Novel Once-Weekly Echinocandin Rezafungin (CD101) for Prevention and Treatment of Pneumocystis Biofilms Melanie T. Cushion,1 Margaret S. Collins,1 Jeffrey B. Locke,2 Voon Ong,2 Ken Bartizal2 1University of Cincinnati College of Medicine and the Cincinnati Veterans Affairs Medical Center, Cincinnati, OH 2Cidara Therapeutics, San Diego, CA

Background Results Rezafungin (formerly CD101) is a novel long-acting echinocandin with Table 1. Inhibitory activity of rezafungin against suspension, pharmacokinetics that achieve high, front-loaded plasma exposure and established biofilm, and nascent biofilm cultures of P. carinii allow once-weekly dosing. Rezafungin, currently in development for % reduction in ATP vs. media prophylaxis against Candida, Aspergillus, and Pneumocystis, has Culture Conc. Drug control (average of 2 assays) minimal potential for the myelosuppression and drug-drug interactions type (µg/ml) that often complicate today’s antifungal prophylaxis in patients Day 1 Day 3 Day 7 undergoing bone marrow transplant (BMT). ampicillin 10 3.66 0 3.47 pentamidine 1 77.63 91.2 89.62 Pneumocystis is an opportunistic fungal pathogen deserving of attention 1 16.83 1.88 27.75 as it continues to cause morbidity and mortality for BMT recipients, suspension particularly when prophylaxis is absent or inadequate. Interrupted 10 14.61 2.22 42.48 rezafungin prophylaxis and poor compliance are among risk factors in high-risk 100 59.23 85.1 99.3 populations.(1) Rezafungin has demonstrated in vivo efficacy in blocking Day 3 IC50 = 6.58 µg/ml formation of both trophic and asci forms of Pneumocystis.(2) ampicillin 10 5.13 8.21 0 Biofilm formation by fungi is considered a survival mechanism as biofilms pentamidine 1 73.78 46.59 53.64 are notoriously difficult to treat. It has been hypothesized that the established 1 27.21 26.49 0 morphology of Pneumocystis in mammalian lungs is akin to biofilms. In biofilm 10 21.86 27.15 20.21 the present study, in vitro biofilm formation by P. carinii and P. murina rezafungin 100 37.5 82.76 97.11 was used to explore the ability of rezafungin to prevent biofilm formation and to treat mature biofilms. Day 3 IC50 = 16.75 µg/ml Methods ampicillin 10 0.85 0 11.52 pentamidine 1 83.6 64.7 71.89 • Biofilms using P. carinii isolated from rat lung tissue were grown in nascent 1 0 2.36 23.41 Millicell culture inserts in multi-well plates with supplemented RPMI biofilm 10 0 11.75 36.57 1640 medium (Figure 1), as previously described.(3) rezafungin 100 36.68 57.13 97.76 • Mature biofilms were grown for 7 days prior to treatment; nascent Day 3 IC = 8.53 µg/ml biofilm inserts received test compounds at the time of inoculation. 50 Media alone and ampicillin were used as negative controls while • Against suspension cultures of P. carinii, where non-β-1,3-D-glucan- pentamidine was used as positive control. producing trophic forms are most abundant, rezafungin had moderate activity by day 7 and a day 3 IC of 6.58 µg/ml. • The entire contents of 3 inserts per time point were harvested for 50 assessment of ATP content using a luciferin-luciferase based assay • When exposed to difficult-to-treat established biofilms, the rezafungin at 1, 3, and 7 days of treatment. day 3 IC50 increased to 16.75 µg/ml. Figure 1. Pneumocystis in vitro biofilm model system • However, against nascent biofilm cultures, which measure the ability of the drug to prevent biofilm formation, rezafungin displayed moderate Day 1 5 10 activity with an IC50 of 8.53 μg/ml. • Previous data generated in this biofilm prevention assay demonstrated identical inhibitory activity for the 100 µg/ml dose of anidulafungin (98%), yet in striking contrast, caspofungin had little (14%) and micafungin, no effect (0%) on biofilm formation.(5) A Conclusion • Rezafungin was efficacious in preventing biofilm formation and reducing the viability of mature biofilms of P. carinii. 1 cm • These findings, in conjunction with previously published data demonstrating effective Pneumocystis, Candida, and Aspergillus prophylaxis in immunosuppressed preclinical animal models, further suggest utility of rezafungin in the setting of BMT. References B 1. Cordonnier C, et al. 2016. Pneumocystis jirovecii pneumonia: still a concern in patients with haematological malignancies and stem cell transplant recipients. J Antimicrob Chemother 71:2379-85. 2. Cushion MT, et al. 2016. Efficacy of CD101, a Novel Echinocandin, in Prevention of Pneumocystis Pneumonia (PCP): Thwarting the Biphasic Life Cycle of Pneumocystis. ASH Annual Meeting. Poster 3396. Macroscopic biofilm formation by P. carinii. (A) Duplicate wells showing biofilm 3. Cushion MT, Collins MS, Linke MJ. 2009. Biofilm formation by Pneumocystis spp. Eukaryot formation after 1, 5, and 10 days on a Lab Tek II chamber slide. (B) PTFE Cell 8:197-206. inserts (12 mm) without inocula (left) and after 14 days of biofilm formation 4. Ong V, et al. 2018. CD101, ’A Perfect Storm’ Against Aspergillus: In Vitro Microbiology, In Vivo (right). Scale bar = 1 cm. Tissue Distribution, and Front-Loaded Treatment and Prophylactic Efficacy in Mouse Disseminated and Pulmonary Aspergillosis Infection Models. BMT Tandem. Poster 554. • The in vitro inhibitory activity of rezafungin was evaluated against 5. Cushion MT, Collins MS. 2011. Susceptibility of Pneumocystis to echinocandins in suspension suspension and biofilm (nascent and established) cultures of P. carinii and biofilm cultures. Antimicrob Agents Chemother 55:4513-4518. at 1, 10 and 100 µg/ml. While unbound systemic concentrations at doses currently being studied do not reach 100 µg/ml, such levels Acknowledgements and Disclosures may be achievable with extensive partitioning in epithelial lining M. Cushion: research funding, Cidara Therapeutics; NIH HHSN2722011000181. Task order fluid.(4) HHSN27200008. Subcontractor. Patterson T, PI. In vitro screening for antifungal activity; US Veterans Affairs Merit Review • The percent reduction in ATP vs. media control was measured at day M. Collins: none to disclose J. Locke: employee and ownership interest, Cidara Therapeutics 1, 3 and 7 (averages of 2 replicate assays are reported) and IC50 V. Ong: employee and ownership interest, Cidara Therapeutics values were calculated for rezafungin at day 3. K. Bartizal: employee and ownership interest, Cidara Therapeutics #EBMT18 www.ebmt.org