Objectives: Olorofim (OLO) is a new antifungal agent with a novel mechanism of action, targeting dihydroorotate dehydrogenase (DHODH) in the de novo pyrimidine biosynthesis pathway. It is active against both azole-susceptible and azole-resistant strains of Aspergillus fumigatus. Thus, OLO may be an important treatment option in patients with Aspergillus disease, including azole-resistant cases. Azole resistance selection occurs in >10% of patients with chronic pulmonary aspergillosis during itraconazole (ITZ) therapy. In this study, we analysed the frequency of resistance mutation induction of OLO in A. fumigatus in three different strains using two different methods. The underlying OLO resistance mechanism was also investigated. Methods: Method 1: From two A. fumigatus isolates, AZN 8196 and V139-36, 10 single colonies were separately inoculated onto Sabouraud agar and incubated at 37°C for 96h. From each culture 1 x 108 conidia were applied to 6 RPMI agar plates containing either 0.5 mg/L OLO or 8 mg/L ITZ.. Plates were incubated at 37°C for up to 7 days. Colonies growing on drug-containing plates were subcultured on RPMI agar containing 0.5 mg/L OLO or 8 mg/L ITZ to confirm resistance. Method 2: Spore stocks of A. fumigatus strain Af293 were prepared and inoculated onto yeast nitrogen base with glucose agar (YNBG) containing 0.25 mg/L OLO. A total of 8 x 109 spores were inoculated into 12 x 100 ml YNBG-OLO agar plates that were subsequently incubated for 5 days at 35°C. Colonies growing on drug-containing plates were subcultured on YNBG-OLO to confirm resistance. The mean rate of resistance was calculated for each isolate. The pyrE gene which codes for DHODH was sequenced for initial OLO-susceptible strains and -resistant progeny isolates and analysed for mutations. Results: Method 1: The resistance rate of OLO was 1 in 5 x 107 (frequency of 2 x 10-8 ) for AZN 8196 compared to 1 in 5 x 106 (2 x 10-7 ) for ITZ. For V139-36 the resistance rate of OLO was 1 in 9 x 107 (1.1 x 10-8 ) while the rate for ITZ was 1 in 8 x 106 (1.3 x 10-7 ) Method 2: Resistance to OLO in Af293 occurred at a rate of 1 in 6 x 108 (frequency of 1.7 x 10-9 ). Sequencing of pyrE revealed a hotspot for resistance mutations in the pyrE gene for OLO resistance in A. fumigatus at locus Gly119. Four amino acid substitutions were found; G119V, G119C, G119S and G119A. A single OLO-resistant mutant was obtained that carried the wild type DHODH sequence, but this mutant was slow growing compared with parent. Conclusion: We demonstrate that OLO resistance can be selected in A. fumigatus and can be mediated by mutations in the pyrE gene at locus G119. The frequency of resistance varied from approximately 2 x 10-8 to 1.7 x 10-9 for spontaneous mutations. The resistance rate of OLO was 5 to 10-fold lower compared to ITZ. The low resistance rate found in these experiments indicates that resistance selection may be less frequent in patients with Aspergillus diseases treated with OLO.
Full conference title:
- TIMM (2019)