In contrast, there were significant increases in MICs of voriconazole after ~15 passages.Currently, on treatment, resistance to olorofim has not been seen clinically, although only a small number of IFI patients (n = 203) have been fully evaluated to date.

TABLE 1
Relative growth rates in the presence of serial dilutions olorofim and voriconazole a