Pharmacology

Efficacy of Olorofim (F901318) against Aspergillus fumigatus, A. nidulans, and A. tanneri in Murine Models of Profound Neutropenia and Chronic Granulomatous Disease

S. Seyedmousavi, Y. C. Chang, D. Law, M. Birch, J. H. Rex, K. J. Kwon-Chung
DOI: 10.1128/AAC.00129-19

ABSTRACT

The emergence of azole resistance in Aspergillus fumigatus as well as an increasing frequency of multiresistant cryptic Aspergillus spp. necessitates exploration of new classes of antifungals. Olorofim (formerly F901318) is a new fungicidal agent that prevents the growth of ascomycetous mold species via inhibition of de novo pyrimidine biosynthesis, a mechanism of action distinct from that of currently available antifungal drugs. We studied the in vivo efficacy of olorofim intraperitoneal therapy (15 mg/kg of body weight every 8 h for 9 days) against infection with A. fumigatus, A. nidulans, and A. tanneri in both neutropenic CD-1 mice and mice with chronic granulomatous disease (CGD) (gp91−/− phox mice). In the neutropenic mouse model, 80% to 88% of treated mice survived for 10 days, and in the CGD group, 63% to 88% of treated mice survived for 10 days, depending on the infecting species, while less than 10% of the mice in the control groups survived for 10 days. In the olorofim-treated groups, galactomannan levels were significantly suppressed, with lower organ fungal DNA burdens being seen for all three Aspergillus spp. Histopathological slides revealed a limited number of inflammatory foci with or without detectable fungal elements in the kidneys of neutropenic CD-1 mice and in the lungs of CGD mice. Furthermore, the efficacy of olorofim was unrelated to the triazole MICs of the infecting Aspergillus spp. These results show olorofim to be a promising therapeutic agent for invasive aspergillosis.

TEXT

Invasive aspergillosis (IA) is one of the most serious fungal diseases encountered in immunocompromised patients worldwide (1, 2). The patient populations at the highest risk for IA are those with prolonged neutropenia from intensive myeloablative chemotherapy and those with inborn errors of immunity that impair key innate anti-Aspergillus host defense pathways, such as chronic granulomatous disease (CGD) (3, 4). The primary etiologic agent of IA in both neutropenic and CGD patient populations is Aspergillus fumigatus (5–7). CGD patients may also suffer from infections due to less common species, such as A. nidulans (8) and A. tanneri (9).

Azole antifungals, such as voriconazole (VRC), posaconazole (POS), and isavuconazole, are currently recommended first-choice drugs for the treatment and prophylaxis of aspergillosis (10, 11). However, the management of IA has become more complicated due to the emergence of azole resistance in A. fumigatus (12). In addition, IA remains the primary cause of morbidity and mortality in CGD patients and is often due to non-fumigatus Aspergillus spp., infections with which are harder to diagnose and may require longer durations of therapy (9, 13–16).

Alternative treatments with a new class of antifungals with a novel mechanism of action may improve therapeutic outcomes in patients with IA, even for those cases that are caused by Aspergillus spp. that are refractory to currently available antifungals (17). Olorofim (formerly F901318) is a new class of antifungal, the orotomides, that inhibits de novo pyrimidine biosynthesis by preventing the catalytic activity of dihydroorotate dehydrogenase (DHODH) (18). Olorofim demonstrates potent inhibitory activity against Aspergillus spp. in vitro, including azole-resistant isolates of A. fumigatus (19). The potent activity of olorofim has also been demonstrated in experimental animal models of disseminated infections caused by A. fumigatus (20) and A. flavus (21). In the present study, we aimed to assess the in vivo efficacy of olorofim against A. fumigatus and two other Aspergillus spp., A. nidulans and A. tanneri, which are either more refractory to or nonresponsive to currently available antifungal treatment (16).

(A preliminary version of this article was presented at the 8th Advances against Aspergillosis Conference [AAA2018], 1 to 3 February 2018, Lisbon, Portugal [22].)

RESULTS

In vitro antifungal drug susceptibilities of Aspergillus spp.Table 1 shows the antifungal susceptibility profiles of seven Aspergillus spp. that have been documented from CGD patients with cases of IA (16). Olorofim showed potent and consistent in vitro activity against all seven Aspergillus spp. tested. The highest MICs of the majority of antifungals, including the triazoles (itraconazole [ITC], VRC, POS) and the polyene (amphotericin B [AMB]), were demonstrated for A. tanneri NIH1004. The olorofim MIC ranges for the seven clinical Aspergillus spp. (0.008 to 0.062 μg/ml) were the lowest among the MIC ranges of the antifungals tested, including those of AMB (0.5 to >16 μg/ml), ITC (0.5 to 4 μg/ml), VRC (0.25 to 4 μg/ml), POS (0.125 to 1 μg/ml), and terbinafine (0.25 to 4 μg/ml) (Table 1).

View this table:
TABLE 1

MICs of six antifungals for Aspergillus species

PK of olorofim.To determine the pharmacokinetics (PK) of intraperitoneally administered olorofim, a total of 60 samples from 60 mice (2 mice per time point, 6 time points, 5 different dosages) were analyzed following administration of single doses of olorofim. All 60 mice were alive at the time of sample collection. The corresponding pharmacokinetic parameters are tabulated in Table 2. We found that all the dosing regimens were well tolerated. The pharmacokinetics were linear in the range of 2.5 mg/kg of body weight to 20 mg/kg, and the area under the concentration-time curve (AUC) correlated significantly with the dose in a linear fashion (R = 0.96). The time after administration that the maximum plasma concentration (Cmax) was reached (Tmax) occurred rapidly, within 0.5 h of dosing, between doses of 2.5 mg/kg and 15 mg/kg. The minimum plasma concentration (Cmin) values at the 10-, 15-, and 20-mg/kg doses were above the level (100 to 300 ng/ml) required for efficacy seen in other murine models (20, 21).

View this table:
TABLE 2

Pharmacokinetic parameters of olorofim in CD-1 mice

Olorofim therapy is active in neutropenic CD-1 mice infected with all three Aspergillus spp.In the neutropenic CD-1 mouse model, olorofim therapy (15 mg/kg every 8 h [q8h]) significantly improved survival compared to that in the untreated controls; 81%, 88%, and 80% of treated mice survived infection by A. fumigatus, A. nidulans, and A. tanneri, respectively (Fig. 1A1, B1, and C1). In contrast, less than 10% of the mice in the control groups (mice treated with phosphate-buffered saline [PBS]) survived IA within 10 days postinfection. Olorofim therapy suppressed galactomannan (GM) levels (Fig. 1A2, B2, and C2) and also significantly reduced the fungal DNA burden of the kidney throughout the experimental period (Fig. 1A3, B3, and C3). Histopathology of kidney sections prepared from the olorofim-treated CD-1 mice at 3 days postinfection showed no or a limited number of fungal filaments (Fig. 1A4, B4, and C4, right images, top and bottom) and a few inflammatory foci (data not shown). In contrast, the kidneys of PBS-treated control CD-1 mice showed abundant fungal filaments (Fig. 1A4, B4, and C4, left images, top and bottom) with severe inflammatory infiltrations and prominent necrosis (data not shown). Thus, olorofim treatment of neutropenic mice controlled the infections caused by all three Aspergillus spp.

FIG 1
FIG 1

Efficacy of olorofim against A. fumigatus, A. nidulans, and A. tanneri in CD-1 mice. The CD-1 mice were treated for 9 days with either 15 mg/kg olorofim or PBS every 8 h via intraperitoneal administration, starting at 6 h postinfection (i.v.) with A. fumigatus (1 × 104 conidia/mouse) (A), A. nidulans (2 × 104 conidia/mouse) (B), and A. tanneri (1 × 106 conidia/mouse) (C). (A1, B1, C1) The survival of the mice (n = 11) was monitored for 10 days. (A2, B2, C2) On day 3 postinfection, blood samples were collected from olorofim-treated and untreated mice (n = 3) to measure galactomannan (GM) in centrifuged sera. GM levels were also determined from olorofim-treated surviving mice at day 9 (n = 3). (A3, B3, C3) In addition, kidneys were harvested and fungal burdens were estimated by measuring fungal DNA by qPCR. ****, P ≤ 0.0001; *, P ≤ 0.05. (A4, B4, C4) GMS-stained histology sections were prepared on day 3 postinfection. (Top and bottom left) Representative histological sections of vehicle-treated control mice; (top and bottom right) representative histological sections of olorofim-treated mice. Bars, 1 mm (A4, B4, and C4, top) and 20 μm (A4, B4, and C4, bottom); magnifications, ×25 (A4, B4, and C4, top) and ×400 (A4, B4, and C4, bottom).

Olorofim is active in CGD mice infected with all three species.To examine the activity of olorofim in a CGD mouse model of IA against infection with all three Aspergillus spp., the same regimen of olorofim used in neutropenic CD-1 mice was used: intraperitoneal administration of olorofim (15 mg/kg) three times daily. As shown in Fig. 2A1, B1, and C1, 88%, 75%, and 63% of the mice dosed with olorofim were still alive on day 10 postinfection with A. fumigatus, A. nidulans, and A. tanneri, respectively. In the control group, however, less than 40% of the mice survived infection with A. fumigatus and A. nidulans and no mice infected with A. tanneri survived during the same period. Therefore, olorofim therapy was also efficacious in treating CGD mice with IA caused by the three Aspergillus spp. The day 3 GM levels were significantly higher in the untreated control CGD mice than in those mice receiving olorofim treatment (Fig. 2A2, B2, and C2). Similarly, fungal DNA loads were substantially higher in the lungs of the control group mice than in the lungs of the olorofim-dosed mice (Fig. 2A3, B3, and C3), suggesting that the growth of Aspergillus in host tissue was hampered in vivo by olorofim therapy. Lung sections of the CGD mice in the control group prepared on day 3 postinfection showed extensive granulomas with necrotic debris and hyphae of all three Aspergillus spp. (Fig. 2A4, B4, and C4, top images). The mice treated with olorofim showed significantly reduced granulomas (Fig. 2A4, B4, and C4, bottom images), and only a few fungal elements were scattered throughout the pulmonary architecture (data not shown), suggesting that olorofim treatment controlled pulmonary infection caused by these aspergilli in CGD mice.

FIG 2
FIG 2

Efficacy of olorofim against A. fumigatus, A. nidulans, and A. tanneri in CGD mice. CGD mice were treated for 9 days with either 15 mg/kg olorofim or PBS every 8 h via intraperitoneal administration, starting at 6 h postinfection with A. fumigatus (5 × 103 conidia/mouse) (A), A. nidulans (5 × 103 conidia/mouse) (B), and A. tanneri (2.5 × 106 conidia/mouse) (C), aspirated from the pharynx. (A1, B1, C1) The survival of mice (n = 11) was monitored for 10 days. (A2, B2, C2) On day 3 postinfection, blood samples were collected from olorofim-treated and untreated mice (n = 3) to measure galactomannan (GM) in centrifuged sera. GM levels were also determined from olorofim-treated mice that survived at day 9 (n = 3). (A3, B3, C3) Lungs from infected mice were taken at 3 days postinfection (n = 3), and fungal burdens were estimated. **, P ≤ 0.01; ***, P ≤ 0.001. (A4, B4, C4) Montaged images of H&E-stained lung sections taken at 3 days postinfection show the size and frequency of infectious foci in control mice (top) and olorofim-treated mice (bottom). Bars, 2 mm; magnifications, ×25.

Comparative efficacy of olorofim versus VRC against A. fumigatus, A. nidulans, and A. tanneri.VRC is currently recommended as the first-choice treatment for IA (10). We compared the therapeutic efficacy between VRC and olorofim in CGD mice challenged with three Aspergillus species in multiple independent experiments. Treatment with VRC resulted in 100% survival of mice infected with A. fumigatus and relatively moderate antifungal efficacy (60% survival) against A. nidulans, whereas the rate of survival of vehicle-treated controls was less than 10% (Fig. 3A1 and B1). However, all A. tanneri-infected mice receiving VRC and untreated control mice succumbed to infection at similar rates (Fig. 3C1), indicating that A. tanneri is intrinsically resistant to VRC. In contrast, treatment of CGD mice with olorofim resulted in 80% survival in A. fumigatus-, A. nidulans-, and A. tanneri-infected animals. Of note, the survival rate in A. tanneri-infected CGD mice receiving olorofim (80% survival) was slightly higher than that mentioned above (63% survival) (Fig. 2C1). In histopathology sections of the lungs prepared on day 3 postinfection from mice treated with VRC or olorofim, no granulomatous foci or fungal elements were detected in the A. fumigatus-infected group (Fig. 3A3 and A4), and only mild infection with few granulomatous foci (Fig. 3B3 and B4) was found in the A. nidulans-infected group. In the A. tanneri-infected mice, however, numerous granulomatous foci containing many fungal elements were observed in both the VRC-treated mice and the untreated controls (Fig. 3C2 and C4), while only a few small lesions were observed in the lungs of olorofim-treated animals (Fig. 3C3). These results suggest that olorofim but not VRC reduced the growth of A. tanneri in the lungs of CGD mice.

FIG 3
FIG 3

Efficacy of olorofim versus voriconazole against A. fumigatus, A. nidulans, and A. tanneri. CGD mice were randomized into groups of 14, and the efficacy of either olorofim (total daily dose, 45 mg/kg of body weight) or VRC (total daily dose, 20 mg/kg of body weight) treatment was assessed by their survival rate for 10 days and histopathological analysis. (A1, B1, C1) Survival curves of vehicle (PBS)-treated controls versus olorofim- or VRC-treated animals infected with A. fumigatus, A. nidulans, and A. tanneri, respectively. (A2, B2, C2) Representative histological sections of lungs of vehicle-treated mice infected with A. fumigatus, A. nidulans, and A. tanneri, respectively. (A3, B3, C3) Representative histological sections of lungs of olorofim-treated animals infected with A. fumigatus, A. nidulans, and A. tanneri, respectively. (A4, B4, C4) Representative histological sections of VRC-treated animals infected with A. fumigatus, A. nidulans, and A. tanneri, respectively. Bars, 2 mm; magnifications, ×25.

DISCUSSION

Olorofim is currently an investigational compound under clinical development. It belongs to the orotomides, a new class of antifungal antibiotics with a new mechanism of action targeting DHODH, an enzyme essential in the pathway of de novo pyrimidine biosynthesis (18). This is a novel target, and the compound has shown potent in vitro and in vivo activity against several medically important molds, including several Aspergillus spp. (19–21, 23), the Scedosporium/Pseudallescheria species complex and Lomentospora spp. (24, 25), certain species of Fusarium, Penicillium spp., and Talaromyces marneffei (18), as well as the dimorphic human pathogens Coccidioides spp. (26).

We showed that olorofim controls the growth of seven Aspergillus spp. that have been documented from CGD patients with cases of IA, in contrast to several other commonly used antifungals. More importantly, olorofim therapy was effective in reducing mortality, pathology, GM levels, and fungal DNA loads in two different murine models of IA caused by three different Aspergillus spp.: A. fumigatus, A. nidulans, and A. tanneri. The efficacy of olorofim was unrelated to the Aspergillus species tested, the triazole MIC, or the nature of the mouse immune status. This is of significant importance, since the prevalence of azole-resistant A. fumigatus is increasing (27), and the emergence of drug-resistant non-fumigatus Aspergillus spp. as a cause of IA in CGD patients is a growing concern (16).

CGD is a rare inherited immunodeficiency disorder characterized by specific defects in the NADP (NADPH)-oxidase complex. Subjects with CGD are at a significant risk of aspergillosis. A. fumigatus is the primary etiologic agent (estimated to be ∼55%), followed by A. nidulans (∼35%), a species which is almost exclusively associated with IA in CGD patients (5). Various less well known species, such as A. fumisynnematus, A. quadrilineatus, A. pseudoviridinutans, A. subramanianii, A. udagawae, and A. tanneri, are infrequent causes of infection, with some of these species almost exclusively being reported from CGD patients. Among these Aspergillus spp., several species, including A. tanneri, consistently demonstrate high MICs compared to the proposed epidemiological cutoff values for wild-type A. fumigatus and A. nidulans (see Table S2 in the supplemental material) (28, 29), and the MIC values observed for our collection of clinical isolates are consistent with published experience. Although VRC is currently the recommended first-choice treatment for IA (10), the drug showed no therapeutic efficacy against infections caused by some of these emerging Aspergillus spp. (16). Since olorofim demonstrated potent in vivo efficacy against A. tanneri, we predict that olorofim will be effective in treating IA caused by other Aspergillus spp. known to be refractory to currently available therapy.

In the current study, the absorption and distribution of olorofim into the plasma of mice produced good pharmacokinetic profiles following intraperitoneal administration. Our results are consistent with those of a recent study in which intravenously (i.v.) administered olorofim displayed linear pharmacokinetics when it was administered at doses over the range of 4 to 15 mg/kg q8h, and all the dosing regimens were consistently well tolerated (20, 21). Our pharmacokinetics study also showed a reasonable dose proportionality between 2.5 mg/kg and 20 mg/kg. Of note, we measured the PK with a single dose administered by intraperitoneal injection, and there was a limitation since it is unknown if the PK would be linear following multiple administrations. However, the linearity of the PK parameters following multiple i.v. injections of olorofim has already been shown in two previously published studies (20, 21).

In the present study, the intraperitoneal dosing regimen used (administration of 15 mg/kg olorofim three times daily) demonstrated clear efficacy in both neutropenic and CGD mouse models of IA, indicating that olorofim may have reached the much higher trough levels required for effective drug exposure in both the i.v. and intraperitoneal dosing regimens. Similarly, the exposure-response relationships of olorofim have been defined in immunosuppressed murine and rabbit models of A. fumigatus infection, for which a very strong relationship between the pharmacokinetic (PK)/pharmacodynamic (PD) index Cmin/MIC ratio and treatment outcome was observed. A Cmin/MIC ratio of 9.1 was most closely associated with efficacy against both triazole-susceptible and triazole-resistant A. fumigatus strains (20). In that study, treatment of mice with an olorofim regimen of 15 mg/kg q8h i.v. significantly increased the rate of survival at 10 days postinfection (20). The efficacy of olorofim has also been investigated in an immunosuppressed murine model of acute sinopulmonary infection caused by four clinical A. flavus isolates (MIC range, 0.015 to 0.06 μg/ml) (21). Olorofim therapy demonstrated impressive antifungal activity against A. flavus infection, leading to prolonged survival and a concentration-dependent decline in circulating GM levels. There was also histological clearance of lung tissue that was consistent with the effects of olorofim on the GM levels. A nearly maximal reduction of all A. flavus strains used to challenge the mice was observed in mice receiving 15 mg/kg q8h. The Cmin/MIC values of 9 to 19 (mean, 13.38) were equivalent to or exceeded the efficacy observed by the triazole POS (AUC = 47 ng · h/ml) at the upper boundary of its expected human exposures (21).

Overall, our data presented here indicate that olorofim is efficacious in the treatment of invasive aspergillosis in both profoundly neutropenic and CGD murine models. Because the MICs of olorofim are consistently low and its mechanism of action is entirely different from the mechanisms of action of currently available antifungals, it is predicted that the drug will be effective against many Aspergillus spp. refractory to the commonly used antifungals. As support for such a prediction, we showed the efficacy of olorofim therapy against two azole-resistant species, A. nidulans and A. tanneri. In conclusion, our data provide additional promising results which suggest the value of olorofim for clinical application to treat IA in CGD patients.

MATERIALS AND METHODS

Fungal strains.Isolates of seven pathogenic Aspergillus spp. obtained from patients with disseminated aspergillosis were used (Table 1). A. fumigatus B5233 (30), A. nidulans M24 (30), and A. tanneri NIH1004 (9) were used for both in vitro and in vivo studies. A. fumisynnematus (CFN1891), A. pseudoviridinutans (NIHAV1), A. subramanianii (DI 16-475), and A. udagawae (F41) were also clinical isolates, and they were used only for the in vitro susceptibility tests. The isolates were stored in 10% glycerol at −80°C and were revived on malt extract agar (MEA) at 37°C for 5 to 7 days. All isolates were freshly cultured on MEA for 5 to 7 days at 37°C (up to 14 days at 30°C for A. tanneri) for the preparation of conidial suspensions.

In vitro antifungal susceptibility testing.In vitro antifungal susceptibility testing was performed for isolates of the seven Aspergillus spp. reported from CGD patients (Table 1) by using the Clinical and Laboratory Standards Institute (CLSI) reference method for broth dilution antifungal susceptibility testing of filamentous fungi (31). Drugs were provided by F2G Ltd. (olorofim) or purchased from Sigma (all other agents; St. Louis, MO). The MIC was defined as the lowest concentration that completely inhibited growth in comparison to the growth in the drug-free well (control), as assessed by visual inspection. All incubations were performed in three independent replicates on different days.

Animals and husbandry.The efficacy of olorofim monotherapy was determined in CD-1 mice (32) (4- to 5-week-old females) from Charles River Laboratories and in gp91−/− phox CGD mice (33) (6- to 7-week-old males) from The Jackson Laboratory, USA. Animals were randomized into groups of 17, and the efficacy of olorofim treatment was assessed by measurement of serum galactomannan (GM) using a Platelia Aspergillus GM enzyme immunoassay (EIA; Bio-Rad Laboratories), fungal DNA burdens in the kidneys (CD-1 mice) or lungs (CGD mice) using quantitative real-time PCR (qPCR), and survival to day 10 by the log-rank test and by histopathological analysis. Animals were housed under standard conditions, with drink and feed supplied ad libitum. All experiments for each strain were performed using at least two independent replicates. To avoid selection bias, animals were preassigned at the time of infection to groups to be assessed for survival rate, fungal load, and histopathology.

Animal infection models.It is known that invasive aspergillosis primarily occurs in patients with secondary immunodeficiencies in the form of disseminated pulmonary complications. However, several Aspergillus species have been known to affect only patients with CGD, a form of primary immune deficiency. We therefore investigated the efficacy of olorofim against Aspergillus in two different murine models, the neutropenic and CGD murine models. For the neutropenic model, we chose the tail vein infection method, which has been described previously (20). For the CGD model, the aspiration technique was chosen to mimic the common route of infection. Neutropenic CD-1 mice were infected via the tail vein with 100 μl of a freshly prepared conidial suspension corresponding to the 90% lethal dose of each species (1 × 104 CFU/mouse for A. fumigatus, 2 × 104 CFU/mouse for A. nidulans, and 1 × 106 CFU/mouse for A. tanneri). To render the CD-1 mice neutropenic, cyclophosphamide (150 mg/kg on days −4 and +4 and 100 mg/kg on day −1) was administered as described previously (20). CGD mice were infected with 30 μl a freshly harvested Aspergillus conidial suspension (5 × 103 CFU/mouse for A. fumigatus and A. nidulans and 2.5 × 106 CFU/mouse for A. tanneri) by the intrapharyngeal aspiration technique, as described previously (34). In all survival studies, experienced individuals blind to the animal treatment monitored the infected mice at least twice daily.

Study drug and dosing regimen.For the in vivo studies, olorofim intraperitoneal therapy was started 6 h after infection with a total daily dose of 45 mg/kg of body weight (15 mg/kg every 8 h) for 9 days. To prepare working concentrations, the desired amount of drug was weighed, added to dimethyl sulfoxide (DMSO), and vortexed until it was fully dissolved. Subsequently, polyethylene glycol 400 (PEG 400) was added and the mixture was vortexed. A solution of 35.3% hydroxylpropyl cyclodextrin (HPBCD) was prepared in water. The solution of olorofim in DMSO-PEG 400 and the HPBCD solution were mixed to give a clear solution. Appropriate volumes of olorofim solutions were prepared and stored at −20°C for daily use. Prior to use, drug-containing vials were fully thawed and vortexed. The control mice received phosphate-buffered saline (PBS) intraperitoneally. A commercially available, pharmaceutical-grade formulation of VRC (Vfend; Pfizer, USA) was purchased from a local pharmacy and used as a positive control in some experiments. An intraperitoneal dose of VRC of 20 mg/kg/body weight per day had previously been demonstrated to yield the maximum effect (100% survival) in a murine model of IA (35). The efficacy of olorofim treatment was assessed by monitoring of survival for 10 days and measurement of serum galactomannan, tissue histopathology, and the fungal burdens in the kidneys (CD-1 mice) or lungs (CGD mice) at 3 days postinfection. The establishment of a time point of 3 days postinfection for sampling allowed us to ensure accurate and timely data collection before animals started to die or be euthanized to prevent unnecessary animal pain and distress due to the Aspergillus infection.

Galactomannan enzyme immunoassay.The Bio-Rad Laboratories Platelia Aspergillus enzyme immunoassay was used to detect GM in centrifuged serum samples according to the manufacturer's instructions, and the results are reported as the galactomannan index (GMI).

Determination of fungal burden.On the days postinfection indicated above, DNA was extracted from the kidneys (CD-1 mice) or lungs (CGD mice) using a Fast DNA Spin kit (MP Biomedicals, Santa Ana, CA) as described previously (36). The concentration of total DNA isolated from the lungs was measured by use of a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Aspergillus loads were determined by quantitative real-time (qPCR) using primers and a probe (6-carboxyfluorescein labeled) targeting the 28S rRNA-ITS2 region of the ribosomal subunit gene of each Aspergillus sp., as described previously (36). The sequences of the primers and probes used for qPCR assays are shown in Table S1 in the supplemental material. The qPCRs were run with 250 ng of total DNA isolated from the kidneys or lungs of infected mice. To determine the Aspergillus load in each organ sample (in picograms per nanogram of total DNA isolated), the fungal DNA concentration was calculated from a standard curve derived from an 8-fold dilution series of the genomic DNA of each Aspergillus sp.

Histopathological analysis.The kidneys (CD-1 mice) and lungs (CGD mice) were harvested from the surviving animals for histopathological analysis using hematoxylin and eosin (H&E) and Gomori's methenamine-silver (GMS) staining.

Pharmacokinetic analysis of intraperitoneal olorofim administration in mice.Outbred CD-1 female mice (age, 4 to 5 weeks; weight, 20 to 22 g) were used for the PK experiments. Cyclophosphamide-immunosuppressed mice were infected with the A. fumigatus B5233 isolate through the lateral tail vein, and after 6 h, single doses of olorofim were given at 2.5, 5, 10, 15, and 20 mg/kg of body weight intraperitoneally. Blood samples were drawn from the mice for each individual predefined sampling time point (6 time points in total): immediately before administration of drugs (0.0 h) and subsequently at 0.5, 1, 2, 4, and 8 postdose. The blood samples were drawn through the orbital vein or by heart puncture and placed into lithium-heparin-containing tubes, cooled, and centrifuged for approximately 10 min at 1,000 × g within 30 min of collection. Plasma was aspirated, transferred to two 2-ml plastic tubes, and stored at −80°C until future analysis. The olorofim concentrations in plasma were measured by a high-performance liquid chromatography–mass spectrometry (HPLC-MS) method using a Kinetex 2.6-μm XB-C18 100-Å 50- by 2.1-mm column (Phenomenex, Cheshire, UK) and a 5-μl injection volume. A standard curve encompassing 0.01 to 20 mg/liter was constructed from stock solutions of olorofim at 1,000 mg/liter in DMSO that were further diluted in methanol (Fisher Scientific, Loughborough, United Kingdom). Details of the analytical assay have been described elsewhere (20).

The corresponding pharmacokinetic parameters (area under the concentration-time curve from time zero to 8 h postinfection [AUC0–8], the maximum plasma concentration [Cmax], the minimum plasma concentration [Cmin], and the time after administration that the maximum plasma concentration was reached [Tmax]) were calculated. Peak concentrations in serum were directly observed from the data. The area under the plasma concentration-time curve from time zero to 8 h postinfection was determined by use of the log-linear trapezoidal rule. The elimination rate constant was determined by linear regression of the terminal points of the log-linear plasma concentration-time curve. The terminal half-life was defined as ln2 divided by the elimination rate constant.

Ethics statement.The Institutional Animal Care and Use Committee of the National Institute of Allergy and Infectious Diseases approved all animal studies (LCIM-5E). Studies were performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Research Council (37).

Statistical analysis.All data analyses were performed using GraphPad Prism (version 7) software (GraphPad Software, San Diego, CA). The observed plasma concentrations, GM levels, and fungal DNA loads were log10 transformed (if necessary) to approximate a normal distribution prior to statistical analysis. Mortality data were analyzed by the log-rank test. Student's t test and two-way analysis of variance (ANOVA) followed by a multiple-comparison test were used to define whether there were significant differences between indicated samples. Statistical significance for comparisons was defined as a P value of ≤0.05 (two-tailed).

ACKNOWLEDGMENTS

This work was supported by the intramural program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. Olorofim (F901318) powder was provided by F2G Ltd.

S.S., Y.C., and K.J.K.-C. declare no conflict of interests related to this publication. D.L, M.B., and J.H.R. are employed by and own stock in F2G Ltd.

FOOTNOTES

    • Received 24 January 2019.
    • Returned for modification 17 February 2019.
    • Accepted 14 March 2019.
    • Accepted manuscript posted online 18 March 2019.

  • Supplemental material for this article may be found at https://doi.org/10.1128/AAC.00129-19.

REFERENCES

  1. 1.↵
    1. Segal BH,
    2. Romani LR
    . 2009. Invasive aspergillosis in chronic granulomatous disease. Med Mycol 47:S282–S290. doi:10.1080/13693780902736620.
    OpenUrlCrossRefPubMed
  2. 2.↵
    1. Brown GD,
    2. Denning DW,
    3. Gow NA,
    4. Levitz SM,
    5. Netea MG,
    6. White TC
    . 2012. Hidden killers: human fungal infections. Sci Transl Med 4:165rv13. doi:10.1126/scitranslmed.3004404.
    OpenUrlFREE Full Text
  3. 3.↵
    1. Segal BH,
    2. Leto TL,
    3. Gallin JI,
    4. Malech HL,
    5. Holland SM
    . 2000. Genetic, biochemical, and clinical features of chronic granulomatous disease. Medicine (Baltimore) 79:170–200. doi:10.1097/00005792-200005000-00004.
    OpenUrlCrossRefPubMedWeb of Science
  4. 4.↵
    1. Segal BH,
    2. Holland SM
    . 2000. Primary phagocytic disorders of childhood. Pediatr Clin North Am 47:1311. doi:10.1016/S0031-3955(05)70273-X.
    OpenUrlCrossRefPubMedWeb of Science
  5. 5.↵
    1. Henriet S,
    2. Verweij PE,
    3. Holland SM,
    4. Warris A
    . 2013. Invasive fungal infections in patients with chronic granulomatous disease. Adv Exp Med Biol 764:27–55.
    OpenUrlPubMed
  6. 6.↵
    1. Lanternier F,
    2. Cypowyj S,
    3. Picard C,
    4. Bustamante J,
    5. Lortholary O,
    6. Casanova JL,
    7. Puel A
    . 2013. Primary immunodeficiencies underlying fungal infections. Curr Opin Pediatr 25:736–747. doi:10.1097/MOP.0000000000000031.
    OpenUrlCrossRefPubMed
  7. 7.↵
    1. Antachopoulos C
    . 2010. Invasive fungal infections in congenital immunodeficiencies. Clin Microbiol Infect 16:1335–1342. doi:10.1111/j.1469-0691.2010.03289.x.
    OpenUrlCrossRefPubMedWeb of Science
  8. 8.↵
    1. Segal BH,
    2. DeCarlo ES,
    3. Kwon-Chung KJ,
    4. Malech HL,
    5. Gallin JI,
    6. Holland SM
    . 1998. Aspergillus nidulans infection in chronic granulomatous disease. Medicine (Baltimore) 77:345–354. doi:10.1097/00005792-199809000-00004.
    OpenUrlCrossRefPubMedWeb of Science
  9. 9.↵
    1. Sugui JA,
    2. Peterson SW,
    3. Clark LP,
    4. Nardone G,
    5. Folio L,
    6. Riedlinger G,
    7. Zerbe CS,
    8. Shea Y,
    9. Henderson CM,
    10. Zelazny AM,
    11. Holland SM,
    12. Kwon-Chung KJ
    . 2012. Aspergillus tanneri sp. nov., a new pathogen that causes invasive disease refractory to antifungal therapy. J Clin Microbiol 50:3309–3317. doi:10.1128/JCM.01509-12.
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    1. Patterson TF,
    2. Thompson GR, III,
    3. Denning DW,
    4. Fishman JA,
    5. Hadley S,
    6. Herbrecht R,
    7. Kontoyiannis DP,
    8. Marr KA,
    9. Morrison VA,
    10. Nguyen MH,
    11. Segal BH,
    12. Steinbach WJ,
    13. Stevens DA,
    14. Walsh TJ,
    15. Wingard JR,
    16. Young JA,
    17. Bennett JE
    . 2016. Practice guidelines for the diagnosis and management of aspergillosis: 2016 update by the Infectious Diseases Society of America. Clin Infect Dis 2016 63:e1–e60. doi:10.1093/cid/ciw326.
    OpenUrlCrossRefPubMed
  11. 11.↵
    1. Ullmann AJ,
    2. Aguado JM,
    3. Arikan-Akdagli S,
    4. Denning DW,
    5. Groll AH,
    6. Lagrou K,
    7. Lass-Florl C,
    8. Lewis RE,
    9. Munoz P,
    10. Verweij PE,
    11. Warris A,
    12. Ader F,
    13. Akova M,
    14. Arendrup MC,
    15. Barnes RA,
    16. Beigelman-Aubry C,
    17. Blot S,
    18. Bouza E,
    19. Bruggemann RJM,
    20. Buchheidt D,
    21. Cadranel J,
    22. Castagnola E,
    23. Chakrabarti A,
    24. Cuenca-Estrella M,
    25. Dimopoulos G,
    26. Fortun J,
    27. Gangneux JP,
    28. Garbino J,
    29. Heinz WJ,
    30. Herbrecht R,
    31. Heussel CP,
    32. Kibbler CC,
    33. Klimko N,
    34. Kullberg BJ,
    35. Lange C,
    36. Lehrnbecher T,
    37. Loffler J,
    38. Lortholary O,
    39. Maertens J,
    40. Marchetti O,
    41. Meis JF,
    42. Pagano L,
    43. Ribaud P,
    44. Richardson M,
    45. Roilides E,
    46. Ruhnke M,
    47. Sanguinetti M,
    48. Sheppard DC,
    49. Sinko J,
    50. Skiada A
    , et al. 2018. Diagnosis and management of Aspergillus diseases: executive summary of the 2017 ESCMID-ECMM-ERS guideline. Clin Microbiol Infect 24:e1–e38. doi:10.1016/j.cmi.2018.01.002.
    OpenUrlCrossRef
  12. 12.↵
    1. Seyedmousavi S,
    2. Verweij PE
    . 2017. Azole resistance in Aspergillus fumigatus: mechanisms, route of resistance selection and clinical implications, p 403–421. In Gotte M, Berghuis A, Matlashewski G, Wainberg MA, Sheppard D (ed), Handbook of antimicrobial resistance. Springer, New York, NY.
  13. 13.↵
    1. Vinh DC,
    2. Shea YR,
    3. Jones PA,
    4. Freeman AF,
    5. Zelazny A,
    6. Holland SM
    . 2009. Chronic invasive aspergillosis caused by Aspergillus viridinutans. Emerg Infect Dis 15:1292–1294. doi:10.3201/eid1508.090251.
    OpenUrlCrossRefPubMed
  14. 14.↵
    1. Vinh DC,
    2. Shea YR,
    3. Sugui JA,
    4. Parrilla-Castellar ER,
    5. Freeman AF,
    6. Campbell JW,
    7. Pittaluga S,
    8. Jones PA,
    9. Zelazny A,
    10. Kleiner D,
    11. Kwon-Chung KJ,
    12. Holland SM
    . 2009. Invasive aspergillosis due to Neosartorya udagawae. Clin Infect Dis 49:102–111. doi:10.1086/599345.
    OpenUrlCrossRefPubMedWeb of Science
  15. 15.↵
    1. Natarajan M,
    2. Hsu AP,
    3. Weinreich MA,
    4. Zhang Y,
    5. Niemela JE,
    6. Butman JA,
    7. Pittaluga S,
    8. Sugui J,
    9. Collar AL,
    10. Lim JK,
    11. Zangeneh T,
    12. Carr T,
    13. Oler AJ,
    14. Similuk M,
    15. Rosen LB,
    16. Desai JV,
    17. Freeman AF,
    18. Holland SM,
    19. Kwon-Chung KJ,
    20. Milner JD,
    21. Lionakis MS
    . 2018. Aspergillosis, eosinophilic esophagitis, and allergic rhinitis in signal transducer and activator of transcription 3 haploinsufficiency. J Allergy Clin Immunol 142:993–997.e3. doi:10.1016/j.jaci.2018.05.009.
    OpenUrlCrossRef
  16. 16.↵
    1. Seyedmousavi S,
    2. Lionakis MS,
    3. Parta M,
    4. Peterson SW,
    5. Kwon-Chung KJ
    . 2018. Emerging Aspergillus species almost exclusively associated with primary immunodeficiencies. Open Forum Infect Dis 5:ofy213. doi:10.1093/ofid/ofy213.
    OpenUrlCrossRef
  17. 17.↵
    1. Seyedmousavi S,
    2. Rafati H,
    3. Ilkit M,
    4. Tolooe A,
    5. Hedayati MT,
    6. Verweij P
    . 2017. Systemic antifungal agents: current status and projected future developments. Methods Mol Biol 1508:107–139. doi:10.1007/978-1-4939-6515-1_5.
    OpenUrlCrossRef
  18. 18.↵
    1. Oliver JD,
    2. Sibley GE,
    3. Beckmann N,
    4. Dobb KS,
    5. Slater MJ,
    6. McEntee L,
    7. Du Pre S,
    8. Livermore J,
    9. Bromley MJ,
    10. Wiederhold NP,
    11. Hope WW,
    12. Kennedy AJ,
    13. Law D,
    14. Birch M
    . 2016. F901318 represents a novel class of antifungal drug that inhibits dihydroorotate dehydrogenase. Proc Natl Acad Sci U S A 113:12809–12814. doi:10.1073/pnas.1608304113.
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    1. Buil JB,
    2. Rijs A,
    3. Meis JF,
    4. Birch M,
    5. Law D,
    6. Melchers WJG,
    7. Verweij PE
    . 2017. In vitro activity of the novel antifungal compound F901318 against difficult-to-treat Aspergillus isolates. J Antimicrob Chemother 72:2548–2552. doi:10.1093/jac/dkx177.
    OpenUrlCrossRef
  20. 20.↵
    1. Hope WW,
    2. McEntee L,
    3. Livermore J,
    4. Whalley S,
    5. Johnson A,
    6. Farrington N,
    7. Kolamunnage-Dona R,
    8. Schwartz J,
    9. Kennedy A,
    10. Law D,
    11. Birch M,
    12. Rex JH
    . 2017. Pharmacodynamics of the orotomides against Aspergillus fumigatus: new opportunities for treatment of multidrug-resistant fungal disease. mBio 8:e01157-17. doi:10.1128/mBio.01157-17.
    OpenUrlAbstract/FREE Full Text
  21. 21.↵
    1. Negri CE,
    2. Johnson A,
    3. McEntee L,
    4. Box H,
    5. Whalley S,
    6. Schwartz JA,
    7. Ramos-Martin V,
    8. Livermore J,
    9. Kolamunnage-Dona R,
    10. Colombo AL,
    11. Hope WW
    . 2018. Pharmacodynamics of the novel antifungal agent F901318 for acute sinopulmonary aspergillosis caused by Aspergillus flavus. J Infect Dis 217:1118–1127. doi:10.1093/infdis/jix479.
    OpenUrlCrossRef
  22. 22.↵
    1. Seyedmousavi S,
    2. Chang YC,
    3. Law D,
    4. Birch M,
    5. Rex JH,
    6. Kwon-Chung KJ
    . 2018. Abstr 8th Advances against Aspergillosis Conf, Lisbon, Portugal, poster 144.
  23. 23.↵
    1. Lackner M,
    2. Birch M,
    3. Naschberger V,
    4. Grässle D,
    5. Beckmann N,
    6. Warn P,
    7. Gould J,
    8. Law D,
    9. Lass-Flörl C,
    10. Binder U
    . 2018. Dihydroorotate dehydrogenase inhibitor olorofim exhibits promising activity against all clinically relevant species within Aspergillus section Terrei. J Antimicrob Chemother 73:3068–3073. doi:10.1093/jac/dky329.
    OpenUrlCrossRef
  24. 24.↵
    1. Wiederhold NP,
    2. Law D,
    3. Birch M
    . 2017. Dihydroorotate dehydrogenase inhibitor F901318 has potent in vitro activity against Scedosporium species and Lomentospora prolificans. J Antimicrob Chemother 72:1977–1980. doi:10.1093/jac/dkx065.
    OpenUrlCrossRef
  25. 25.↵
    1. Biswas C,
    2. Law D,
    3. Birch M,
    4. Halliday C,
    5. Sorrell TC,
    6. Rex J,
    7. Slavin M,
    8. Chen SC
    . 2018. In vitro activity of the novel antifungal compound F901318 against Australian Scedosporium and Lomentospora fungi. Med Mycol 56:1050–1054. doi:10.1093/mmy/myx161.
    OpenUrlCrossRef
  26. 26.↵
    1. Wiederhold NP,
    2. Najvar LK,
    3. Jaramillo R,
    4. Olivo M,
    5. Birch M,
    6. Law D,
    7. Rex JH,
    8. Catano G,
    9. Patterson TF
    . 2018. The orotomide olorofim is efficacious in an experimental model of central nervous system coccidioidomycosis. Antimicrob Agents Chemother 27 62:e00999-18. doi:10.1128/AAC.00999-18.
    OpenUrlAbstract/FREE Full Text
  27. 27.↵
    1. Fisher MC,
    2. Hawkins NJ,
    3. Sanglard D,
    4. Gurr SJ
    . 2018. Worldwide emergence of resistance to antifungal drugs challenges human health and food security. Science 360:739–742. doi:10.1126/science.aap7999.
    OpenUrlAbstract/FREE Full Text
  28. 28.↵
    1. Espinel-Ingroff A,
    2. Diekema DJ,
    3. Fothergill A,
    4. Johnson E,
    5. Pelaez T,
    6. Pfaller MA,
    7. Rinaldi MG,
    8. Canton E,
    9. Turnidge J
    . 2010. Wild-type MIC distributions and epidemiological cutoff values for the triazoles and six Aspergillus spp. for the CLSI broth microdilution method (M38-A2 document). J Clin Microbiol 48:3251–3257. doi:10.1128/JCM.00536-10.
    OpenUrlAbstract/FREE Full Text
  29. 29.↵
    1. Espinel-Ingroff A,
    2. Cuenca-Estrella M,
    3. Fothergill A,
    4. Fuller J,
    5. Ghannoum M,
    6. Johnson E,
    7. Pelaez T,
    8. Pfaller MA,
    9. Turnidge J
    . 2011. Wild-type MIC distributions and epidemiological cutoff values for amphotericin B and Aspergillus spp. for the CLSI broth microdilution method (M38-A2 document). Antimicrob Agents Chemother 55:5150–5154. doi:10.1128/AAC.00686-11.
    OpenUrlAbstract/FREE Full Text
  30. 30.↵
    1. Sugui JA,
    2. Peterson SW,
    3. Figat A,
    4. Hansen B,
    5. Samson RA,
    6. Mellado E,
    7. Cuenca-Estrella M,
    8. Kwon-Chung KJ
    . 2014. Genetic relatedness versus biological compatibility between Aspergillus fumigatus and related species. J Clin Microbiol 52:3707–3721. doi:10.1128/JCM.01704-14.
    OpenUrlAbstract/FREE Full Text
  31. 31.↵
    Clinical and Laboratory Standards Institute. 2017. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi, 3rd ed. M38-A2. Clinical and Laboratory Standards Institute, Wayne, PA.
  32. 32.↵
    1. Seyedmousavi S,
    2. Mouton JW,
    3. Melchers WJ,
    4. Verweij PE
    . 2015. Posaconazole prophylaxis in experimental azole-resistant invasive pulmonary aspergillosis. Antimicrob Agents Chemother 59:1487–1494. doi:10.1128/AAC.03850-14.
    OpenUrlAbstract/FREE Full Text
  33. 33.↵
    1. Pollock JD,
    2. Williams DA,
    3. Gifford MA,
    4. Li LL,
    5. Du X,
    6. Fisherman J,
    7. Orkin SH,
    8. Doerschuk CM,
    9. Dinauer MC
    . 1995. Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production. Nat Genet 9:202–209. doi:10.1038/ng0295-202.
    OpenUrlCrossRefPubMedWeb of Science
  34. 34.↵
    1. Rao GV,
    2. Tinkle S,
    3. Weissman DN,
    4. Antonini JM,
    5. Kashon ML,
    6. Salmen R,
    7. Battelli LA,
    8. Willard PA,
    9. Hoover MD,
    10. Hubbs AF
    . 2003. Efficacy of a technique for exposing the mouse lung to particles aspirated from the pharynx. J Toxicol Environ Health A 66:1441–1452. doi:10.1080/15287390306417.
    OpenUrlCrossRefPubMedWeb of Science
  35. 35.↵
    1. Seyedmousavi S,
    2. Bruggemann RJ,
    3. Melchers WJ,
    4. Rijs AJ,
    5. Verweij PE,
    6. Mouton JW
    . 2013. Efficacy and pharmacodynamics of voriconazole combined with anidulafungin in azole-resistant invasive aspergillosis. J Antimicrob Chemother 68:385–393. doi:10.1093/jac/dks402.
    OpenUrlCrossRefPubMedWeb of Science
  36. 36.↵
    1. Seyedmousavi S,
    2. Davis MJ,
    3. Sugui JA,
    4. Pinkhasov T,
    5. Moyer S,
    6. Salazar AM,
    7. Chang YC,
    8. Kwon-Chung KJ
    . 2018. Exogenous stimulation of type I interferon protects mice with chronic granulomatous disease from aspergillosis through early recruitment of host-protective neutrophils into the lung. mBio 9:e00422-18. doi:10.1128/mBio.00422-18.
    OpenUrlAbstract/FREE Full Text
  37. 37.↵
    National Research Council. 2011. Guide for the care and use of laboratory animals, 8th ed. National Academies Press, Washington, DC.
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KEYWORDS

Aspergillus fumigatus
Aspergillus nidulans
Aspergillus tanneri
F901318
olorofim
chronic granulomatous disease
invasive aspergillosis
neutropenia

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