In vitro and in vivo Evaluation of the Antifungal Activity of APX001A/APX001 Against Candida auris

20 Pages·2017·0.59 MB·English

AAC Accepted Manuscript Posted Online 8 January 2018
Antimicrob. Agents Chemother. doi:10.1128/AAC.02319-17
Copyright © 2018 Hager et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
1 Title: In vitro and in vivo Evaluation of the Antifungal Activity of APX001A/APX001 Against Candida
2 auris
3 Running title: Activity of APX001/APX001A Against Candida auris
4 Byline: Christopher L. Hager,1* Emily L. Larkin,1* Lisa Long,1 Fatima Z. Abidi,1 Karen J. Shaw,2
5 Mahmoud A. Ghannoum1#
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6 Affiliation: Center for Medical Mycology, University Hospitals Cleveland Medical Center and Case w
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7 Western Reserve University, Cleveland, Ohio, USA1; Amplyx Pharmaceuticals, San Diego, California, lo
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8 USA2 ed

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9 *CLH and ELL contributed equally to the work and should be considered joint first authors o
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10 # Corresponding author: h
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11 Mahmoud A Ghannoum, PhD, MBA, FIDSA //
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12 Center for Medical Mycology c
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13 Department of Dermatology m
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14 Case Western Reserve University and rg
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15 University Hospitals Cleveland Medical Center n

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16 Email: [email protected] n
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17 Phone: [phone] r
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20 Key words: APX001, APX001A, susceptibility testing, yeast, efficacy, Candida auris, infection model, b
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21 brain penetration, survival u
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23 [This accepted manuscript was published on 8 January 2018 with a standard copyright line (“© 2018
24 American Society for Microbiology. All Rights Reserved.”). The authors elected to pay for open access
25 for the article after publication, necessitating replacement of the original copyright line, and this change
26 was made on 22 January 2018.]
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28 ABSTRACT
29 Candida auris is an emerging multidrug-resistant yeast that has been responsible for invasive
30 infections associated with high morbidity and mortality. C. auris strains often demonstrate high
31 fluconazole and amphotericin B minimum inhibitory concentration values, and some strains are D
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32 resistant to all 3 major antifungal classes. Here we evaluated the susceptibility of 16 C. auris n
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33 clinical strains, isolated from a wide geographical area, to 10 antifungal agents including d
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34 APX001A, a novel agent that inhibits the fungal protein Gwt1 (GPI-anchored wall transfer fr
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35 protein 1). APX001A demonstrated significantly lower MIC50 and MIC90 values (0.004 µg/mL h
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36 and 0.031 µg/mL, respectively) than all other agents tested. :/
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37 The efficacy of the prodrug APX001 was evaluated in an immunocompromised murine c
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38 model of disseminated C. auris infection. Significant efficacy (80%-100% survival) was m
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39 observed in all three APX001 treatment groups versus 50% survival for the anidulafungin g
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40 treatment group. In addition, APX001 showed a significant log reduction in colony forming

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41 units (CFU) counts in kidney, lung and brain tissue (1.03 to 1.83) versus the vehicle control. u
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42 Anidulafungin also showed a significant log reduction in CFU in kidney and lung (1.5 and 1.62,
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43 respectively), but did not impact brain CFU. These data support further clinical evaluation of this
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44 new antifungal agent. 9

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45 g
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46 INTRODUCTION s
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47 Candida auris is an emerging multidrug-resistant fungal pathogen associated with
48 therapeutic drug failure and mortality (1). In a recent study, the mortality rate of patients with C.
49 auris infections was 59%, with patients dying either with, or as a result of the C. auris infection
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50 (2). A contributing factor is widespread resistance to fluconazole, and variable susceptibility to
51 amphotericin B, echinocandins, and other azoles. Examination of this collection of 54 isolates
52 from Pakistan, India, South Africa and Venezuela revealed that the isolates were resistant to
53 fluconazole (93%), voriconazole (54%), amphotericin B (35%), echinocandins (7%) and
54 flucytosine (6%) using stringent breakpoints inferred from other Candida species (2). Resistance D
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55 to echinocandins, the first-line treatment for Candida infections, is particularly problematic. In n
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56 the same study, two of the isolates were triply resistant to azoles, echinocandins and d
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57 amphotericin B, resulting in a dearth of treatment options among the three approved major drug fr
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58 classes. C. auris can persist in the environment for at least 2 weeks (3), and patients with C. auris h
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59 have been colonized for several months after the infection has resolved (1). This has led to :/
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60 environmental spread and transmission within health care facilities that are difficult to c
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61 decontaminate, and outbreaks that are difficult to treat (2, 4). m
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62 Contributing to the health threat is that C. auris is often misdiagnosed as Candida g
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63 haemulonii, Candida famata, or Rhodotorula glutinis and cannot be accurately identified using

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64 commercial yeast identification systems such as Vitek® 2 or API® 20C AUX. Instead, diagnosis nu
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65 relies upon matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) or
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66 molecular methods based upon sequencing the D1-D2 region of the 28s rDNA or the Internal
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67 Transcribed Region (ITS) of rDNA (1). This is problematic since many laboratories do not have 9

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68 rapid access to these technologies.
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69 C. auris was originally identified in a patient in Japan in 2009 (5). Since then, it appears s
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70 to have emerged simultaneously in several countries, resulting in the identification of four
71 distinct clades (2). International travel has expedited the spread of this species, which has now
72 been identified in numerous countries around the globe, including Japan, South Korea, South
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73 Africa, Kuwait, Kenya, United Kingdom, India, Pakistan, Colombia, Venezuela and the US (2,
74 6-9).
75 APX001 (formerly E1211) is a first-in-class small molecule antifungal drug candidate. It
76 is a water-soluble phosphate prodrug which is rapidly metabolized by systemic phosphatases to
77 the active moiety, APX001A (formerly E1210). APX001A has a novel mechanism of action that D
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78 is different than the other five classes of antifungal agents. APX001A targets the highly n
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79 conserved fungal enzyme Gwt1. This conserved enzyme catalyzes the glycosylphosphatidyl d
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80 inositol (GPI) post-translational modification that anchors eukaryotic cell surface proteins to the fr
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81 cell membrane (10, 11). In yeasts, GPI mediates cross-linking of cell wall mannoproteins to β- h
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82 1,6-glucan. Inhibition of this enzyme in both Candida albicans and Saccharomyces cerevisiae :/
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83 results in inhibition of maturation and localization of GPI-anchored mannoproteins. Inhibiting c
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84 Gwt1 blocks the inositol acylation step during synthesis of GPI anchored proteins of the fungal m
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85 cell wall which compromises cell wall integrity, biofilm formation, germ tube formation, and g
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86 produces severe fungal growth defects (10, 12). The closest mammalian ortholog of Gwt1 is the

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87 PIG-W protein, which is not sensitive to inhibition by APX001A (10). u
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88 APX001A has broad in vitro activity against major fungal pathogens, including, Candida,
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89 Cryptococcus, Aspergillus, Scedosporium, Fusarium and members of the Mucorales order (13-
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90 17). It maintains its activity against azole-resistant and echinocandin-resistant strains, which is 9

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91 consistent with the distinct mechanism of action. In invasive fungal infection animal models,
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92 administration of APX001 (or APX001A) resulted in high survival rates and reduced colony s
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93 counts of fungi in the lungs, kidney and brain of infected mice (18-21). The latter is particularly
94 important given the brain dissemination associated with several invasive fungal infections (22).
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95 In this study we evaluated the activities of APX001A and APX001 against a panel of 16
96 diverse C. auris isolates and in a disseminated candidiasis infection model, respectively. These
97 isolates were previously used to evaluate the activity of two compounds in clinical development,
98 the glucan synthesis inhibitor SCY-078 and the echinocandin CD101 (23, 24). The strains
99 included clinical isolates from Germany, Japan, Korea and India (23) and many demonstrated D
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100 elevated minimum inhibitory concentration (MIC) values for amphotericin B and fluconazole. n
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101 d
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102 RESULTS fr
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103 Antifungal susceptibility profile. APX001A was highly active against all 16 strains of h
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104 C. auris with markedly lower MIC50 and MIC90 values (concentration that inhibits 50 and 90% ://
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105 of the tested isolates, respectively) than the other tested antifungals with a MIC of 0.004 c
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106 µg/mL, a MIC of 0.031 µg/mL, and a range of values between 0.002–0.063 µg/mL (Table 1). m
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107 These values are equal to or lower than what has been previously observed for the activity of g
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108 APX001A against other Candida species (14, 16). The next most active agent was anidulafungin

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109 with MIC50 and MIC90 values of 0.125 and 0.25 µg/mL, respectively. Other compounds u
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110 demonstrated MIC90 values of 1 or 2 µg/mL, including flucytosine, caspofungin, micafungin, 1
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111 and the 3 azoles itraconazole, posaconazole and voriconazole. Amphotericin B was slightly less
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112 active, with MIC values of 4 µg/mL or 8 µg/mL when read at 24 h or 48 h, respectively (Table 9
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113 1). The latter values are above the amphotericin B epidemiological cutoff of 2 µg/mL for the 5
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114 other Candida species with Clinical and Laboratory Standards Institute (CLSI) interpretive s
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115 breakpoints, suggesting a trend for C. auris towards elevated MIC values or resistance (25).
116 Fluconazole was the least active drug tested with both an MIC and MIC >64 µg/mL,
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117 consistent with widespread resistance that has been reported (2).
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118 Survival in a disseminated C. auris infection model. A neutropenic mouse model of
119 disseminated candidiasis was developed to enable the in vivo assessment of the efficacy of
120 antifungal compounds against C. auris. To determine the optimal inoculum that would cause a
121 disseminated infection, immunocompromised mice (n = 5) were inoculated with 1×106 to 1×108
122 blastospores in 0.1 ml of phosphate buffered saline (PBS) (via the tail vein) of clinical isolates D
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123 CBS 12766 (India) and CBS 12777 (India). Survival was monitored as a marker of disease for n
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124 14 days. Based on these preliminary experiments, an inoculum of 3×107 blastospores of CBS d
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125 12766 was found to be optimal for use in subsequent efficacy studies (unpublished observations). fr
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126 APX001 intraperitoneal (IP) dosing regimens were chosen that included both twice daily h
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127 (BID) and thrice daily (TID) for two different dose levels (78 mg/kg and 104 mg/kg). These :/
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128 dose levels were chosen based upon doses successfully used in a previous animal model (20). c
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129 Phase 1 studies of APX001 have demonstrated a long half-life in man (~2 ½ days) (26, 27); m
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130 however, the half-life in mice is short (1 to 1.8 h depending upon route of administration), g
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131 necessitating multiple daily doses (20, 28). The anidulafungin dose (10 mg/kg BID IP or 20

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132 mg/kg/24 h) was chosen since it is above the dose associated with killing of 1 log colony u
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133 forming units (CFUs) of several strains of C. albicans (1.95-13 mg/kg/24 h) and Candida
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134 glabrata (3.31-11.8 mg/kg/24 h), which were previously determined for anidulafungin in similar
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135 murine neutropenic disseminated candidiasis models (29). 9

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136 In this disseminated C. auris infection model, 100% mortality was observed for the
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137 vehicle BID-treated control group by day 5 (Figure 1). Percent survival on day 16 for mice in s
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138 the APX001 78mg/kg BID, 78 mg/kg TID, 104 mg/kg BID, and anidulafungin 10 mg/kg BID
139 groups were 90%, 100%, 80%, and 50%, respectively. Mice in all APX001 treatment groups
140 had a significantly higher percent survival when compared to the anidulafungin (P = 0.034) and
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141 vehicle groups (P <0.0001). There was no statistically significant difference between the three
142 APX001-treated groups. The anidulafungin treated mice also had a significantly higher percent
143 survival when compared to the vehicle group (P < 0.0001).
144 Fungal burden: CFU reductions. In a subsequent study of disseminated C. auris
145 infection, mice were sacrificed at 48 h for determination of lung, kidney, and brain fungal burden D
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146 after IP dosing of APX001 (78 mg/kg BID, 78 mg/kg TID) and anidulafungin (10 mg/kg BID) n
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147 versus vehicle control. The 48 h time point was chosen to ensure 100% survival at the time of d
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148 sacrifice. Both doses of APX001 and anidulafungin demonstrated a significant log10 reduction in fro
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149 kidney CFUs/g (1.16 to 1.62) and lung CFUs/g (1.49 to 1.74) at 48 h versus the vehicle control h
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150 group (P = 0.0003) (Figure 2). There was no statistically significant difference in the reduction :/
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151 of fungal burden in lung and kidney for the two APX001-treated groups. c
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152 Since Candida infections can be hematogenously disseminated to other organs including m
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153 the brain, CFU counts were also enumerated from brain tissue at 48 h. The APX001 78 mg/kg g
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154 BID treatment group demonstrated a 1.83 and 1.48 log reduction when compared to both the

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155 vehicle control and the anidulafungin -treated group (P values of 0.0004 and 0.005, u
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156 respectively). The APX001 78 mg/kg BID and TID treatment groups were not statistically
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157 different from each other, although the APX001 TID treatment group did not reach statistical
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158 significance versus the vehicle control (P = 0.0691). 9

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159 At the 48 h time point, APX001 demonstrated a significant reduction in kidney, lung, and
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160 brain fungal burden, whereas anidulafungin demonstrated a significant reduction in only kidney s
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161 and lung fungal burden (Figure 2). These findings are consistent with poor central nervous
162 system (CNS) penetration observed for the echinocandins (30).
163
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164 DISCUSSION
165 The Center for Disease Control (CDC) has identified C. auris as a serious global health
166 threat that can cause serious invasive infections
167 (https://www.cdc.gov/fungal/diseases/candidiasis/candida-auris-qanda.html). C. auris is often
168 multidrug resistant, difficult to diagnose, and has caused outbreaks in healthcare settings. As a D
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169 result, new treatment options are required to combat this emerging health care threat, especially n
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170 for multi-drug resistant strains. In this study, we evaluated the in vitro activity of APX001A d
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171 against a panel of 16 diverse C. auris isolates as well as the in vivo activity of the prodrug fr
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172 APX001 in a disseminated candidiasis model that utilized one of the isolates that originated from h
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173 India (CBS 12766). :/
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174 APX001A demonstrated an MIC value that was 8-fold lower than the next most active c
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175 drug, anidulafungin, and >30 fold lower than all other compounds tested (Table 1). In addition m
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176 to the improved C. auris microbiological activity versus currently marketed drugs, APX001A g
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177 also demonstrated a lower MIC value than two other agents in clinical development, SCY-078
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178 and CD101, that were evaluated versus the same 16 strains; the MIC90 values for SCY-078 and u
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179 CD101 were 1 µg/mL and 0.25 µg/mL, respectively (23, 24). Recently, other compounds in
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180 development have been evaluated against a panel of 100 strains housed at the CDC representing
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181 the four C. auris clades that included triply-resistant isolates. Against this panel, MIC values 9
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182 for VT-1598, a fungal CYP51 inhibitor, and SCY-078 were both 1 µg/mL (31, 32).
g
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183 APX001 was evaluated in a disseminated C. auris infection model in s
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184 immunocompromised mice. The echinocandin anidulafungin was chosen as a control since it
185 was the second most microbiologically active agent and represents the class of agents most likely
186 to be used as first-line therapy (1, 9). The dose of anidulafungin that was chosen has been
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187 previously shown in pharmacokinetic/pharmacodynamic modeling experiments to be higher than
188 both the stasis dose and the dose resulting in 1 log kill of C. albicans and C. glabrata (29). Thus,
189 it was anticipated that this dose regimen would be effective in this model. The doses of
190 APX001 used in this model are greater than or equal to doses that have been previously shown to
191 be effective in animal models of candidiasis or other invasive fungal infections (18, 20, 28) and D
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192 achieve area under the concentration-time curve (AUC) after 24 h values in mice that are several n
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193 fold (~3) lower than the steady state AUC values anticipated to be used in Phase 2 studies in d
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194 man (26, 27). In the current model of disseminated C. auris infection, APX001 resulted in fr
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195 significantly better survival than anidulafungin. Both APX001 and anidulafungin demonstrated h
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196 significant and equivalent decreases in kidney and lung CFUs at 48 h. However, only APX001 :/
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197 demonstrated a reduction in brain CFUs, consistent with brain penetration observed in 14C- c
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198 APX001 distribution studies where radioactivity was detected in tissues associated with invasive m
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199 fungal infections (33). This is contrasted to the poor CNS penetration observed for the g
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200 echinocandins (30). It is possible that better brain penetration (or other unexamined

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201 compartments that show a differentiated distribution pattern such as eye) may have contributed u
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202 to better overall survival in this model.
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203 One limitation to the study is that only the APX001 78 mg/kg BID dosing, and not the 78
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204 mg/kg TID dosing, demonstrated a statistically significant reduction in brain CFUs. It is possible 9

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205 that larger numbers of animals in each treatment group would allow resolution of this
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206 discrepancy, or alternatively, evaluating fungal brain burden at a time point greater than 48 h. In s
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207 addition, the efficacy of APX001 administration in a delayed therapy model requires further
208 investigation.
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209 In this study, we demonstrated that APX001A and its prodrug form, APX001, has potent
210 activity against C. auris both in vitro and in vivo, respectively. APX001 may be a viable
211 treatment option for infections attributable to the emerging nosocomial pathogen C. auris,
212 especially due to strains that are resistant to multiple drug classes.
213 D
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214 n
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215 MATERIALS AND METHODS d
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216 Isolates tested. A total of 16 clinical isolates of C. auris from a wide geographic area fr
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217 (Germany, Japan, S. Korea, and India) were tested for their susceptibility to 10 different h
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218 antifungal agents. 14 of the C. auris isolates were obtained from the CBS-KNAW Fungal :/
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219 Biodiversity Centre, Utrecht, the Netherlands (CBS # 10913, 12372, 12373, 12766-68, 12770- c
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220 77) and the other two strains were from the Center for Medical Mycology Culture Collection m
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221 previously characterized (23, 24). Candida krusei ATCC 6258 and Candida parapsilosis ATCC g
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222 22019 were used as quality control strains in susceptibility testing. One of the clinical isolates of

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223 C. auris (CBS 12766, isolated in India) was used as the infecting strain in the animal model of u
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224 disseminated infection.
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225 Antifungal agents. The activities of 10 antifungals against C. auris were tested,
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226 including amphotericin B, anidulafungin (Pfizer Pharmaceuticals, New York, NY, USA), 9

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227 caspofungin (Merck Co., Kenilworth, NJ USA), fluconazole, flucytosine (Astellas Pharma US,
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228 Inc., Northbrook, IL, USA), itraconazole, micafungin (Astellas Pharma, Inc., Tokyo, Japan), s
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229 posaconazole and voriconazole (AMB, FLC, 5FC, ITC, POS, AND VRC were obtained from
230 Sigma-Aldrich, St. Louis, MO, USA). APX001A was originally synthesized at Eisai Co. Ltd.,
231 Tokyo, Japan and obtained from Amplyx Pharmaceuticals.
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