1 1 2 NB-002, a Novel Nanoemulsion with Broad Antifungal Activity Against Dermatophytes, 3 ...

30 Pages·2009·4.45 MB·English

AAC Accepts, published online ahead of print on 11 May 2009
Antimicrob. Agents Chemother. doi:10.1128/AAC.00218-09
Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
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3 NB-002, a Novel Nanoemulsion with Broad Antifungal Activity Against Dermatophytes,
4 Other Filamentous Fungi and Candida albicans
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6 J. Pannu,1 A. McCarthy,1 A. Martin,1 T. Hamouda,1 S. Ciotti,1A. Fothergill,2 and J. Sutcliffe1* lo
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8 NanoBio Corporation, 2311 Green Road, Suite A, Ann Arbor, MI 48105,1 and Fungal Testing ro
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9 Laboratory, University of Texas Health Science Center, San Antonio, TX 782292 h
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11 Running Title: Antifungal activity of NB-002 .
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12 Key words: nanoemulsion, antifungal, dermatophyte, fungicidal, cutaneous infections
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14 Corresponding Author: New Contact Info: N
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15 Joyce Sutcliffe Joyce Sutcliffe e
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16 VP, Research SVP, Research e
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17 NanoBio Corporation Tetraphase Pharmaceuticals, Inc. 6
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18 2311 Green Rd., Suite A 480 Arsenal St., Suite 110 1
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19 Ann Arbor, MI 48105 Watertown, MA 02472 y

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20 Email: [email protected] Email : [email protected] e
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21 Phone: [phone] Phone : [phone]
22 Fax : [phone] Fax: [phone]
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23 Abstract
24 NB-002 is an oil-in-water emulsion designed for use in skin, hair and nail infections. NB-002
25 was compared to available antifungal drugs against the major dermatophytes responsible for
26 cutaneous infections, Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton
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27 floccosum and Microsporum spp., as well as 12 other genera of filamentous fungi. NB-002 o
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28 consistently displayed fungicidal activity against all dermatophytes. The comparator compounds lo
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29 were either fungistatic or fungicidal and for some strain/drug combinations, tolerance was e
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30 observed. Assessment of spontaneous resistance development in different dermatophyte species o
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31 yielded few stably resistant mutants. For filamentous non-dermatophyte fungi, the MIC range h
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32 varied from ≤0.03-0.5 µg/ml for Alternaria spp. to 2-8 µg/ml for Paecilomyes spp. NB-002 had //
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33 activity against yeast, both azole-susceptible and –resistant Candida albicans isolates, with .
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34 MIC /MFC values of 2/4 and 2/8 µg/ml, respectively. The kinetics of fungicidal activity of
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35 NB-002 against T. rubrum isolates compared to other antifungal drugs was assessed. NB-002 /

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36 killed both mycelia and microconidia even when the fungal forms were dormant or not actively N
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37 growing. Electron micrographs of mycelia and spores treated with NB-002 showed significant e
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38 disruption of the fungal structure. The in vitro broad coverage of NB-002 against filamentous e
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39 fungi, dermatophytes and C. albicans as well as its rapid fungicidal activity warrant further 6
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40 investigations to ascertain if NB-002 would be useful for the treatment of cutaneous mycoses. 1
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41 INTRODUCTION
42 Superficial fungal infections are found in the top layers of the skin and mucous membranes, the
43 hair, and nails. Examples of fungal infections of the skin and other external surfaces include
44 athlete's foot, jock itch, ringworm, and other tinea infections. Most of these infections are caused
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45 by three genera of dermatophytes, Trichophyton, Epidermophyton and Microsporum spp. (5, 6, o
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46 22, 31, 45, 47). lo
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47 Filamentous fungi that are normal soil saprophytes have also emerged as major opportunistic
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48 fungi, especially in immunosuppressed patients (35, 53). Such organisms include Aspergillus

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49 spp. (3, 20, 35, 42, 49), Fusarium spp. (20, 24, 30, 35, 37, 49), Scedosporium spp. (20, 35), tp
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50 Paecilomyces spp. (25, 26), Scopulariopsis spp (20), Scytalidium spp. (13),Chaetomium spp.(15) a
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51 Alternaria spp. (4, 20, 49), Acremonium spp. (20) and Curvularia spp. (49). Yeast such as C. s
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52 albicans also cause skin infections, generally at sites between the fingers, toes, around the anus
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53 and on the penis or at sites of abrasion where the skin is continuously moist (46). o
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54 Most cutaneous skin infections are treated with topical antifungals containing naftidine, e
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55 tolnaftate, terbinafine or itraconazole. Oral therapies of griseofulvin, terbinafine and e
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56 itraconazole are used to treat tinea capitis. Nail infections can be treated with oral agents as well 6
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57 as the topical agent ciclopirox. Terbinafine and azole-like compounds carry the risk of liver and 0
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58 cardiac side effects and drug-drug interactions (1, 2, 16). Topical therapies for inflammatory b
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59 dermatomycoses often combine an azole and a corticosteroid to rapidly reduce inflammatory
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60 symptoms and to increase the bioavailability of the antifungal agent (32).
61 Antimicrobial nanoemulsions are highly stable oil-in-water emulsions composed of nanometer-
62 sized, positively-charged droplets that have broad-spectrum activity against enveloped viruses,
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63 fungi and bacteria (7, 19, 27-29, 36, 43, 48). NB-002 contains the cationic quaternary
64 ammonium compound cetylpyridinium chloride (CPC) oriented at its oil-water interface that
65 stabilizes the nanoemulsion droplets, contributes to the anti-infective activity and serves as a
66 marker for the activity.
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67 Studies with NB-002 containing fluorescein have shown that the nanodroplets permeate human w
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68 cadaver skin using a transfollicular route (8). Using a modified Franz cell apparatus, NB-002 a
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69 was also shown to diffuse laterally from hair follicles and sebaceous glands along tissue planes
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70 to reach concentrations in excess of 200 µg/g in human cadaver epidermis as far as 1 cm from

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71 the site of application (9). This concentration is significantly above the MIC /MFC or p
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72 MIC/MFC range values determined in this work, ≤4 µg /ml for Trichophyton spp., Microsporum a
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73 spp., and Epidermophyton floccosum. s
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74 These studies assessed the microbiological activity of NB-002 against fungal pathogens of /

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75 cutaneous skin infections. Further, we show that the fungicidal activity of NB-002 is rapid, N
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76 killing both microconidia and mycelia of the dermatophyte T. rubrum. Consistent with this “kill- e
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77 on-contact” mechanism of action, the majority of dermatophyte isolates spontaneously resistant e
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78 to NB-002 had MICs ± 2-fold of the initial MIC . 6
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79 MATERIALS AND METHODS 8

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80 Nanoemulsion manufacturing and potency. NB-002 is an oil-in-water emulsion manufactured u
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81 from ingredients that are on the FDA list of recognized inactive ingredients in drug substances.
82 The emulsion is formed from highly purified oil, ethanol, polysorbate 20, cetylpyridinium
83 chloride and water. The average nanoemulsion droplet size is 180 nm as measured by dynamic
84 light scattering using a Malvern Zetasizer Nano 3600 (Malvern Instruments Ltd., Worcestershire,
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85 UK). The relative activity of NB-002 is expressed in terms of the concentration of cationic
86 surfactant present as we have done previously in clinical trials with NB-001, a formulation of the
87 same components, for treatment of herpes labialis (33). Thus, the antifungal activity of NB-002
88 is expressed in µg CPC/ml.
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89 Source of comparator compounds. Amphotericin B, ciclopirox, itraconazole, terbinafine and
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90 tolnaftate were purchased from Sigma Chemicals (catalog numbers A4888, C0415, I6657, T8826 a
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91 and T6638 respectively.) Naftifine (catalog number 1450404) was obtained from United States
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92 Pharmacopeia (USP).

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93 Source of fungal isolates. Dermatophytes T. rubrum, T. mentagrophytes and E. floccosum were //
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94 clinical trial specimens, from either untreated subjects under evaluation for participation in .
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95 clinical trials or from blinded study-treated subjects in ongoing trials undergoing treatment with
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96 an azole or allylamine for onychomycosis, tinea pedis, tinea corporis, and tinea unguium /

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97 (Mycology Consultants Laboratory, Holland, Michigan). The terbinafine-resistant T. rubrum N
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98 isolates were from The Center for Medical Mycology Case Western Reserve University, e
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99 Cleveland, Ohio (21). The Microsporum spp. isolates (5 each of M. canis, M. nanum and M. e
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100 gypseum) were from the American Type Culture Collection. A total of 65 clinical isolates of 6
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101 filamentous fungi included five Aspergillus spp. (A. sydowii (1), A. terreus (3), A. niger (1)), four 1
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102 Paecilomyces lilacinus, four Fusarium oxysporum, five Fusarium solani, one Fusarium y

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103 semitectum, , three Trichophyton soudanense, three Trichopyton verrucosum and three e
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104 Epicoccum nigrum. Other isolates that were tested with identification to genus level only
105 include five Acremonium spp., five Scopulariopsis spp., five Scedosporium spp., ten Scytalidium
106 spp. ,three Alternaria spp., three Curvularia spp., Phoma spp. and Chaetomium spp.. All these
107 filamentous fungi were recovered from dermatologic sources by The Fungus Testing Laboratory,
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108 University of Texas Health Science Center, San Antonio, Texas. C. albicans isolates were
109 obtained from David Stevens (Stanford University, Stanford,California) and are described in

110 White, et al. (51).
111 MIC/MFC determinations. MICs were determined using microtiter broth dilution
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112 methodology in RPMI 1640 medium as described in CLSI method for filamentous fungi (11). w
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113 For dermatophytes a hemacytometer count of conidia was done to check the initial inoculum a
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114 count. In most of the dermatophyte studies, the inoculum level was increased from 1-3 x 103 d
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115 cfu/ml to 1-3 x 104cfu/ml to allow for determination of MFCs by accepted statistical methods (10, m

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116 44). Pre-made microtiter plates containing each of the drugs at twice the desired final tp
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117 concentration serially diluted as described in the Clinical Laboratory Standards Institute (CLSI) a
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118 document for testing of filamentous fungi were inoculated with twice the final desired inoculum. s
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119 The final volume in each well was 200 µl. Plates were incubated at 35°C for 4 days and then
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120 examined visually for 80% (100% for NB-002) growth inhibition endpoints. T. rubrum MYA- o
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121 4498 and T. mentagrophytes MYA-4439, the two dermatophyte quality control strains o
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122 recommended in CLSI document M38-A2, were included on each day of testing.
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123 For molds, antifungal susceptibility testing followed the CLSI methodology that used RPMI- 6
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124 1640 with glutamine and without bicarbonate, an inoculum size of 0.4-5 × 104, and incubation at 0
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125 35°C for 48 hours (11, 12). Paecilomyces variotii ATCC MYA-360-3630 was the quality b
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126 control isolate. Testing of amphotericin B was conducted in Antibiotic Medium 3 (Difco,
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127 Detroit, MI). The MIC was defined as the lowest concentration that resulted in an 80% reduction
128 in turbidity as compared to a drug-free control for each isolate/drug combination for terbinafine
129 and ciclopirox; NB-002, amphotericin B and itraconazole were read at the lowest concentration
130 that was optically clear for non-dermatophyte species. The MIC and the MIC are the lowest
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131 concentration of antifungal agent that collectively inhibits the growth of 50% or 90% of the
132 isolates tested, respectively.
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134 For minimum fungicidal concentration (MFC) determinations, 100 µl was removed from each
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135 well at the MIC and at least four concentrations above the MIC and plated onto Sabourard w
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136 Dextrose Agar. To avoid antifungal carryover, the aliquots were allowed to soak into the agar a
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137 and then were streaked for isolation once dry, thus removing the cells from the drug source.
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138 MFC was defined as the lowest concentration of drug that reduced the initial inoculum by ≥3

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139 logs. A compound works by a fungicidal mechanism if the MFC/MIC ratio is ≤4; if the tp
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140 compound is fungistatic, then the MFC/MIC ratio will be >4 (10). The MFC and the MFC a
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141 are the lowest concentration of antifungal agent that collectively kills (≥3 log reduction) 50% or s
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142 90% of the isolates tested, respectively. Tolerance was defined as a MBC/MIC ratio of ≥32 (10,
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143 44). o
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144 e
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145 Susceptibility studies with C. albicans isolates were determined using microtiter broth dilution r

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146 methodology as specified by CLSI (12). A change to a denser inoculum (1-4 x 104 cfu/ml) was ,
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147 done to allow for the MFC determination upon removal of 100 µl from each well where there 8

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148 was no growth (at the MIC) and at least four concentrations above the MIC (10, 44). Colony
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149 counts were determined on Sabouraud dextrose agar (SDA) after incubation at 35°C for 4 days. s
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150 Both Candida parapsilosis ATCC22019 and Candida krusei ATCC6258 were included as
151 quality control isolates and tests were validated by the quality ranges for fluconazole,
152 amphotericin B, and itraconazole as described in the CLSI guidance (12).
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153 Susceptibility of terbinafine-resistant T. rubrum to NB-002. Clinical isolates with decreased
154 susceptibility to terbinafine were part of The Center for Mycology collection (Case Western
155 Reserve University, Cleveland, Ohio) (21) and were tested in the laboratory of Mahmoud
156 Ghannoum for susceptibility to NB-002 and terbinafine according to recent CLSI standards (11).
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157 Determination of spontaneous resistance frequencies. RPMI 1640 agar plates containing 4X, w
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158 8X or 16X the respective MIC of NB-002, itraconazole, terbinafine or ciclopirox were inoculated a
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159 with 107 – 108 cfu of E. floccosum (two isolates), T. mentagrophytes (five isolates) and T. rubrum d
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160 (five isolates) and incubated for 7-8 days at 35ºC. Colonies recovered from the plates were

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161 cloned to RPMI 1640 agar plates containing the compound at the selecting concentration. tp
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162 Microtiter broth or agar-based MICs were determined for recovered isolates by CLSI a
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163 methodology (11) except that a higher inoculum (1-4 x 104 cfu/ml) was used. To ensure that s
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164 drug selective pressure was maintained, putative drug-resistant isolates were cultured on RPMI
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165 1640 agar plates containing the drug concentration to which they were initially selected during o
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166 the outgrowth of the frozen cultures. The percentage of spontaneous resistance for an o
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167 isolate/drug combination was calculated from the number of colonies that grew on plates
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168 containing drug versus the number of colonies that grew on unsupplemented agar.
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169 Time-kill studies and electron microscopy. For time-kill experiments and electron 0
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170 `micrographs of mechanism of action, microconidia were harvested from 7 day-old cultures of T. b
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171 rubrum growing on potato dextrose agar using sterile distilled water and adjusted to a u
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172 concentration of 106 conidia/ml (34, 44). Part of the conidial suspension was pelleted and
173 resuspended in RPMI 1640 medium and grown for 16-18 hours overnight at room temperature to
174 allow germination of microconidia (34). After germination, the hyphae were collected by
175 centrifugation and resuspended in distilled water. After mixing with different concentrations of
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176 NB-002 or a comparator compound, the rate of killing of microconidia and mycelia was
177 followed for up to 24 hours by plating 0.1 ml of 10-1, 10-2 and 10-3 dilutions onto SDA. Colony-
178 forming units were counted after four days of incubation at 35°C. Control experiments
179 determined that samples containing NB-002 had to be diluted 1:100 to remove residual activity
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180 (data not shown). o
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181 For scanning electron microscopy (SEM), samples (450 µl) from different time points during a a
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182 time-kill study with NB-002 were mixed with 113 µl of fixative (10% aqueous solution of
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183 glutaraldehyde). Mixtures were vortexed and placed at 4°C for at least 18 hours. Samples were

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184 fixed in 1.0 % OsO in Sorenson’s buffer, taken through a gradual series of dehydration steps p
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185 with ethanol, and mounted on SEM stubs using the mixture of colloidal graphite and duco a
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186 cement. Samples for electron microscopy were sputter-coated with gold using the Polaron s
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187 sputter coater (Quorum Technologies, UK) and examined on an Amray 1910 FE Scanning r
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188 Electron Microscope and digitally imaged using Xstream imaging software (SEMTech Solutions, n

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189 Inc. North Billerica, Mass.).
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190 RESULTS e
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191 NB-002 is fungicidal for dermatophytes. The antifungal activity of NB-002, its topical 2
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192 comparators ciclopirox, tolnaftate and naftifine, and oral comparators terbinafine, itraconazole 8

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193 and griseofulvin were evaluated using clinical isolates of dermatophytes. NB-002 was the only g
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194 antifungal that was consistently fungicidal against isolates of T. rubrum, T. mentagrophytes, T. s
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195 tonsurans, E. floccosum and Microsporum spp. (Table 1). Ciclopirox, itraconazole, tolnaftate
196 naftifine and griseofulvin had MICs that ranged from 0.125-2 µg/ml, 0.031-0.5 µg/ml, 0.016-1.0
197 µg/ml, 0.016-0.25 µg/ml and 0.25-4 µg/ml, respectively, but none were fungicidal for any
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198 species. Terbinafine was fungicidal for T. rubrum isolates only. Fungicidal activity was not
199 determined for T. soundanense, but the two most active compounds were NB-002 and
200 terbinafine, with MIC ranges of ≤0.03-0.06 µg/ml and ≤0.004-0.015 µg/ml, respectively.
201 NB-002 was tested against five clinical isolates of T. rubrum characterized as having elevated
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202 MICs to terbinafine by The Center for Medical Mycology (Case Western Reserve University, w
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203 Cleveland, Ohio) (Table 1) (21). The MIC and MFC ranges were 2 – 4 µg/ml and 2 – 8 µg/ml a
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204 for NB-002, respectively. All isolates had a MIC/MFC of >8 µg/ml for terbinafine.
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205 Antibiotic susceptibilities of filamentous fungi. The antifungal activity of NB-002 against 12 h
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206 genera of filamentous fungi was assessed (Table 2). Nearly all isolates were susceptible to ≤4 :/
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207 µg/ml NB-002, with an overall range of MICs of ≤0.03-8 µg/ml. Against certain genera, other c
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208 compounds were either uniformly inactive or had mixed potency. NB-002 distinguished itself
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209 from amphotericin B, itraconazole and terbinafine because of its potency against Scopulariopsis g
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210 spp. and Scedosporium spp. and was superior to ciclopirox because of its activity against
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211 Fusarium spp. and Paecilomyces spp. e
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212 Antibiotic susceptibilities of C. albicans to NB-002 and comparators. NB-002 was r

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213 unvaryingly fungicidal against the panel of C. albicans clinical isolates (Tables 3A and 3B). The
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214 MIC and MFC values for the 34 isolates were 2 and 8 µg/ml, respectively. The strains were 8
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215 susceptible to amphotericin B with MIC and MFC values of 2 µg/ml. The majority of isolates
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216 were resistant to the two azoles tested, fluconazole and itraconazole, with MIC values of >64 s
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217 and >16 µg/ml, respectively. Ciclopirox had a MIC of 1 µg/ml, but its MFC value and range
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218 indicated that it was uniformly fungistatic against the isolates. NB-002 had nearly identical
219 MIC /MFC values for both azole-susceptible (2/4 µg/ml) and –resistant (2/8 µg/ml) isolates.
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