In vitro and in vivo antifungal profile of a novel and long acting inhaled azole, PC945, on

43 Pages·2017·1.45 MB·English

AAC Accepted Manuscript Posted Online 21 February 2017
Antimicrob. Agents Chemother. doi:10.1128/AAC.02280-16
Copyright © 2017 Colley et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
AAC (Research Article, T.Colley)
1 Title
2 In vitro and in vivo antifungal profile of a novel and long acting inhaled azole,
3 PC945, on Aspergillus fumigatus infection
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6 Thomas Colley1, Alexandre Alanio2, Steven L. Kelly3, Gurpreet Sehra1, Yasuo Kizawa4, Andrew lo
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7 G.S. Warrilow3, Josie E. Parker3, Diane E. Kelly3, Genki Kimura4, Lauren Anderson-Dring1, e
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8 Takahiro Nakaoki4, Mihiro Sunose5, Stuart Onions5, Damien Crepin5, Franz Lagasse5, Matthew ro
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9 Crittall5, Jonathan Shannon5, Michael Cooke5, Stéphane Bretagne, John King-Underwood6, John ht
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10 Murray1, Kazuhiro Ito1#, Pete Strong1 and Garth Rapeport1 //a
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12 1Pulmocide Ltd, London, UK, SW7 2BP, 2Unit of Molecular Mycology, Institut Pasteur, Paris, .
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13 France; URA 3012, The French National Centre for Scientific Research (CNRS), Paris, France; /

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14 Sorbonne Paris Cité, Université Paris Diderot, Paris, France; Hôpital Saint-Louis, Paris, France, A
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15 3Centre for Cytochrome P450 Biodiversity, Institute of Life Science, Swansea University il
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16 Medical School, Wales, UK, SA2 8PP, 4Nihon University, Chiba, Japan, 5Sygnature Discovery 20
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17 Ltd, Nottingham, UK, NG1 1GF, 6 Compchem Resource, Pendock, UK, GL19 3PW. b
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21 Running title: Antifungal activity of novel azole PC945
22 Key words: Aspergillus fumigatus, azole, inhalation, CYP51, azole resistant
23 #Address correspondence to Kazuhiro Ito: [email protected]
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24 ABSTRACT
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26 The profile of PC945, a novel triazole antifungal, designed for administration via inhalation, has
27 been assessed in a range of in vitro and in vivo studies. PC945 was characterized as a potent,
28 tight-binding inhibitor of Aspergillus fumigatus sterol 14α-demethylase (CYP51A and CYP51B)
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29 activity (IC , 0.23 μM and 0.22 μM, respectively), with characteristic type II azole binding w
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30 spectra. Against 96 clinically isolated A. fumigatus strains, the MIC values of PC945 ranged a
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31 from 0.032~>8 μg/ml, whilst those of voriconazole ranged from 0.064~4 μg/ml. d

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32 Spectrophotometric analysis of the effects of PC945 against itraconazole-susceptible and - m

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33 resistant A. fumigatus growth, yielded IC50 (OD) values between 0.0012~0.034 μg/ml, whereas tp
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34 voriconazole (0.019~>1 μg/ml) was less effective than PC945. PC945 was effective against a a
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35 broad spectrum of pathogenic fungi (MIC ranged from 0.0078~2 μg/ml) including Aspergillus s
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36 terreus, Trichophyton rubrum, Candida albicans, Candida glabrata, Candida krusei, o
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37 Cryptococcus gattii, Cryptococcus neoformans and Rhizopus oryzae (1~2 isolates each). In o
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38 addition, when A. fumigatus hyphae or human bronchial cells were treated with PC945, and then p
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39 washed, PC945 was found to be quickly absorbed into both target and non-target cells and to 1
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40 produce persistent antifungal effects. In temporarily neutropenic immunocompromised mice 1
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41 infected with A. fumigatus intranasally, 50% of the animals survived until day 7 when treated y

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42 intranasally with PC945 at 0.56 µg/mouse, while posaconazole showed similar effects (44%) at e
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43 14 µg/mouse. This profile affirms that topical treatment with PC945 should provide potent
44 antifungal activity in the lung.
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47 INTRODUCTION
48
49 The current management of the three major forms of aspergillosis: invasive aspergillosis
50 (IA), chronic pulmonary aspergillosis (CPA) and allergic bronchopulmonary aspergillosis
51 (ABPA) (1-4), involves prophylactic or therapeutic administration of triazoles and, infrequently,
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52 surgical intervention (5). Existing antifungal medicines are predominantly dosed either orally or w
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53 systemically. These frequently exploited routes of delivery are poor for treating airway disease, a
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54 since drug concentrations achieved at the site of infection tend to be lower than those in other, d

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55 healthy organs. This is especially so for the liver, which is a site of triazole toxicity: up to 15% of m

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56 patients treated with voriconazole experience raised transaminase levels (6, 7). Exposure of the tp
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57 liver also results in significant drug interactions arising from triazole inhibition of hepatic P450 a
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58 enzymes (8, 9). s
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59 It is evident that there is an unmet clinical need for improved antifungal therapies which elicit o
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60 fewer drug interactions; show reduced toxicity; achieve higher and more sustained pulmonary o
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61 drug concentrations and also demonstrate potent activity against azole-resistant Aspergillus p
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62 strains. Thus, there are several advantages of topical treatment over oral/systemic treatment 1
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63 which alter the risk benefit ratio of treatment favourably. An optimised compound for topical 1
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64 delivery should have prolonged lung tissue residence with limited systemic exposure to display a y

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65 better adverse effect profile and eradicate invasive aspergillosis due to high concentration e
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66 exposure. We have undertaken an extensive lead optimization program in order to identify potent
67 azole antifungal agents with optimal properties for topical administration to the lung including
68 tissue retention and physicochemical properties required for formulation. In this report we
69 disclose the in vitro and in vivo activity of PC945, which is: 4‐[4‐(4‐{[(3R,5R)‐5‐(2,4‐
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70 difluorophenyl)‐5‐(1H‐1,2,4‐triazol‐1‐ylmethyl)oxolan‐3‐yl]methoxy}‐3‐
71 methylphenyl)piperazin‐1‐yl]‐N‐(4‐fluorophenyl)benzamide (Structure in Fig. 1A); a novel,
72 triazole, antifungal agent designed specifically for inhaled administration (10).
73
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74 MATERIALS AND METHODS
75 Antifungal agents. For in vitro antifungal assays, stock solutions of test agents were prepared in
76 DMSO (2000 μg/ml). For in vivo studies test agents were suspended in physiological saline.
77 PC945 was synthesised by Sygnature Discovery Ltd (Nottingham, UK), and voriconazole
78 (Tokyo Chemical Industry UK Ltd., Oxford, UK), posaconazole (Apichem Chemical
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79 Technology Co., Ltd., Zhejiang, China), itraconazole (Arkopharma, Carros, France), w
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80 amphotericin B (Selleckchem, Munich, Germany) and caspofungin (Selleckchem, Munich, a
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81 Germany) were procured from commercial sources. d

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82 m

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83 A. fumigatus CYP51 binding assay and enzyme inhibitory activity. A. fumigatus CYP51 tp
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84 binding properties were determined as described by Warrilow et al. (11). Test agents were a
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85 titrated against 4 μM recombinant A. fumigatus CYP51A or CYP51B proteins and binding s
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86 saturation curves were constructed from the change in the absorbance between the spectral peak o
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87 and the trough. A rearrangement of the Morrison equation was used to determine the dissociation o
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88 constant (Kd) values when ligand binding was tight (12). p
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89 A CYP51 reconstitution assay system was used to determine 50% inhibitory (IC ) 1
50 ,

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90 concentrations (13). Test agent was added to a mixture of 0.5 μM CYP51, 1 μM A. fumigatus 1
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91 cytochrome p450 reductase isoenzyme 1 (AfCPR1), 50 μM eburicol, 4% (w/v) 2-hydroxypropyl- y

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92 β-cyclodextrin, 0.4 mg ml-1 isocitrate dehydrogenase, 25 mM trisodium isocitrate, 50 mM NaCl, e
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93 5 mM MgCl and 40 mM 3-(N-morpholino) propanesulfonic acid (MOPS) (pH ~7.2). The
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94 mixtures were then incubated at 37°C for 10 minutes prior to initiation with 4 mM β-NADPHNa
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95 followed by shaking for 20 minutes at 37°C. Sterol metabolites were recovered by extraction
96 with ethyl acetate followed by derivatisation with 0.1 ml N,O-
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97 bis(trimethylsilyl)trifluoroacetamide : trimethylchlorosilane (99:1) and 0.3 ml anhydrous
98 pyridine prior to analysis by gas chromatography mass spectrometry.
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100 A. fumigatus sterol analysis. A working suspension of A. fumigatus spores was prepared in
101 filter-sterilised MOPS RPMI-1640 (RPMI-1640 containing 2 mM L-glutamine, 2% glucose,
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102 0.165 M MOPS, buffered to pH 7 with NaOH) at a final concentration of 8 x 106 spores ml-1. To o
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103 each 100 mm Petri dish, 10 ml of the working suspension was added and the dishes were lo
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104 incubated for 4 h at 35°C and 5% CO2. Samples for baseline determinations were collected by d

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105 scraping, pelleted by centrifugation at 2000 rpm for 5 minutes and stored at -80°C. Test m

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106 compounds or DMSO (50 µL) were added to the remaining dishes, which were subsequently tt
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107 gently rocked by hand to disperse the compounds. Dishes were incubated for 2 h at 35°C and 5% a
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108 CO . Samples were collected and processed as described above. Posaconazole and PC945 a
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109 concentrations of 0.0001, 0.001, 0.01, 0.1 and 1 µg ml-1 were tested. These samples were .o
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110 prepared in the laboratory at Pulmocide Ltd., and sent to the laboratory in the Centre for
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111 Cytochrome P450 Biodiversity, Institute of Life Science, Swansea University Medical School,
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112 for experimentation. il 1
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113 Non-saponifiable lipids were extracted as previously reported (14) and were derivatised 0
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114 with 0.1 ml N,O-bis(trimethylsilyl)trifluoroacetamide : trimethylchlorosilane (99:1) and 0.3 ml b
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115 anhydrous pyridine (2 h at 80°C) prior to analysis by gas chromatography mass spectrometry u
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116 (15). Sterol composition was calculated using peak areas from the gas chromatograms and the
117 mass fragmentation pattern compared to known standards were used to confirm sterol identity.
118 The sterol content of A. fumigatus (basal) and treated A. fumigatus (either DMSO, posaconazole
119 or PC945) were determined.
120
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121 A. fumigatus cell based ergosterol assay. Growth medium (RPMI-1640, 2 mM L-glutamine,
122 2% glucose, 0.165 M MOPS, 0.5% BSA, pH 7.0) was added across a 96-well plate and test
123 agents were added in duplicate. A. fumigatus (NCPF2010) conidia were added across the plate at
124 a final concentration of 1 x 104 ml-1. After incubation for 24 h at 35°C, media was removed from
125 all wells and replaced with reaction buffer (Amplex red cholesterol assay kit, ThermoFisher,
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126 A12216) and Amplex red solution. Plates were incubated for 30 minutes at 37°C, protected from
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127 light, after which fluorescence was quantified using a spectrophotometer. Media was removed lo
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128 from all wells and replaced with crystal violet solution (1% v/v), and plates were incubated at d

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129 room temperature on a shaker for 30 minutes. Plates were washed three times with PBS, and m

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130 sodium dodecyl sulfate solution (0.1% v/v) was added across the plate to lyse the cells. After ttp
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131 incubation at room temperature for 1 h, absorbance was measured at OD using a a
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132 spectrophotometer. a
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133 .o
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134 Fluorescent imaging of A. fumigatus infected cells. Human alveolar epithelial cells (A549)
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135 were seeded onto collagen-coated coverslips and incubated at 37°C and 5% CO for 24 h. Cells
2 p
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136 were incubated in the presence of test agents for 2 h, after which the media was replaced and the il 1
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137 coverslips were incubated at 37°C, and 5% CO for 24 h. CellTracker red CMTPX dye 0
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138 (ThermoFisher, C34552) was added to cell media for 30 minutes, wells were washed with PBS, b
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139 and GFP-A. fumigatus conidia, [a kind gift from Professor William Hope, University of u
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140 Liverpool], were added to wells at a final concentration of 1 x 103 spores ml-1. After 24 h
141 incubation at 35°C and 5% CO , coverslips were washed and affixed to slides using
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142 Fluoroshield™ with DAPI (Sigma, F6057).
143
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144 In vitro antifungal activity against A. fumigatus. Assessment of antifungal activity against a
145 selection of A. fumigatus laboratory/clinical strains (NCPF2010 [National Collection of
146 Pathogenic Fungi (NCPF), Bristol, UK], AF72 [NCPF, Bristol, UK], AF91 [NCPF, Bristol, UK],
147 AF293 [NCPF, Bristol, UK], AF294 [NCPF, Bristol, UK], TR34-L98H [Professor Bretagne, St
148 Louis Hospital, Paris, France]) was performed according to the European Committee on
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149 Antimicrobial Susceptibility Testing (EUCAST) definitive document EDef 9.3 (16), with the
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150 following exceptions: (i) 0.5% bovine serum albumin (BSA) was added to the growth medium to lo
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151 avoid any loss of lipophilic compounds by adherence to plastic plate surfaces and (ii) 384-well d

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152 plates were used rather than 96-well plates. Growth medium (RPMI-1640, 2mM L-glutamine, m

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153 2% glucose, 0.165 M MOPS, 0.5% BSA, pH 7.0) was added across the plate, test agents were tt
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154 added in quadruplicate and the DMSO concentration was identical across the plates. Conidia a
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155 were added across the plate at a final concentration of 1 x 105 ml-1. Plates were incubated for 48 .a
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156 h at 35°C after which turbidity was assessed by measuring optical density (OD) at 530 nm using .o
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157 a spectrophotometer, and the IC and IC values were calculated from the concentration-
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158 response curve generated for each test compound using a four-parameter logistic equation
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159 (Dotmatics, Bishops Stortford, UK). A. fumigatus ATCC204305 was used as the assay control. il 1
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160 Determination of antifungal activity against 50 A. fumigatus clinical isolates from St Louis 0
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161 Hospital (Paris, France) was performed with 96-well plates using the modified EUCAST method b
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162 in the presence of 0.5% BSA as described above. Antifungal susceptibility testing for 46 A. u
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163 fumigatus isolates [obtained from the North West England Mycology Reference Centre] was
164 performed by Evotec (UK) Ltd (Manchester, UK) according to EUCAST guidelines. Assessment
165 of the antifungal activity of four of the A. fumigatus strains (ATCC1028, ATCC10894,
166 ATCC13073, and ATCC16424) was performed according to methodology described by the
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167 Clinical and Laboratory Standards Institute (CLSI) by Eurofins Panlabs Taiwan Ltd. (Taipei,
168 Taiwan).
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170 In vitro antifungal activity against other fungal species. For the measurement of activity
171 against Cryptococcus gattii, the method described in EUCAST definitive document EDef 7.2
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172 was used and assay plates were incubated statically at 37°C in ambient air for 24 h (± 2 h) unless
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173 poor growth necessitated further incubation to 36 or 48 h (17). Antifungal potency against lo
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174 Aspergillus flavus, Aspergillus niger and Aspergillus terreus, was determined as set out in d

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175 EUCAST definitive document EDef 9.2 and assay plates were incubated at 37°C for 48 h (18). m

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176 These tests were conducted at Evotec (UK) Ltd (Manchester, UK). Measurement of activity tt
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177 against other fungi was performed by Eurofins Scientific according to methodology described by a
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178 the Clinical and Laboratory Standards Institute (CLSI) (CLSI M38-A (19) or M27-A2 (20), a
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179 www.eurofinspanlabs.com). .o
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180
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181 In vitro determination of persistence of action on A. fumigatus hyphae. The persistence of
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182 action of test agents was calculated in A. fumigatus hyphae (NCPF2010). Conidia were diluted in il 1
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183 growth media (RPMI-1640, 2 mM L-glutamine, 2 % glucose, 0.165 M MOPS, pH 7.0) and 0
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184 added across a 384-well plate at a final concentration of 1x103/well. After incubation at 35oC for b
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185 exactly 6 h, test and reference articles or neat DMSO (as vehicle) (0.5 µl /well) were added to the u
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186 appropriate wells to give a final concentration of 0.5% DMSO. The plates were incubated for
187 exactly 20 minutes at 35oC and 5% CO . After the incubation time had elapsed all wells on the
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188 designated washout plate were aspirated and growth media (100 µl/well) was added across the
189 plate. For the non-washout plate, after compounds were added to hyphae, no media change was
190 applied. Resazurin (0.04% diluted in growth media) was added to all wells of both non-washout
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191 and washout plates (5 µl/well) to give a final concentration of 0.002% resazurin. The plates were
192 incubated at 35oC and 5% CO for 16 h. Subsequently fluorescence in each well was measured at
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193 λex/λem 545/600 nm using a multiscanner (Clariostar: BMG, Buckinghamshire, UK). The
194 percentage inhibition for each well was calculated and the IC value was calculated from the
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195 concentration-response curve generated for each test compound using a four-parameter logistic
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196 equation (Dotmatics, Bishops Stortford, UK).
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198 In vitro determination of persistence of action on bronchial epithelial cells. The persistence d

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199 of action of test agents was evaluated in immortalised, bronchial, epithelial cells (BEAS2B). m

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200 Each experiment consisted of one non-washout plate (96-well) and a parallel washout plate into tt
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201 which BEAS2Bs were seeded at a concentration of 3x104 cells/well in growth media (RPMI- /a
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202 1640, 2 mM L-glutamine, 10% FCS), and incubated for 24 h at 37oC, 5% CO . Test and .a
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203 reference articles or neat DMSO (as vehicle) (0.5 µl /well) were added to the appropriate wells of .o
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204 the washout plate to give a final concentration of 0.5% DMSO. The plate was incubated for
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205 exactly 1 h at 37oC and 5% CO . After the incubation time had elapsed all wells on the washout A
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206 plate were aspirated and growth media (100 µl/well) was added across the plate. After 24 h il 1
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207 incubation at 37°C, test and reference articles or neat DMSO (as vehicle) (0.5 µl /well) were 0
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208 added to the appropriate wells of the non-washout plate to give a final concentration of 0.5% b
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209 DMSO. The plate was incubated for exactly 1 h at 37oC and 5% CO after which A. fumigatus u
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210 conidia were added across both plates at a final concentration of 1 x 103/well. Fungal growth was
211 determined after a further 24 h incubation at 35°C, 5% CO , by measuring galactomannan (GM)
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212 concentrations, using Platelia GM-EIA kits (Bio-Rad Laboratories, 62794). The percentage
213 inhibition for each well was calculated and the IC value was calculated from the concentration-
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