In Vitro and In Vivo Antifungal Profile of a Novel and Long-Acting Inhaled Azole, PC945, on Aspergillus fumigatus Infection.

PHARMACOLOGY crossm In Vitro and In Vivo Antifungal Profile of a Novel and Long-Acting Inhaled Azole, PC945, on Aspergillus fumigatus Infection Thomas Colley,a Alexandre Alanio,b,c,d Steven L. Kelly,e Gurpreet Sehra,a Yasuo Kizawa,f Andrew G. S. Warrilow,e Josie E. Parker,e Diane E. Kelly,e Genki Kimura,f Lauren Anderson-Dring,a Takahiro Nakaoki,f Mihiro Sunose,g Stuart Onions,g Damien Crepin,g Franz Lagasse,g Matthew Crittall,g Jonathan Shannon,g Michael Cooke,g Stéphane Bretagne,b,c,d John King-Underwood,h John Murray,a Kazuhiro Ito,a Pete Strong,a Garth Rapeporta Pulmocide Ltd., London, United Kingdoma; Institut Pasteur, CNRS, Molecular Mycology Unit, French National Reference Center for Invasive Mycoses & Antifungals, URA3012, Paris, Franceb; Paris Diderot, Sorbonne Paris Cité University, Paris, Francec; Parasitology-Mycology Laboratory, Saint Louis Hospital, Assistance PubliqueHôpitaux de Paris (APHP), Paris, Franced; Centre for Cytochrome P450 Biodiversity, Institute of Life Science, Swansea University Medical School, Wales, United Kingdome; Nihon University, Chiba, Japanf; Sygnature Discovery Ltd., Nottingham, United Kingdomg; Compchem Resource, Pendock, United Kingdomh The profile of PC945, a novel triazole antifungal designed for administration via inhalation, was assessed in a range of in vitro and in vivo studies. PC945 was characterized as a potent, tightly binding inhibitor of Aspergillus fumigatus sterol 14␣-demethylase (CYP51A and CYP51B) activity (50% inhibitory concentrations [IC50s], 0.23 ␮M and 0.22 ␮M, respectively) with characteristic type II azole binding spectra. Against 96 clinically isolated A. fumigatus strains, the MIC values of PC945 ranged from 0.032 to ⬎8 ␮g/ml, while those of voriconazole ranged from 0.064 to 4 ␮g/ml. Spectrophotometric analysis of the effects of PC945 against itraconazolesusceptible and -resistant A. fumigatus growth yielded IC50 (determined based on optical density [OD]) values of 0.0012 to 0.034 ␮g/ml, whereas voriconazole (0.019 to ⬎1 ␮g/ml) was less effective than PC945. PC945 was effective against a broad spectrum of pathogenic fungi (with MICs ranging from 0.0078 to 2 ␮g/ml), including Aspergillus terreus, Trichophyton rubrum, Candida albicans, Candida glabrata, Candida krusei, Cryptococcus gattii, Cryptococcus neoformans, and Rhizopus oryzae (1 or 2 isolates each). In addition, when A. fumigatus hyphae or human bronchial cells were treated with PC945 and then washed, PC945 was found to be absorbed quickly into both target and nontarget cells and to produce persistent antifungal effects. Among temporarily neutropenic immunocompromised mice infected with A. fumigatus intranasally, 50% of the animals survived until day 7 when treated intranasally with PC945 at 0.56 ␮g/mouse, while posaconazole showed similar effects (44%) at 14 ␮g/ mouse. This profile affirms that topical treatment with PC945 should provide potent antifungal activity in the lung. ABSTRACT Received 24 October 2016 Returned for modification 21 November 2016 Accepted 14 February 2017 Accepted manuscript posted online 21 February 2017 Citation Colley T, Alanio A, Kelly SL, Sehra G, Kizawa Y, Warrilow AGS, Parker JE, Kelly DE, Kimura G, Anderson-Dring L, Nakaoki T, Sunose M, Onions S, Crepin D, Lagasse F, Crittall M, Shannon J, Cooke M, Bretagne S, KingUnderwood J, Murray J, Ito K, Strong P, Rapeport G. 2017. In vitro and in vivo antifungal profile of a novel and long-acting inhaled azole, PC945, on Aspergillus fumigatus infection. Antimicrob Agents Chemother 61:e02280-16. https://doi.org/10.1128/AAC.02280-16. Copyright © 2017 Colley et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Kazuhiro Ito, . KEYWORDS Aspergillus fumigatus, azole, inhalation, CYP51, azole resistant, long acting T he current management of the three major forms of aspergillosis, invasive aspergillosis (IA), chronic pulmonary aspergillosis (CPA), and allergic bronchopulmonary aspergillosis (ABPA) (1–4), involves prophylactic or therapeutic administration of triazoles and, infrequently, surgical intervention (5). Existing antifungal medicines are predominantly dosed either orally or systemically. These frequently exploited routes of delivery are poor for treating airway disease, since drug concentrations achieved at the May 2017 Volume 61 Issue 5 e02280-16 Antimicrobial Agents and Chemotherapy aac.asm.org 1 Colley et al. Antimicrobial Agents and Chemotherapy FIG 1 Efficacy of PC945 as an inhibitor of A. fumigatus sterol 14␣-demethylases (CYP51 enzymes). (A) Structure of PC945. (B) Type II azole binding spectra for A. fumigatus CYP51A and CYP51B. Each experiment was performed 4 to 6 times, although data for only one replicate are shown. (C) Azole IC50 determinations for posaconazole () and PC945 (Œ). Mean relative velocity values are shown with standard deviations. (D) Sterol composition of A. fumigatus treated with PC945. The relative levels of lanosterol and eburicol are shown. site of infection tend to be lower than those in other, healthy organs. This is especially so for the liver, which is a site of triazole toxicity: up to 15% of patients treated with voriconazole experience raised transaminase levels (6, 7). Exposure of the liver also results in significant drug interactions arising from triazole inhibition of hepatic P450 enzymes (8, 9). It is evident that there is an unmet clinical need for improved antifungal therapies which elicit fewer drug interactions, show reduced toxicity, achieve higher and more sustained pulmonary drug concentrations, and also demonstrate potent activity against azole-resistant Aspergillus strains. Thus, there are several advantages of topical treatment over oral/systemic treatment which alter the risk-benefit ratio of treatment favorably. An optimized compound for topical delivery should have prolonged lung tissue residence with limited systemic exposure to display a better adverse effect profile and to eradicate invasive aspergillosis due to a high-concentration exposure. We have undertaken an extensive lead optimization program in order to identify potent azole antifungal agents with optimal properties for topical administration to the lung, including tissue retention and physicochemical properties required for formulation. In this report, we disclose the in vitro and in vivo activity of PC945, which has the chemical formula 4-[4-(4-{[(3R,5R)-5-(2,4-difluorophenyl)-5-(1H-1,2,4-triazol-1-ylmethyl)oxolan-3yl]methoxy}-3-methylphenyl)piperazin-1-yl]-N-(4-fluorophenyl)benzamide (Fig. 1A) and is a novel triazole antifungal agent designed specifically for inhaled administration (10). RESULTS CYP51-binding properties. PC945 produced type II difference spectra when titrated against purified Aspergillus fumigatus CYP51A and CYP51B enzymes (AfCYP51A and AfCYP51B) and bound to CYP51A with an affinity similar to that of posaconazole (Table 1; Fig. 1B). In con (...truncated)