Olorofim: Discovery and development of a novel IV and oral orotomide class candidate for systemic mould infections Dr Emma Harvey Global Head of Medical Affairs F2G Ltd Olorofim_RICAI_December 2019 1
Disclaimer Olorofim is not currently approved for use in any market Olorofim_RICAI_December 2019 2
Agenda Olorofim … ▪ Is the first orotomide antifungal ▪ Stops growth quickly; Cell rupture follows ▪ Is active in vivo: PK-PD driven by Cmin ▪ Target levels readily achieved in man both PO and IV ▪ Phase 2 Open-Label study has recruited > 40 patients Olorofim_RICAI_December 2019 3
Olorofim structure Previously known as F901318 MW = 499 Formula = C 28 H 27 FN 6 O 2 > 99% protein bound in all species F2G, Ltd., data on file Olorofim_RICAI_December 2019 4
Orotomide Mechanism of Action ▪ Olorofim is a potent selective inhibitor of fungal dihydroorotate dehydrogenase (DHODH) ▪ DHODH is a key enzyme involved in pyrimidine biosynthesis ▪ Humans also have this enzyme ▪ But, > 2000-fold difference in IC 50 between human and fungal enzymes ▪ Pyrimidine inhibition has profound effects as it interferes with ▪ DNA synthesis and cell cycle regulation ▪ RNA synthesis and protein production ▪ Cell wall synthesis ▪ Phospholipid synthesis Oliver et al. PNAS 113:12809-14, 2016. Olorofim_RICAI_December 2019 5
DHODH Protein Homology Taxonomy Note the clustering of (PEZIZOMYCOTA) activity within the (BACTERIAL) OLOROFIM Pezizomycota: SENSITIVE DHODH structurally conserved across these species OLOROFIM Work in collaboration INTRINSIC RESISTANCE with Paul Boyer, Univ Olorofim_RICAI_December 2019 6 Manchester
Aspergillus: In vitro ▪ Olorofim MICs are tightly clustered ▪ MIC 90 s are 0.03-0.06 mg/L (CLSI and EUCAST) ▪ MICs are of the same order for all Aspergillus spp. tested ▪ >2900 isolates from 35 species including : ▪ >2300 isolates of 4 most common species 1 ▪ 220 isolates of 16 cryptic species 2 ▪ No induction of resistance with serial passage ▪ Spontaneous resistance seen at low frequency ▪ Cross-resistance is not seen ▪ MICs of the same order in azole-resistant isolates ▪ MICs of the same order in amphotericin-resistant species 1. A. fumigatus, flavus, niger, and terreus 2. A. alliaceus, aureoterreus, calidoustus, carneus, citrinoterreus, fumigatiaffinis, hiratsukae, hortai, insuetus, keveii, lentulus, ochraceus, pseudofischerii, sclerotiorum, tubingensis, and udagawae Beckmann et al. ICAAC 2015; Fothergill et al. ICAAC 2015; Buil et al.JAC 2017; Rivero- 7 Olorofim_RICAI_December 2019 Menéndez et al ECCMID 2017; Oliver et al PNAS 2016; Jorgensen et al. TIMM 2017
In vitro activity against other fungi ▪ Scedosporium spp., L. prolificans and numerous other moulds* and endemic dimorphic fungi ▪ MICs similar to Aspergillus ▪ Fusarium spp ▪ Mixed and method-dependent results ▪ Further investigative work underway ▪ Mucorales, Candida spp ., Cryptococcus neoformans ▪ Not active (structurally different DHODH enzyme) *Tested to date: Acremonium persicinum , Acrophialophora fusispora , Rasamsonia spp., Phaeoacremonium spp., Sarocladium kilienses , Scopulariopsis brevicaulis , Microascus spp., Sporothrix schenkii , Trichoderma spp., Ramichloridium (Myrmecridium) schulzeri , Paecilomyces spp., Pleurostomophora richardsiae , Verruconis gallopava, Chaetomium spp., and Penicillium spp. (including P. marneffiii ) . Wiederhold et al. JAC 2107, Biswas et al. Med Mycol 2018; Alastruey et al. TIMM 8 Olorofim_RICAI_December 2019 2017; Oliver et al. PNAS 2016; F2G Ltd, data on file
Across the related fungi, the MICs are tightly clustered >2900 Aspergillus isolates >300 Scedosporium isolates >100 Lo Pro isolates Olorofim_RICAI_December 2019 9 F2G Ltd, data on file
Agenda Olorofim … ▪ Is the first orotomide antifungal ▪ Stops growth quickly; Cell rupture follows ▪ Is active in vivo: PK-PD driven by Cmin ▪ Target levels readily achieved in man both PO and IV ▪ Phase 2 Open-Label study has recruited > 40 patients Olorofim_RICAI_December 2019 10
Fungicidal: Growth inhibition begins at ~30 minutes and leads to killing ▪ 16-hour-old A. fumigatus hypha Time-lapse video (48h compressed to ~ 15 seconds) ▪ Actively growing ▪ Treated with 0.1 mg/L olorofim ▪ Growth inhibition at ≈ 30 mins ▪ Cell rupture at ≈ 24 hours Shown here are TEM views of the effect of olorofim at 24h Untreated hyphae 0.1 mg/L olorofim (24h) Note normal nucleus, cytoplasmic Note absence of cytoplasmic content, and continuous cell wall content, broken/thin cell wall Olorofim_RICAI_December 2019 11 du Pre S et al. Antimicrob Agents Chemother 62:e00231-18, 2018.
Cell rupture is then seen ▪ Cell viability marker (Green = Dead) ▪ DiBac: Bis-(1,3-Dibutyl barbituric Acid) Trimethine Oxonol 100 75 % lysed 50 25 0 24h 48h 72h 96h 120h Olorofim_RICAI_December 2019 12 du Pre S et al. Antimicrob Agents Chemother 62:e00231-18, 2018.
Effects on conidia as well Conidia treated for 24h with olorofim Untreated (0.1 µg/mL). Note breaks in cell wall. A. fumigatus conidia were incubated at 37C in Vogel’s minimal medium. Olorofim_RICAI_December 2019 13 du Pre S et al. Antimicrob Agents Chemother 62:e00231-18, 2018.
Agenda Olorofim … ▪ Is the first orotomide antifungal ▪ Stops growth quickly; Cell rupture follows ▪ Is active in vivo: PK-PD driven by Cmin ▪ Target levels readily achieved in man both PO and IV ▪ Phase 2 Open-Label study has recruited > 40 patients Olorofim_RICAI_December 2019 14
In vivo activity ▪ In vivo activity (mostly murine, some rabbit) shown for ▪ A. fumigatus, A. flavus, A. terreus, A. nidulans, A. tanneri ▪ Scedosporium apiospermum ▪ Lomentospora prolificans ▪ Scedosporium (Pseudallescheria) boydii ▪ Coccidioides immitis ▪ In vivo activity has been shown PO & IV ▪ Endpoints: Survival, tissue burden, and GM ▪ Works equally well on azole-and amphotericin B-resistant Aspergillus isolates: ▪ Azole-resistant ( A. fumigatus, A. tanneri, L. prolificans ) ▪ Amphotericin B-resistant ( A. terreus, A. tanneri ) Hope et al. mBio 8:1-17, 2017; Seyedmousavi et al. Antimicrob Ag Chemother 63:e00129-19, 2019; J Lackner et al. J Antimicrob Chemo 73:3068-73, 2018; Seyedmousavi et al. Trends in Medical Mycology (abstract), 2019; Wiederhold et al. AAC 2018;62(9):e00999-18. 15 Olorofim_RICAI_December 2019
Olorofim activity against A. fumigatus A. fumigatus (NIH 4215, azole wild-type) Susceptible to OLO, POSA, and VORI Control study, wild- 100 type strain: OLO and POSA are 80 POSA both active. % survival 60 OLO R 40 Reminder: It’s tricky to study voriconazole in the mouse due its rapid metabolism. 20 Control This model used IV dosing using Drug 0 posaconazole not voriconazole. 0 2 4 6 8 10 Study Day Neutropenic mouse model, inhalation IA, drug given IV starting 6h after infection through Day 3; Olorofim MIC 0.03 mg/L for both isolates; Posaconazole (POSA) MIC 0.125 mg/L (NIH 4215) and 0.5 mg/L (11628); VORI MIC 0.5 (NIH 4215) and 16 mg/L (11628). Hope et al. mBio 8:1-17, 16 Olorofim_RICAI_December 2019 2017; Jeans et al. J Infect Dis, 206:442-52, 2012; Simitsopoulou et al. Antimicrob Ag Chemother 52:3301-6, 2008.
Olorofim is effective in vivo vs. azole-resistant A. fumigatus A. fumigatus (11628, G138C mutant) Susceptible to OLO, Resistant to POSA and VORI 100 OLO 80 60 POSA 40 Control 20 Drug 0 0 2 4 6 8 10 Study Day Neutropenic mouse model, inhalation IA, drug given IV starting 6h after infection through Day 3; Olorofim MIC 0.03 mg/L for both isolates; Posaconazole (POSA) MIC 0.125 mg/L (NIH 4215) and 0.5 mg/L (11628); VORI MIC 0.5 (NIH 4215) and 16 mg/L (11628). Hope et al. mBio 8:1-17, 17 Olorofim_RICAI_December 2019 2017; Jeans et al. J Infect Dis, 206:442-52, 2012; Simitsopoulou et al. Antimicrob Ag Chemother 52:3301-6, 2008.
Olorofim activity vs A. tanneri in CGD mouse model A. tanneri (NIH 1004). Susceptible to OLO, When tested against Resistant to VORI, POSA, and AmB 100 AmB-resistant A. tanneri , OLO remains 80 active but the azole fails OLO 60 40 Control 20 Control VORI Drug 0 0 2 4 6 8 10 CGD= chronic granulomatous disease Study Day Survival curves for mice with oxidative-deficient (CGD) neutrophils. Infection by tail vein injection, drug IP starting 6h after infection through Day 9. MICs for B5213 and NIH 1004 were 0.01 and 0.06 mg/L (OLO), 0.5 and 4 mg/L (VORI), 0.125 and 0.5 mg/L (POSA), and 0.5 and > 16 mg/L 18 Olorofim_RICAI_December 2019 (AmB). Seyedmousavi et al. Antimicrob Ag Chemother 63:e00129-19, 2019.
Olorofim activity in vivo vs. amphotericin-resistant A. terreus OLO, iv 100 OLO, po Similar results 80 for olorofim dosed IV and PO 60 % survival 40 20 Ampho Drug Control 0 0 2 4 6 8 10 Study Day Neutropenic mouse model, intravenous IA, drug dosed from Day 1 to Day 9; A. terreus with OLO MIC of 0.01 mg/L, amphotericin B 19 MIC of 2 mg/L; Lackner et al. J Antimicrob Chemo 73:3068-73, 2018. Olorofim_RICAI_December 2019
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