OBSERVATION
A large proportion of deaths caused by human fungal pathogens are related to infections caused by species of the genus
Aspergillus (
1). Up to today, the repertoire of antifungal agents for the treatment of aspergillosis is limited to three major drug classes with azole antifungals being the first-line treatment of choice (
2,
3). Over the past years, the worldwide distribution and emergence of azole resistance have raised concerns about their future clinical use (
4,
5), which stresses the demand for novel, potent antifungal agents with novel mechanisms of action. Several antifungals with promising anti-
Aspergillus activities are currently in clinical development including olorofim (previously named F901318) (
6–9), which is the first representative of the new antifungal drug class, the orotomides, that entered clinical phase III in 2022 (
https://clinicaltrials.gov/ct2/show/NCT05101187). Olorofim inhibits dihydroorotate dehydrogenase, a crucial enzyme in the
de novo biosynthesis of pyrimidines (
8). Previous work demonstrated that olorofim was highly active against major pathogenic
Aspergillus species including
Aspergillus fumigatus,
Aspergillus flavus, and
Aspergillus niger with mean minimum inhibitory concentration (MIC) levels <0.031 µg/mL. In addition to
Aspergillus spp., its spectrum of activity comprises various further filamentous and pathogenic dimorphic fungi (
8–10).
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 Susceptibility 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 concentrations. 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 antifungal effects also at low doses if drug levels fall below targeted therapeutic concentrations during standard dosing. However, it is important to note that population pharmacokinetic modeling (in-house data) has predicted that, with the standard dosing regimen, ≥94% of patients with invasive fungal infection (IFI) will have plasma concentrations 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.