Comparison of in vitro antifungal activities of efinaconazole and. currently available antifungal agents against a variety of pathogenic

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AAC Accepts, published online ahead of print on 14 January 2013 Antimicrob. Agents Chemother. doi:10.1128/aac.02056-12 Copyright 2013, American Society for Microbiology. All Rights Reserved. 1 2 3 Comparison of in vitro antifungal activities of efinaconazole and currently available antifungal agents against a variety of pathogenic fungi associated with onychomycosis 4 5 6 William J. Jo Siu 1#, Yoshiyuki Tatsumi 2, Hisato Senda 2, Radhakrishnan Pillai 1, Takashi Nakamura 2, Daisuke Sone 2, Annette Fothergill 3 7 8 9 10 11 12 13 1 Dow Pharmaceutical Sciences, a Division of Valeant Pharmaceuticals North America LLC, 1330 Redwood Way, Petaluma, CA 94954, U.S.A. 2 Kaken Pharmaceutical Company, Ltd., 14, Shinomiya, Minamigawara-cho, Yamashinaku, Kyoto 607-8042, Japan 3 University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, U.S.A. 14 15 16 17 18 # Corresponding author Tel.: 707-793-2600 Fax: 707-793-0145 Email: wjo@dowpharmsci.com 19 20 21 Running Title: In vitro antifungal activity of efinaconazole Keywords: efinaconazole, triazole antifungal, tinea unguium, onychomycosis 22 1

23 Abstract 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Onychomycosis is a common fungal nail infection in adults that is difficult to treat. The in vitro antifungal activity of efinaconazole, a novel triazole antifungal, was evaluated in recent clinical isolates of T. rubrum, T. mentagrophytes and C. albicans, common causative onychomycosis pathogens. In a comprehensive survey of 1493 isolates, efinaconazole minimum inhibitory concentrations (MIC) against T. rubrum and T. mentagrophytes ranged from 0.002 to 0.06 µg/ml, with 90% of isolates inhibited (MIC 90 ) at 0.008 and 0.015 µg/ml, respectively. Efinaconazole MICs against 105 C. albicans isolates ranged from 0.0005 to >0.25 µg/ml, with 50% of isolates inhibited (MIC 50 ) by 0.001 and 0.004 µg/ml at 24 and 48 hours, respectively. Efinaconazole potency against these organisms was similar or greater compared to antifungal drugs currently used in onychomycosis, including amorolfine, ciclopirox, itraconazole and terbinafine. In 13 T. rubrum toenail isolates from onychomycosis patients who were treated daily with topical efinaconazole for 48 weeks, there were no apparent increases in susceptibility, suggesting low potential for dermatophytes to develop resistance to efinaconazole. The activity of efinaconazole was further evaluated in other 8 dermatophyte, 15 nondermatophyte and 10 yeast species (109 isolates total from research repositories). Efinaconazole was active against Trichophyton, Microsporum, Epidermophyton, Acremonium, Fusarium, Paecilomyces, Pseudallescheria, Scopulariopsis, Aspergillus, Cryptococcus, Trichosporon and Candida, and compared favorably to other antifungal drugs. In conclusion, efinaconazole is a potent antifungal 2

45 46 with broad spectrum of activity that may have clinical applications in onychomycosis and other mycoses. 47 48 Introduction 49 50 51 52 53 54 55 Onychomycosis (tinea unguium) is a chronic fungal infection of the nail characterized by nail discoloration, thickening and deformity (1). The disease, although not lifethreatening, can markedly affect quality of life and well-being (1). It is the most common nail disorder in older adults, frequently involving several nails and affecting the toenail in 80% of cases (2). Onychomycosis can be caused by dermatophytes, nondermatophytes and yeasts, and the species prevalence in nail infections varies with geographical location, climate and migration (3). 56 57 58 59 Dermatophytes, mainly Trichophyton rubrum and Trichophyton mentagrophytes, are the predominant causative agents of fingernail and toenail onychomycosis, accounting for approximately 50-90% of cases, with infection prevalence in toenails much higher than in fingernails (3-6). 60 61 62 63 64 65 66 The role of nondermatophytes and yeasts as sole causative agents of onychomycosis has not been fully established and, in several cases, these organisms are considered to be colonizing organisms or contaminants rather than pathogens (6, 7). However, in recent years, yeasts have been increasingly recognized as pathogens in fingernail infections, as well as other nondermatophytes (6, 8). Isolation of an organisms from infected nail tissue is not proof that it is the causative pathogen, and positive potassium hydroxide and histopathological examination demonstrating fungus invasion of the nail plate are usually 3

67 required to confirm disease etiology (8). 68 69 70 71 72 Among yeasts, Candida albicans and Candida parapsilosis are most commonly implicated in onychomycosis (4, 5). Candida species have a high incidence in fingernail infections, present in as many as 75% of cases, and are more prevalent than dermatophytes (4). In contrast, the incidence of yeasts in toenail infections is much lower, approximately 2-10% of cases. 73 74 75 76 77 78 79 The incidence of nondermatophytic fungi such as Scopulariopsis, Scytalidium, Acremonium, Fusarium and Aspergillus in onychomycotic nails is lower, 2 21% of cases (5, 6). Infections attributed to nondermatophytes are estimated to be 2-6% of cases although higher rates, 15%, have been reported (8, 9). Nondermatophyte onychomycosis is seen most frequently in the elderly, in patients with skin disease that affect the nails and in immunocompromised patients, and is more frequent in toenails than in fingernails (6, 8). 80 81 82 83 84 85 86 87 88 Clinical management of onychomycosis includes prescription systemic and topical antifungal treatment. Currently used antifungals include terbinafine (an allylamine), amorolfine (a morpholine), itraconazole (a triazole) and ciclopirox (a hydroxypiridone). Ciclopirox, itraconazole and terbinafine are approved in the U.S.A. and around the world for onychomycosis treatment while amorolfine and fluconazole are approved in Europe. In spite of the availability of several therapeutic options, onychomycosis remains difficult to treat, and new drugs with improved safety and efficacy are needed. Efinaconazole (also known as KP-103) is a novel triazole antifungal that inhibits lanosterol-14α demethylase and blocks fungal membrane ergosterol biosynthesis, a mechanism of action 4

89 90 91 92 93 94 95 96 97 98 99 100 101 102 consistent with other azole antifungals (Tatsumi et al., under peer review). Efinaconazole is active in vitro against dermatophytes including Trichophyton, Microsporum and Epidermophyton species, responsible for tinea unguium, tinea corporis, tinea pedis and tinea capitis (10). It is also effective against C. albicans and other Candida species responsible for nail and skin candidiasis, and Malassezia species responsible for tinea versicolor. Further, efinaconazole administered topically is effective in reducing fungal burden in guinea pig models of tinea unguium and tinea pedis (11). In this study, we aimed to characterize the in vitro antifungal activity of efinaconazole. We conducted a comprehensive assessment of efinaconazole s activity against recent clinical isolates recovered from onychomycosis patients. We further compared its activity to that of terbinafine, ciclopirox, itraconazole and amorolfine in T. rubrum, T. mentagrophytes and C. albicans isolates. In addition, we characterized efinaconazole s spectrum of activity against various fungal pathogenic species, many of which are associated with onychomycosis and other superficial mycoses. 103 104 Materials and methods 105 106 107 108 109 110 Antifungal Agents. Efinaconazole was supplied by Kaken Pharmaceutical Co., Ltd., (Kyoto, Japan). Efinaconazole, amorolfine (Tokyo Chemical Industry Co. Ltd., Tokyo, Japan; 3B Pharmachem International (Wuhan) Co. Ltd., Libertyville, IL), ciclopirox (Sigma Aldrich, St. Louis, MO), itraconazole (Sigma Aldrich) and terbinafine (Tokyo Chemical Industry Co. Ltd) were dissolved in dimethyl sulfoxide (DMSO) to make 100x 5

111 112 or 200x stock solutions. Stocks were freshly prepared before use or stored frozen until use. 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 Fungal isolates. A total of 1751 clinical isolates were used for in vitro susceptibility testing. One hundred and six T. mentagrophytes and 1387 T. rubrum isolates were recovered from toenail onychomycosis patients in the U.S.A., Canada and Japan, and an additional 44 T. mentagrophytes clinical isolates from U.S.A. patients were obtained from the Fungus Testing Laboratory collection, University of Texas Health Science Center at San Antonio, Texas, U.S.A. One hundred and five C. albicans clinical isolates recovered from skin, nails and oral and vaginal mucosa, were obtained from the Fungus Testing Laboratory collection. The T. mentagrophytes, T. rubrum and C. albicans isolates were recovered between 2009 and 2011. One hundred and nine research repository isolates of other dermatophyte (Tricophyton, Microsporum and Epidermophyton), nondermathophyte (Acremonium, Fusarium, Paecilomyces, Pseudallescheria, Scopulariopsis, and Aspergillus) and other yeast (Candida, Trichosporon and Cryptococcus) species were obtained from several Japanese bio-resource centers and from the American Type Culture Collection (ATCC). These included a total of 33 species, with the number of isolates per species ranging from 1 to 13. The collection date for these isolates was not available. The reference strains T. mentagrophytes ATCC- MYA-4439, T. rubrum ATCC-MYA-4438, C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 were used for assay validation. 132 6

133 134 135 136 137 138 Fungal isolates post-efinaconazole therapy. Thirteen T. rubrum clinical isolates from toenail onychomycosis patients treated topically with efinaconazole were tested for efinaconazole susceptibility in vitro. These isolates were obtained from two identical, multi center, multi country, vehicle-controlled Phase 3 clinical trials with topical efinaconazole 10% applied daily to toenails for 48 weeks. Isolates were recovered at the end of treatment (study week 48) and four weeks afterwards (study week 52). 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 In vitro antifungal susceptibility. Testing was conducted using the broth dilution minimum inhibitory concentration (MIC) assay following the standard procedures as described in the Clinical and Laboratory Standards Institute (CLSI) documents M38-A2 and M27-A3. Clinical isolates were tested against efinaconazole, amorolfine, ciclopirox, itraconazole and terbinafine. Drug stock solutions (100x or 200x) in DMSO were serially diluted 1/2 in DMSO to obtain a range of ten concentrations, which were further diluted 1/100 or 1/50 in RPMI media. The final DMSO concentration was 0.5% or 1% in the drug microdilution plates. Concurrent DMSO vehicle control wells were included as the 100% growth controls. The MIC was determined in fungal cultures at 96 hours postinoculation for dermatophytes. For nondermatophytes, the MIC was determined at 48 hours except for A. potronii and S. brumptii (7 days). For yeasts, the MIC was determined at 24 and 48 hours except for C. neoformance (72 hours). Reference strains were assayed concurrently on each test day, and drug MICs compared to CLSI quality control ranges (when established). 154 7

155 156 157 158 159 160 161 162 Statistical analysis. MIC data for each drug were log (base 2) transformed in Excel 2003 (Microsoft Corp., Redmond, WA), and compared using the repeat measure ANOVA and Tukey s multiple comparison tests (α = 0.05) in Prism version 5.04 (Graphpad Software, Inc., La Jolla, CA, U.S.A.). For MICs with values above the highest concentration tested, the next higher multiple of two was used as the MIC in the statistical analysis. For MIC values equal to or below the lowest concentration, the lowest concentration tested was used. Correlation analysis between drug pairs MIC was conducted with the Spearman correlation function in Prism (two-tailed, CI = 95%). 163 164 Results 165 166 167 168 169 170 171 172 173 174 175 176 Susceptibility pattern of efinaconazole against T. mentagrophytes and T. rubrum onychomycosis clinical isolates. The in vitro susceptibility to efinaconazole was determined in recent clinical isolates of T. rubrum (n = 1387) and T. mentagrophytes (n = 106) from onychomycosis patient toenails in the U.S.A., Canada and Japan. Control wells containing culture media and DMSO vehicle alone showed normal dermatophyte growth with no apparent vehicle effect. Efinaconazole inhibited fungal growth at concentrations ranging from 0.002 to 0.06 µg/ml (Table 1), with most T. rubrum and T. mentagrophytes isolates inhibited at 0.008 and 0.015 µg/ml, respectively, based on MIC 90. The MICs of 58% of isolates was 0.002 µg/ml. Overall, efinaconazole activity against T. mentagrophytes and T. rubrum isolates was comparable. There were no differences in susceptibility pattern by geographical location; the MIC range, MIC 50 and 8

177 178 179 180 MIC 90 were comparable between U.S.A., Canada and Japan. Reproducible MIC results were obtained for efinaconazole when using the reference strains T. mentagrophytes ATCC-MYA-4439 and T. rubrum ATCC-MYA-4438 in a series of more than 30 separate sets of assays conducted over 12 months (data not shown). 181 182 183 184 185 186 187 188 189 190 191 192 193 Susceptibility of T. rubrum isolates recovered from toenail onychomycosis patients treated topically with efinaconazole for 48 weeks. In order to evaluate potential changes in fungal susceptibility to efinaconazole after prolonged treatment, the MICs of isolates from onychomycosis patients treated with efinaconazole were determined. A total of 13 T. rubrum isolates were tested, which were recovered from patient toenails following once daily topical application of efinaconazole for 48 weeks (immediately after treatment cessation and four weeks afterwards). The efinaconazole MICs ranged from 0.002 to 0.015 µg/ml and were within the range for isolates from untreated patients. Ten of these 13 isolates had a corresponding isolate collected prior to treatment from the same patient toenail; the MIC of 6 increased minimally at post-treatment relative to screening (highest change was from 0.002 to 0.008 µg/ml), 1 decreased and 3 did not change. 194 195 196 197 198 199 In vitro antifungal activity of efinaconazole and comparative antifungals against the dermatophytes T. rubrum and T. mentagrophytes. The in vitro susceptibility to efinaconazole was compared to amorolfine, ciclopirox, itraconazole and terbinafine in recent isolates of T. rubrum (n = 130) and T. mentagrophytes (n = 129), mostly recovered from onychomycosis patients. Due to the high number of isolates with efinaconazole 9

200 201 202 203 204 205 206 207 208 209 210 211 212 213 MICs 0.002 µg/ml in the large-scale evaluation of clinical isolates as reported above, the test concentration range was decreased by two dilutions and the lowest concentration was 0.0005 µg/ml. Efinaconazole inhibited fungal growth at concentrations ranging from 0.001 to 0.03 µg/ml (Table 2). Efinaconazole activity against T. mentagrophytes and T. rubrum isolates was comparable, and higher than that of the comparator drugs (Figure 1). In general, there was no species selectivity in the antifungal activity of efinaconazole and comparator drugs. The MIC 50 and MIC 90 of efinaconazole were comparable to that of amorolfine and terbinafine (within 4-fold), and 8- to 64-fold lower than ciclopirox and itraconazole (Table 2). The MIC geometric means of efinaconazole in the two dermatophyte species were 2- to 34-fold lower than the comparator drugs. For both T. mentagrophytes and T. rubrum, the efinaconazole MIC values were significantly lower when compared to those of the comparator drugs (p<0.001). 214 215 216 217 218 219 220 221 222 In vitro antifungal activity of efinaconazole and comparative antifungals against C. albicans isolates. The in vitro susceptibility to efinaconazole was evaluated in recent C. albicans isolates (n = 105) recovered primarily from skin, nail, oral or vaginal mucosa of candidiasis patients. Because there is no CLSI recommended reading timepoint for efinaconazole in C. albicans, the MIC for all test drugs was determined after 24 and 48 hours incubation. The MIC of efinaconazole, amorolfine, itraconazole and terbinafine could not be determined for several isolates as it was outside the tested concentration range. However, it was possible to calculate the MIC 90 in most cases, as the number of 10

223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 affected isolates was 10% of the total tested. Efinaconazole inhibited C. albicans growth at concentrations ranging from 0.0005 to >0.25 µg/ml (Table 2), with 50% of isolates inhibited (MIC 50 ) at 0.001 and 0.004 µg/ml at 24 and 48 hours, respectively. The MIC 50 and MIC 90 for all drugs increased at 48 hours relative to 24 hours. Growth control wells showed normal C. albicans growth with no apparent vehicle effect. At 24 hours, efinaconazole was more potent in inhibiting C. albicans growth than amorolfine, ciclopirox, itraconazole and terbinafine. The higher potency of efinaconazole relative to the other drugs was particularly marked in the low and mid-range of the susceptibility distribution spectrum (Figure 2); the MIC 50 of efinaconazole was 8- to 1000-fold lower than the comparator drugs, while the MIC 90 was 2- to 66-fold lower. At 48 hours, efinaconazole was also more potent than the comparator drugs; MIC 50 of efinaconazole was 4- to 1000-fold lower. However, a comparison could not be made based on MIC 90 because it was higher than the top concentration tested for three drugs (Table 2). The efinaconazole MIC values were significantly lower when compared to those of the comparator drugs both at 24 and 48 hours (p<0.001). As expected, terbinafine had the lowest activity in vitro among all test agents evaluated, (i.e., highest MICs) against C. albicans. Among the comparator drugs, terbinafine exhibited the greatest difference with efinaconazole, with MIC 50 and MIC 90 that were 1000- and 66-fold higher, respectively. We further compared the susceptibility patterns of efinaconazole and itraconazole at 48 hours. The breakpoint for itraconazole resistance in C. albicans is >0.5 µg/ml, and a total of 15 isolates tested (14%) were considered resistant. In this case, the susceptibility to these two drugs were well correlated (r = 0.85) (Table 3). 11

246 247 248 249 250 251 252 253 254 255 In vitro antifungal activity against various fungal pathogens. The spectrum of antifungal activity of efinaconazole was characterized in a panel of repository isolates, which included dermatophyte, nondermatophyte and yeast species. Efinaconazole was active against all of the isolates tested with most MICs of 1 µg/ml or lower (Table 4) and generally compared favorably with comparator drugs. Growth control wells showed normal fungal growth with no apparent vehicle effect. Against dermatophytes (excluding T. mentagrophytes and T. rubrum), the MIC range for efinaconazole was 0.002 to 0.5 μg/ml. Efinaconazole MICs were generally similar to terbinafine and lower than amorolfine, ciclopirox and itraconazole. 256 257 258 259 260 Against Candida species (excluding C. albicans), the MIC range for efinaconazole was 0.002 to 0.13 μg/ml. Efinaconazole inhibited the growth of Cryptococcus and Trichosporon species with MICs ranging from 0.002 to 0.031 μg/ml. The MIC and MIC range of efinaconazole was at least one order of magnitude lower than those of terbinafine, ciclopirox, itraconazole and amorolfine. 261 262 263 264 265 The MICs of efinaconazole against nondermatophyte species ranged from 0.0078 to 2 μg/ml. By comparison, the MICs of amorolfine, ciclopirox, terbinafine and itraconazole ranged from 0.063 to >4 μg/ml. Based on the geometric mean MIC or MIC values, the antifungal activity of efinaconazole against these nondermatophyte molds was similar or greater than that of the comparators. 266 12

267 Discussion 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 Efinaconazole is a novel triazole antifungal agent. In this study, we aimed to evaluate the antifungal activity of efinaconazole against T. rubrum, T. mentagrophytes and C. albicans, pathogens most commonly associated with onychomycosis. We also compared its activity to current topical and oral treatments for onychomycosis, including terbinafine, ciclopirox, itraconazole and amorolfine. Dermatophytes, primarily T. mentagrophytes and T. rubrum, are responsible for almost 90% of toenail onychomycosis infections. We conducted a large in vitro survey that included 1493 recent clinical isolates of these two species recovered from toenail onychomycosis patients. Efinaconazole showed potent antifungal activity within a narrow concentration range (i.e., 0.001 to 0.06 µg/ml), with no geographical differences in susceptibility patterns between North American (U.S.A. and Canada) and Japanese isolates. Compared to previously reported efinaconazole MICs in Japanese T. rubrum and T. mentagrophytes clinical isolates (10), the MICs obtained in the current study were approximately 13 to 63 times lower. The higher MICs reported previously may be due to a different methodology, which included use of Sabouraud dextrose broth (ph 5.6) and a longer timepoint for MIC reading (7 days). In contrast, we determined the MIC in accordance with the current CLSI standard, using RPMI 1640 medium (ph 7.0) and endpoint reading at 96 hours (4 days). Based on MIC 50 and MIC 90 data, efinaconazole is at least as effective as the currently available antifungal agents used to treat onychomycosis, and in many cases, more potent. 13

289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 Against T. rubrum, and T. mentagrophytes, efinaconazole has comparable activity (1- to 4-fold) to amorolfine and terbinafine, and higher activity (8- to 64-fold) compared to ciclopirox and itraconazole. Based on MIC geometric means, efinaconazole was more potent than all comparator drugs. Onychomycotic infections can also be caused by yeasts, generally C. albicans. Efinaconazole was active against recent C. albicans isolates, with MIC 50 of 0.001 µg/ml (24 hours) and 0.004 µg/ml (48 hours). Furthermore, efinaconazole was more potent in inhibiting C. albicans growth than terbinafine, ciclopirox, itraconazole and amorolfine. These results were obtained in topical isolates mostly from vaginal and oral candidiasis, and patients may have been treated with topical or systemic azole antifungals. For all drugs, the MIC range of C. albicans isolates was broader than for T. rubrum and T. mentagrophytes. A broad susceptibility range of C. albicans has also been reported for terbinafine and fluconazole in cutaneous isolates (12). The relatively extensive usage of existing antifungals in candidiasis possibly affected the sensitivity of isolates against the drugs tested, including efinaconazole. We further characterized efinaconazole s antifungal spectrum of activity in other 33 fungal species. Overall, efinaconazole was active against all species tested with MIC ranges of 0.002 to 0.5 µg/ml for other dermatophytes, 0.002 to 0.13 µg/ml for other yeasts and 0.0078 to 2 µg/ml for nondermatophyte molds. Efinaconazole was generally more active against these molds than amorolfine, ciclopirox, itraconazole and terbinafine, and its antifungal spectrum was broader than that of the existing drugs. Although efinaconazole and terbinafine had comparable antifungal activity against dermatophytes and nondermatophytes, their activity was markedly different against 14

312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 Candida spp. Efinaconazole was significantly more potent than terbinafine, with MICs mostly <0.1 µg/ml and 1 µg/ml, respectively. Terbinafine was previously found to be inactive against C. glabrata, C. krusei and C. tropicalis when tested at concentrations as high as 128 µg/ml. In contrast, efinaconazole has potent antifungal activity against these species, as well as other yeast in which terbinafine is also active. In vitro studies have reported that some fungal clinical isolates, including Candida species, with reduced susceptibility to one azole antifungal agent may also be less susceptible to other azoles (13). The MICs of efinaconazole against C. albicans correlated well with those of itraconazole, another azole, although efinaconazole showed a one order of magnitude higher potency. There are no breakpoints established for efinaconazole. However, there were 15 resistant C. albicans isolates tested based on a resistant breakpoint of >0.5 µg/ml, 12 of which were also the least susceptible (MIC 0.25 µg/ml) to efinaconazole (Table 3). Thus, it appears that the mechanisms of resistance common to azoles, as discussed in the literature, may also affect fungal susceptibility to efinaconazole. There are few reports of drug resistance in dermatophytes. In our large-scale susceptibility investigation, efinaconazole demonstrated potent antifungal activity with a narrow range of MICs against T. rubrum and T. mentagrophytes. Very few isolates were recovered after 48 weeks with daily topical treatment with efinaconazole and there were no apparent changes in susceptibility compared to isolates from untreated patients. There were no significant increases in MICs in T. rubrum and T. mentagrophytes after serial subculturing in vitro with efinaconazole (Iwata et al., manuscript in preparation). Although limited data, these results suggest a low potential for dermatophytes to develop 15

335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 resistance to efinaconazole in a clinical setting. However, the possibility of emergence of efinaconazole-resistant strains over time, after widespread clinical use, cannot be excluded. Epidermophyton, Microsporum, Acremonium, Fusarium, Scopulariopsis, Scytalidium, Aspergillus and Candida are commonly associated with onychomycosis, as these have been isolated from onychomycotic nails (4-6). Several studies have confirmed the role of these organisms as causative pathogens although their relative incidence compared to Trichophyton infections is low (14-16). Further, mixed fungal infections occur in approximately one fifth of onychomycosis cases and may be a reason for clinical treatment failure (17, 18). Potent antifungal agents with broad spectrum of activity may be advantageous in onychomycosis therapy. Our results show that efinaconazole is not only active against the most common onychomycosis pathogens but also against other pathogenic/colonizing species. Because many infections are of mixed pathogen nature, the broad spectrum of activity of efinaconazole is expected to be useful in onychomycosis treatment. In vitro evaluation of antifungal activity enables comparison between different antimycotic agents, and may clarify the reasons for lack of clinical response and assist the clinician in therapy selection (19). Onychomycosis MIC breakpoints have not been established for any of the agents that we tested, and therefore, it is unclear whether in vitro activity is predictive of clinical outcome. Drug delivery to the nail site of action at effective concentrations represents the main difficulty in developing topical antifungals. Thus, excellent in vitro activity against fungal pathogens may not necessarily translate well into high clinical cure rates in onychomycosis. For example, ciclopirox nail lacquer 16

358 359 360 361 362 363 364 365 366 367 368 369 370 has demonstrated only modest efficacy in onychomycosis, with reported complete cure rates of 5.5-8.5% (20). Other topical agents such as amorolfine nail lacquer and terbinafine nail solution (under development) have been shown to have minimal efficacy (<2% complete cure rates) even though both are reasonably potent antifungals (21). Our in vitro data on efinaconazole are encouraging and the unique properties of the molecule and its topical formulation have led to an extensive clinical program to evaluate its efficacy in onychomycosis (22). In conclusion, efinaconazole has potent in vitro antifungal activity against T. rubrum, T. mentagrophytes and C. albicans. The activity of efinaconazole is at least comparable, and in many cases more potent than current treatments used in onychomycosis. The relevance of these in vitro findings to clinical efficacy has not been established. Furthermore, efinaconazole has a broad spectrum of activity against fungi associated with onychomycosis and other mycoses, and may have additional therapeutic utility. 17

371 Table 1: In vitro susceptibility pattern of efinaconazole in dermatophyte clinical isolates Geographical location (No. of isolates) No. of isolates inhibited at concentration (μg/ml) a MIC (μg/ml) 372 373 374 0.002 0.004 0.008 0.015 0.03 0.06 Range MIC 50 MIC 90 T. mentagrophytes U.S.A. (39) 5 4 17 12 0 1 0.002-0.06 0.008 0.015 Canada (21) 7 4 7 3 0 0 0.002-0.015 0.004 0.015 Japan (46) 27 10 5 3 0 1 0.002-0.06 0.002 0.008 All (106) b 39 18 29 18 0 2 0.002-0.06 0.004 0.015 T. rubrum U.S.A. (963) 588 228 119 25 3 0 0.002-0.03 0.002 0.008 Canada (218) 123 60 28 6 1 0 0.002-0.03 0.002 0.008 Japan (206) 122 40 34 9 1 0 0.002-0.03 0.002 0.008 All (1387) b 833 328 181 40 5 0 0.002-0.03 0.002 0.008 a The test concentration range was 0.002-1 µg/ml; all isolates were inhibited at concentrations 0.06 µg/ml b All = U.S.A. + Canada + Japan 18

375 376 377 378 Table 2: In vitro antifungal activities of efinaconazole and four reference drugs against common onychomycosis causative fungi Organism (No. of isolates) T. rubrum (130) T. mentagrophytes (129) Test agent MIC (μg/ml) Range MIC 50 MIC 90 Geometric mean Efinaconazole 0.001-0.015 0.002 0.008 0.003 Terbinafine 0.004-0.06 0.008 0.015 0.009 Ciclopirox 0.03-0.5 0.125 0.25 0.101 Itraconazole 0.015-0.125 0.03 0.06 0.037 Amorolfine 0.004-0.015 0.008 0.015 0.008 Efinaconazole 0.001-0.03 0.004 0.015 0.005 Terbinafine 0.004-0.5 0.008 0.03 0.010 Ciclopirox 0.03-0.5 0.06 0.25 0.094 Itraconazole 0.03-0.25 0.06 0.125 0.063 Amorolfine 0.004-0.06 0.008 0.015 0.009 C. albicans (105) a Efinaconazole 0.0005->0.25 0.001 0.06 0.0029 Terbinafine 0.06->16 1 4 1.409 Ciclopirox 0.06-0.5 0.125 0.25 0.151 Itraconazole 0.004->2 0.008 0.125 0.014 Amorolfine 0.03-0.5 0.03 0.125 0.041 C. albicans (105) b Efinaconazole 0.0005->0.25 0.004 >0.25 0.0079 a MIC endpoint determined at 24 hours b MIC endpoint determined at 48 hours Terbinafine 0.125->16 4 >16 6.873 Ciclopirox 0.06-0.5 0.25 0.5 0.248 Itraconazole 0.004->2 0.015 >2 0.039 Amorolfine 0.03-8 0.03 1 0.091 379 19

380 Table 3: MIC analysis for efinaconazole and itraconazole against C. albicans isolates 381 Itraconazole MIC (µg/ml) Efinaconazole MIC (µg/ml) 0.0005 0.001 0.002 0.004 0.008 0.015 0.03 0.06 0.125 0.25 >0.25 Total 0.004 8 6 2 1 17 0.008 6 12 5 3 2 28 0.015 7 3 5 1 2 1 19 0.03 3 1 4 0.06 1 2 3 0.125 2 1 2 1 2 8 0.25 1 1 3 5 0.5 4 2 6 1 1 1 2 0 >2 1 1 3 9 14 Total 14 25 10 11 7 7 2 4 3 8 14 105 Spearman correlation coefficient, r = 0.85 382 Comparisons made with MICs determined at 48 hours 20

383 Table 4: In vitro antifungal activities of efinaconazole and four reference drugs against a variety of fungal species a Organism (No. of isolates) Dermatophytes MIC geometric mean [range] (µg/ml) b Efinaconazole Terbinafine Ciclopirox Itraconazole Amorolfine Trichophyton ajelloi (2) 0.044 [0.031-0.063] 0.046 [0.016-0.13] 0.25 [0.25] 0.35 [0.25-0.5] 0.5 [0.25-1] Trichophyton schoenleinii (1) NC [0.0039] NC [0.0039] NC [0.25] NC [0.13] NC [0.016] Trichophyton tonsurans (1) NC [0.016] NC [0.016] NC [0.25] NC [0.13] NC [0.25] Trichophyton verrucosum (1) NC [0.0039] NC [0.016] NC [0.13] NC [0.016] NC [0.25] Microsporum canis (2) 0.18 [0.13-0.25] 0.13 [0.063-025] 0.25 [0.25] 0.35 [0.25-0.5] >4.0 [>4] Microsporum cookei (1) NC [0.5] NC [0.13] NC [0.25] NC [0.5] NC [0.5] Microsporum gypseum (3) 0.010 [0.0039-0.016] 0.050 [0.031-0.063] 0.31 [0.25-0.5] 0.1 [0.031-0.25] 0.08 [0.063-0.13] Epidermophyton floccosum (3) Yeasts 0.005 [ 0.002-0.0078] 0.039 [0.031-0.063] 0.31 [0.25-0.5] 0.08 [0.063-0.13] 0.16 [0.13-0.25] Candida glabrata (7) 0.026 [0.0039-0.13] >8 [>8] 0.13 [0.13] 0.74 [0.25-2] >4.9 [2->8] Candida krusei (10) 0.024 [0.0078-0.063] >8 [>8] 0.21 [0.13-0.25] 0.38 [0.13-0.5] 0.27 [0.13-0.5] Candida parapsilosis (13) 0.0046 [ 0.002-0.28 [0.13-1] 0.22 [0.13-0.5] 0.13 [0.063-0.25] 0.56 [0.13-4] 0.016] Candida tropicalis (10) 0.014 [0.0078-0.063] >8 [>8] 0.5 [0.5] 0.31 [0.063-0.5] NC [ 0.016->8] Candida guilliermondii (1) NC [0.016] NC [1] NC [0.25] NC [0.13] NC [0.25] Candida kefyr (1) NC [ 0.002] NC [2] NC [0.13] NC [0.031] 0.063 Candida lusitaniae (1) NC [0.0039] NC [4] NC [0.25] NC [0.13] 0.5 Cryptococcus neoformans (5) 0.0023 [0.002-0.0039] Trichosporon asahii (3) 0.005 [ 0.002-0.0078] 0.25 [0.063-0.5] 0.031 [ 0.016-0.063] 0.041 [0.031-0.063] 0.064 [ 0.016-0.13] 0.63 [0.5-1] 0.13 [0.13] 0.1 [0.063-0.13] 0.08 [0.063-0.13] 21

384 385 Trichosporon beigeleii (2) 0.016 [0.0078-0.031] 0.5 [0.5] 0.18 [0.13-0.25] 0.18 [0.13-0.25] 0.35 [0.25-0.5] Nondermatophytes Acremonium potronii (3) 0.31 [0.25-0.5] 0.25 [0.13-0.5] 0.25 [0.13-0.5] >2.5 [1->4] 0.26 [0.13-1] Acremonium sclerotigenum (2) 0.18 [0.13-0.25] 0.09 [0.063-0.13] 1.4 [1-2] >4 [>4] 1 [1] Fusarium oxysporum (3) 1 [0.5-2] 2.5 [1-4] 1 [1] >4 [>4] >4 [>4] Fusarium solani (1) NC [0.5] NC [4] NC [>4] NC [>4] NC [>4] Paecilomyces variotii (1) NC [0.0078] NC [0.25] NC [0.25] NC [0.13] NC [>4] Paecilomyces lilacinus (3) 0.031 [0.031] 0.1 [0.063-0.13] 4 [4] 1.6 [1.0-4] 0.25 [0.25] Pseudallescheria boydii (1) NC [0.063] NC [>4] NC [4] NC [>4] NC [4] Scopulariopsis brevicaulis (4) 0.25 [0.13-0.5] 1 [0.5-2] 0.59 [0.5-1] >4 [>4] 0.09 [0.063-0.13] Scopulariopsis brumptii (1) NC [0.13] NC [1] NC [0.5] NC [>4] NC [0.5] Aspergillus fumigatus (4) 0.089 [0.031-0.5] 1.4 [1-2] 0.42 [0.25-0.5] 0.5 [0.25-1] >4 [>4] Aspergillus flavus (4) 0.11 [0.063-0.13] 0.11 [0.063-0.5] >3.4 [2->4] 0.18 [0.13-0.25] >4 [>4] Aspergillus niger (3) 0.2 [0.13-0.25] 0.16 [0.13-0.25] 0.63 [0.5-1] 0.63 [0.5-1] >4 [>4] Aspergillus sydowii (4) 0.037 [0.0078-0.25] 0.076 [0.063-0.13] 0.59 [0.5-1] >0.3 [0.063->4] >4 [>4] Aspergillus terreus (4) 0.09 [0.063~0.13] 0.13 [0.13] 0.5 [0.25-1] 0.21 [0.13-0.25] >4 [>4] Aspergillus nidulans (4) 0.0078 [0.0078] 0.063 [0.063] 1 [0.5-4] 0.089 [0.063-0.25] >4 [>4] a ATCC or Japanese repository isolates b MIC geometric means are calculated for species with at least 2 isolates tested; NC = not calculated 22

386 Figure Legends 387 388 389 Figure 1: Cumulative MIC frequency distribution of efinaconazole and comparator drugs against (A) T. mentagrophytes (n = 129) and (B) T. rubrum (n = 130). 390 391 392 393 Figure 2: Cumulative MIC frequency distribution of efinaconazole and comparator drugs against C. albicans at 24 hours (n = 105). Not all isolates were inhibited at the highest dose tested following treatment with efinaconazole, itraconazole and terbinafine. 394 395 396 Acknowledgements 397 398 399 400 401 The authors would like to thank Linda Mutter and Brian Bulley for manuscript discussions and review. WJS and RP are employees and stockholders in Valeant Pharmaceuticals North America LLC. HS, TN, DS and TY are employees and stockholders in Kaken Pharmaceutical Co., Ltd. 402 403 404 23

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A 100 T. mentagrophytes Cumulative percent of isolates inhibited 80 60 40 20 B 0 0.0001 0.001 0.01 0.1 1 MIC (µg/ml) 100 T. rubrum Cumulative percent of isolates inhibited 80 60 40 20 0 0.0001 0.001 0.01 0.1 1 MIC (µg/ml) Efinaconazole Amorolfine Itraconazole Ciclopirox Terbinafine

100 C. albicans Cumulative percent of isolates inhibited 80 60 40 20 0 0.0001 0.001 0.01 0.1 1 10 100 MIC (µg/ml) Efinaconazole Amorolfine Itraconazole Ciclopirox Terbinafine