Activity of TDT 067 (Terbinafine in Transfersome) against Agents of Onychomycosis, as Determined by Minimum Inhibitory and Fungicidal Concentrations

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1 JOURNAL OF CLINICAL MICROBIOLOGY, May 2011, p Vol. 49, No /11/$12.00 doi: /jcm Copyright 2011, American Society for Microbiology. All Rights Reserved. Activity of TDT 067 (Terbinafine in Transfersome) against Agents of Onychomycosis, as Determined by Minimum Inhibitory and Fungicidal Concentrations Mahmoud Ghannoum, 1 * Nancy Isham, 1 Jacqueline Herbert, 1 William Henry, 2 and Sam Yurdakul 2 Center for Medical Mycology, Case Western Reserve University and University Hospitals Case Medical Center, Cleveland, Ohio, 1 and Celtic Pharma Development Services Europe Ltd., London, United Kingdom 2 Received 14 January 2011/Returned for modification 27 January 2011/Accepted 7 March 2011 TDT 067 is a novel carrier-based dosage form (liquid spray) of 15 mg/ml of terbinafine in Transfersome that has been developed to deliver terbinafine to the nail bed to treat onychomycosis. In this study, we report the in vitro activities of TDT 067 against dermatophytes, compared with those of the Transfersome vehicle, naked terbinafine, and commercially available terbinafine (1%) spray. The MICs of TDT 067 and comparators against 25 clinical strains each of Trichophyton rubrum, T. mentagrophytes, and Epidermophyton floccosum were determined according to the CLSI M38 A2 susceptibility method (2008). Minimum fungicidal concentrations (MFCs) were determined by subculturing visibly clear wells from the MIC microtiter plates. TDT 067 demonstrated potent activity against the dermatophyte strains tested, with an MIC range of to g/ml. Overall, TDT 067 MIC 50 values (defined as the lowest concentrations to inhibit 50% of the strains tested) were 8-fold and 60-fold lower than those of naked terbinafine and terbinafine spray, respectively. The Transfersome vehicle showed minimal inhibitory activity. TDT 067 demonstrated lower MFC values for T. rubrum and E. floccosum than naked terbinafine and terbinafine spray. TDT 067 has more potent antifungal activity against dermatophytes that cause nail infection than conventional terbinafine preparations. The Transfersome vehicle appears to potentiate the antifungal activity of terbinafine. Clinical investigation of TDT 067 for the topical treatment of onychomycosis is warranted. Onychomycosis is a common fungal disease that is responsible for up to 50% of nail disorders (13). This disease affects 3 to 23% of the population in the United States and Europe (13, 28), with higher prevalence ( 20%) among older people and diabetic patients (23, 25). Other predisposing factors include immunosuppression, poor peripheral circulation, nail trauma, and tinea pedis (31). The incidence of onychomycosis is likely to increase due to a growing elderly population and the spread of human immunodeficiency virus infection/aids. The majority of nail infections (90 to 95%) are caused by dermatophytes (Trichophyton rubrum, T. mentagrophytes, or Epidermophyton floccosum), with the remainder being caused by yeasts (Candida spp.) and nondermatophyte molds (11). Though a few dermatophyte species are endemic to certain areas and absent altogether in others, most dermatophytoses worldwide are caused by only a half-dozen species (1). This distribution is constantly changing, however, due to increased population migration and events such as mass tourism and international sports activities which serve to disseminate lesscommon species. The most common dermatophyte isolated in the United States, Canada, Mexico, and Europe is T. rubrum, followed in frequency by T. mentagrophytes and E. floccosum (17, 33, 29). An analysis of the dermatophytes isolated in the British Isles over the last 3 decades indicated that T. rubrum * Corresponding author. Mailing address: Center for Medical Mycology, Department of Dermatology, Case Western Reserve University, Euclid Ave., Cleveland, OH Phone: (216) Fax: (216) mahmoud.ghannoum@case.edu. Published ahead of print on 16 March and T. mentagrophytes together comprised up to 90% of all foot infections (3), whereas the incidence of E. floccosum infection was dramatically reduced. However, E. floccosum is more prevalent in other areas of the world and has recently been reported from 17% of the onychomycosis infections in the Middle East area (34). Treatment of onychomycosis has improved considerably over the past decade as a result of the introduction of oral terbinafine and itraconazole, with terbinafine being regarded generally as the more effective of the two agents (10, 30). In spite of the encouraging mycological cure rates shown after treatment with these antifungal agents (30), many nail infections are notoriously difficult to cure, with rates of recurrence of 11.9% and 33.7% being reported for terbinafine and itraconazole, respectively, after long-term follow-up (26). Moreover, oral therapy has the inherent disadvantages of drug-drug interactions and systemic adverse effects (e.g., liver toxicity) (18, 21), which are serious concerns, given the non-life-threatening nature of onychomycosis. Although the use of topical agents avoids the systemic effects of oral therapy, those available for the treatment of onychomycosis (e.g., tioconazole, amorolfine, and ciclopirox) have limited efficacy. For example, the 28% formulation of tioconazole was reported to have a cure rate of only 20 to 22% (19). A multicenter study of 456 patients demonstrated a 54% clinical response rate for amorolfine nail lacquer; however, patients in this study had no nail bed involvement (27). Ciclopirox, the only FDA-approved topical agent for the treatment of onychomycosis in the United States, achieves drug concentrations in the nail considerably higher than the MIC of ciclopirox 1716

2 VOL. 49, 2011 ACTIVITY OF TDT 067 AGAINST DERMATOPHYTES 1717 for dermatophytes (5). However, poor clinical cure rates have been reported in United States-based studies ( 10% complete cure) (16). Combination therapy involving topical and oral antifungals has recently been introduced. For instance, amorolfine hydrochloride 5% nail lacquer and oral terbinafine was shown to be more effective than oral terbinafine alone for the treatment of onychomycosis (2), although a similar study with oral terbinafine and either ciclopirox or amorolfine failed to demonstrate a significant increase in efficacy over that of oral terbinafine alone (20). The underlying reason for the poor clinical outcomes reported with topical preparations for the treatment of onychomycosis is their inability to penetrate the nail plate and reach the nail bed where the causative fungi reside (24). Different approaches have been investigated to optimize drug penetration through the nail plate, and these include use of (i) potent drugs to ensure that effective drug concentrations are achieved at the site of action, (ii) drugs with optimal physicochemical properties for permeation into the nail plate, (iii) penetration enhancers to facilitate ungual drug permeation, and (iv) appropriate formulations that can aid ungual drug uptake, are easy to use, and stay in contact with nail plates, releasing drugs continuously over long periods of time (24). There has been inconsistency in the results of such studies, and more research is needed in this area (24). TDT 067 is a novel carrier-based dosage form (liquid spray) of 15 mg/ml of terbinafine in Transfersome (developed by Targeted Delivery Technologies Ltd.) that has been developed to deliver terbinafine to the nail plate, nail bed, and surrounding tissues to treat onychomycosis. The Transfersome carrier vesicles are responsive, composite lipid aggregates that are highly deformable and have high surface hydrophilicity. Thus, the strong transport-driving water gradient acts on the vesicles to pull them into pores and into tissue. Transfersomes activate hydrophilic pathways through the skin, ensuring a high level of deep, local drug accumulation, unlike conventional topical products (6). This study compared the in vitro antifungal activity of TDT 067 with those of naked terbinafine (terbinafine not in Transfersome vesicles) and a commercially available terbinafine (1%) spray against dermatophytes known to cause onychomycosis, as measured by MIC and minimum fungicidal concentration (MFC). MATERIALS AND METHODS Fungal isolates. Twenty-five recent clinical strains of each species (T. rubrum, T. mentagrophytes, and E. floccosum), taken from the culture collection at the Center for Medical Mycology, Cleveland, OH, were tested. Although no breakpoints have been defined for terbinafine against dermatophytes, terbinafine resistance is defined here as a terbinafine MIC of 1.0 g/ml (8). T. rubrum MYA 4438 and T. mentagrophytes MYA 4439 were included as quality control isolates on each day of testing. Antifungal agents. TDT 067 liquid formulation (concentration of terbinafine, 15 mg/ml) and Transfersome vehicle (vehicle without terbinafine) liquid formulation were provided by Celtic Pharma Development Services Bermuda Ltd., terbinafine powder (naked terbinafine) was provided by the Center for Medical Mycology, Cleveland, OH, and terbinafine spray (1%) was purchased commercially. Serial dilutions of TDT 067 and Transfersome vehicle were prepared in acetate-ethanol buffer (25 mm acetate buffer, ph 4.0, containing 3% by weight of ethanol). Terbinafine powder was dissolved in dimethyl sulfoxide (DMSO); terbinafine and terbinafine spray were serially diluted in RPMI 1640 buffered with MOPS [3-(N-morpholino) propanesulfonic acid] (Hardy Diagnostics, Santa Maria, CA). Antifungal susceptibility testing. MIC testing was performed according to the CLSI M38 A2 standard methodology for the susceptibility testing of dermatophytes (8, 12). Stock solutions of TDT 067 and Transfersome vehicle were diluted 1:1 with the growth medium/inoculum, and further serial dilutions were prepared in acetate buffer. For TDT 067, this resulted in a final terbinafine concentration range of to 7,500 g/ml. For the Transfersome vehicle, the final concentration range was 0.10 to 50%. Terbinafine powder (dissolved in DMSO) and terbinafine spray were serially diluted with RPMI 1640 to produce a final concentration range of to 0.5 g/ml (terbinafine spray was prepared in a range of 0.1 to 5,000 g/ml for testing terbinafine-resistant T. rubrum strains), while the acetate buffer was serially diluted with RPMI 1640 to produce a final concentration range of 0.10 to 50%. The dilutions (in 100- l aliquots) were added to wells of 96-well microtiter assay plates. Isolates were subcultured on potato dextrose agar (Fisher Scientific, Hampton, NH) or cereal agar (T. rubrum strains) and incubated at 30 C until good conidiation was achieved, usually within 7 days. Conidia were harvested to sterile saline by swabbing the colony surface with a sterile swab and were allowed to settle for 10 to 15 min. Conidial counts were standardized using a hemacytometer, and the suspension was adjusted to to conidia/ml in RPMI 1640 as a growth medium. Inocula (in 100- l aliquots) were combined 1:1 with the test drug solutions in 96-well round-bottom microtiter plates and incubated at 35 C for 4 days. MICs were read visually, and the MIC endpoint was defined as the lowest concentration that inhibited 80% of fungal growth relative to the growth control. MFC determinations were performed according to Canton et al. and Ghannoum and Isham (4, 14). The total contents of each clear well from the MIC assay were subcultured onto potato dextrose agar for determination of the colony count. To avoid antifungal carryover, the aliquots were allowed to soak into the agar and then were streaked for isolation when dry, thus removing the cells from the drug source. RESULTS Five of the 25 strains of T. rubrum tested were defined as terbinafine-resistant (MIC 1.0 g/ml). All other dermatophyte strains tested were defined as susceptible to terbinafine. TDT 067 demonstrated potent activity against all terbinafine-susceptible dermatophyte strains tested, with MIC ranges of to g/ml (Table 1). One strain of T. rubrum with an extremely low MIC was considered an outlier; thus, the range for the remaining terbinafine-susceptible strains (n 69) was to g/ml. The MIC 50 of TDT 067 (defined as the lowest concentration to inhibit 50% of isolates tested) for T. mentagrophytes was g/ml, and that for both E. floccosum and T. rubrum was g/ml. Relative to those of naked terbinafine and terbinafine spray, TDT 067 MIC 50 values were 8-fold and 60-fold lower for T. mentagrophytes, 8-fold and 30-fold lower for E. floccosum, and 4-fold and 30-fold lower for T. rubrum, respectively. The MIC 90 values of TDT 067 (defined as the lowest concentrations to inhibit 90% of isolates tested) were g/ml for T. mentagrophytes and g/ml for both E. floccosum and T. rubrum. Relative to those of naked terbinafine and terbinafine spray, the TDT 067 MIC 90 values were 4-fold and 30-fold lower for T. mentagrophytes, 8-fold and 32-fold lower for E. floccosum, and 4-fold and 15-fold lower for T. rubrum, respectively. MIC values of TDT 067, naked terbinafine, and terbinafine spray for terbinafine-resistant strains were comparable (range, 1.0 to 10 g/ml; Table 1). The Transfersome vehicle demonstrated minimal inhibitory activity compared with TDT 067; 90% of all strains tested were inhibited at a concentration of 1.5%, i.e., five serial dilutions from the highest concentration of vehicle tested (50% solu-

3 1718 GHANNOUM ET AL. J. CLIN. MICROBIOL. Organism (no. of strains) TABLE 1. MICs of TDT 067 and comparators for dermatophytes TDT 067 formulation Naked terbinafine MIC 1% terbinafine spray Transfersome (% solution) Buffer (% solution) a T. mentagrophytes (25) Range MIC MIC E. floccosum (25) Range MIC MIC T. rubrum (20) Range b MIC MIC T. rubrum with terbinafine MIC of 1.0 g/ml (5) Range MIC 50 NA c NA NA NA NA MIC 90 NA NA NA NA NA a 50% maximum concentration tested. b One outlier strain. c NA, not applicable. tion), compared with 20 serial dilutions of the highest concentration of TDT 067 tested (7,500 g/ml solution). Acetateethanol buffer alone showed no inhibition against 90% of isolates tested, with 4 strains inhibited at a concentration of 50% and 3 strains inhibited at a concentration of 25%. TDT 067 demonstrated low MFC values ( 0.25 g/ml) for 96% of the terbinafine-susceptible dermatophyte strains tested (MFC 50 values of g/ml for T. rubrum and E. floccosum and 0.03 g/ml for T. mentagrophytes) (Table 2). TDT 067 MFC 50 values were up to 4-fold and 32-fold lower than those of naked terbinafine and terbinafine spray, respectively, for all strains tested. The MFC 90 values of TDT 067 were g/ml for T. rubrum, 0.06 g/ml for E. floccosum, and 0.25 g/ml for T. mentagrophytes. Relative to naked terbinafine and terbinafine spray, TDT 067 MFC 90 values for E. floccosum were 2-fold and 8-fold lower, respectively, and those for T. rubrum Organism (no. of strains) TABLE 2. Minimum fungicidal concentrations of TDT 067 and comparators for dermatophytes TDT 067 formulation Naked terbinafine MFC 1% terbinafine spray Transfersome (% solution) Buffer (% solution) a T. mentagrophytes (25) Range MFC MFC E. floccosum (25) Range MFC MFC T. rubrum (20) Range MFC MFC T. rubrum with terbinafine MIC of 1.0 g/ml (5) Range 8 7, ,250 5, MFC 50 NA b NA NA NA NA MFC 90 NA NA NA NA NA a 50% maximum concentration tested. b NA, not applicable.

4 VOL. 49, 2011 ACTIVITY OF TDT 067 AGAINST DERMATOPHYTES 1719 were 8-fold and 33-fold lower, respectively. The TDT 067 MFC 90 value was the same as that of naked terbinafine when tested against T. mentagrophytes. None of the formulations demonstrated fungicidal activity against the strains with elevated terbinafine MICs. The Transfersome vehicle showed minimal fungicidal activity against the T. mentagrophytes or E. floccosum strains; however, 90% of T. rubrum strains were killed by Transfersome vehicle at a 50% concentration. DISCUSSION This study demonstrates that TDT 067, a new carrierbased dosage form of terbinafine in Transfersome, has potent antifungal activity against dermatophytes that cause onychomycosis. The inhibitory activity of TDT 067 against the dermatophyte strains tested was greater than that of naked terbinafine (as used in oral therapy) and commercially available topical terbinafine spray, with 4- to 60-fold-lower MIC 50 values and 4- to 32-fold-lower MIC 90 values. It is well established that terbinafine has potent in vitro antifungal activity against dermatophyte strains; a recent study reported terbinafine MICs for dermatophytes in the range of to 0.03 g/ml, which is comparable with the MIC values for naked terbinafine reported here (15). The high-level activity of TDT 067 compared to that of naked terbinafine observed in this study could be a result of the Transfersome potentiating the antifungal activity of terbinafine. This is supported by the observation that the Transfersome vehicle itself had some inhibitory activity. Although TDT 067 was very effective against all the terbinafine-susceptible strains tested in this study, there was a wide range of activity, as evidenced by the broad MIC range compared to that of naked terbinafine. The reason for this variation is unknown, though one possibility would be the presence of trailing growth. However, no trailing growth was observed with TDT 067 in this study. Additional in vitro studies are ongoing to investigate the mechanisms and speed of effect of TDT 067 against dermatophytes. TDT 067 is being developed for topical therapy, which would avoid concerns regarding safety of systemic agents and is also more convenient for patients. Ciclopirox and amorolfine, two of the most widely used antifungal agents available in a topical formulation for onychomycosis, have lower-level antifungal activity against dermatophytes than terbinafine. In a report by Clayton, the mean MIC of amorolfine was similar to that of terbinafine (0.039 g/ml); however, terbinafine demonstrated greater fungicidal activity (7). In 2003, Kokjohn et al. (22) evaluated the in vitro activity of ciclopirox against dermatophytes and yeasts. The MIC range of ciclopirox for the T. mentagrophytes, T. tonsurans, T. rubrum, Microsporum canis, M. gypseum, and E. floccosum strains was 0.03 to 0.25 g/ml. In addition, the available topical agents are not regarded as effective for the treatment of onychomycosis (9). Importantly, TDT 067 demonstrated fungicidal activity against terbinafine-susceptible strains in this study. Relative to those of naked terbinafine and terbinafine spray, the MFC of TDT 067 for E. floccosum was 2-fold and 8-fold lower, respectively, and that for T. rubrum was 8-fold and 33-fold lower, respectively. Fungal infections of the nail are notoriously difficult to treat and often recur after cessation of therapy (26). This is likely to be due to reservoirs of fungal organisms in the nail bed. Terbinafine exhibits potent fungicidal activity against dermatophytes (15), which may be important for preventing relapse of onychomycosis in patients. The rates of relapse reported following treatment with oral terbinafine are lower than corresponding rates for other systemic antifungal agents (26, 32). The potent inhibitory and fungicidal activities of TDT 067, combined with its targeted delivery to the nail bed through the Transfersome technology, could provide a potential clinical benefit for the treatment of onychomycosis. Ongoing clinical studies will provide data on the efficacy and safety of TDT 067 in onychomycosis. ACKNOWLEDGMENTS This work was supported by Celtic Pharma Development Services Bermuda Ltd., Hamilton, Bermuda (an affiliate of Targeted Delivery Technologies Ltd.). M.G. has acted as a consultant or has received grant support from Great Lakes Pharmaceuticals, Merck, Merz, Novartis, Pfizer, and Schering-Plough. REFERENCES 1. Aly, R Ecology and epidemiology of dermatophyte infections. J. Am. Acad. Dermatol. 31(Part 2):S21 S Baran, R., et al A multicentre, randomized, controlled study of the efficacy, safety and cost-effectiveness of a combination therapy with amorolfine nail lacquer and oral terbinafine compared with oral terbinafine alone for the treatment of onychomycosis with matrix involvement. Br. J. Dermatol. 157: Borman, A. M., et al Analysis of the dermatophyte species isolated in the British Isles between 1980 and 2005 and review of worldwide dermatophyte trends over the last three decades. Med. Mycol. 45: Cantón, E., et al Minimum fungicidal concentrations of amphotericin B for bloodstream Candida species. Diagn. Microbiol. Infect. Dis. 45: Ceschin-Roques, C. G., et al Ciclopirox nail lacquer 8%: in vivo penetration into and through nails and in vitro effect on pig skin. Skin Pharmacol. 4: Cevc, G Drug delivery across the skin. Expert Opin. Investig. Drugs. 6: Clayton, Y. M Relevance of broad-spectrum and fungicidal activity of antifungals in the treatment of dermatomycoses. R. J. Dermatol. 130(Suppl.): CLSI Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Approved standard, 2nd ed. CLSI document M38 A2 (ISBN ). CLSI, Wayne, PA. 9. Crawford, F., and S. Hollis Topical treatments for fungal infections of the skin and nails of the foot. Cochrane Database Syst. Rev. 3:CD Crawford, F., et al Oral treatments for toenail onychomycosis: a systematic review. Arch. Dermatol. 138: Foster, K. W., M. A. Ghannoum, and B. E. Elewski Epidemiologic surveillance of cutaneous fungal infection in the United States from 1999 to J. Am. Acad. Dermatol. 50: Ghannoum, M. A., et al Intra- and interlaboratory study of a method for testing the antifungal susceptibilities of dermatophytes. J. Clin. Microbiol. 42: Ghannoum, M. A., et al A large-scale North American study of fungal isolates from nails: the frequency of onychomycosis, fungal distribution, and antifungal susceptibility patterns. J. Am. Acad. Dermatol. 43: Ghannoum, M. A., and N. Isham Voriconazole and caspofungin cidality against non-albicans Candida species. Infect. Dis. Clin. Pract. 15: Ghannoum, M. A., A. Welshenbaugh, Y. Imamura, P. Mallefet, and H. Yamaguchi Comparison of the in vitro activity of terbinafine and lanoconazole against dermatophytes. Mycoses 53: Gupta, A. K., P. Fleckman, and R. Baran Ciclopirox nail lacquer topical solution 8% in the treatment of toenail onychomycosis. J. Am. Acad. Dermatol. 43(4 Suppl.):S70 S Havlickova, A., V. A. Czaika, and M. Friedrich Epidemiological trends in skin mycoses worldwide. Mycoses 51(Suppl. 4): Hay, R. J Risk/benefit ratio of modern antifungal therapy: focus on hepatic reactions. J. Am. Acad. Dermatol. 29:S50 S Hay, R. J., R. M. Mackie, and Y. M. Clayton Tioconazole nail solu-

5 1720 GHANNOUM ET AL. J. CLIN. MICROBIOL. tion an open study of its efficacy in onychomycosis. Clin. Exp. Dermatol. 10: Jaiswal, A., R. P. Sharma, and A. P. Garg An open randomized comparative study to test the efficacy and safety of oral terbinafine pulse as a monotherapy and in combination with topical ciclopirox olamine 8% or topical amorolfine hydrochloride 5% in the treatment of onychomycosis. Ind. J. Dermatol. Venerol. Leprol. 73: Katz, H. I., and A. K. Gupta Oral antifungal drug interactions. Dermatol. Clin. 15: Kokjohn, K., M. Bradley, B. Griffiths, and M. Ghannoum Evaluation of in vitro activity of ciclopiroxolamine, butenafine HCl and econazole nitrate against dermatophytes, yeasts, and bacteria. Int. J. Dermatol. 42: Mayser, P., V. Freund, and D. Budihardja Toenail onychomycosis in diabetic patients: issues and management. Am. J. Clin. Dermatol. 10: Murdan, S Drug delivery to the nail following topical application. Int. J. Pharm. 236: Piérard, G Onychomycosis and other superficial fungal infections of the foot in the elderly: a pan-european survey. Dermatology 202: Piraccini, B. M., A. Sisti, and A. Tosti Long-term follow-up of toenail onychomycosis caused by dermatophytes after successful treatment with systemic antifungal agents. J. Am. Acad. Dermatol. 62: Reinel, D Topical treatment of onychomycosis with amorolfine 5% nail lacquer: comparative efficacy and tolerability of once and twice weekly use. Dermatology 182(Suppl.): Roseeuw, D Achilles foot screening project: preliminary results of patients screened by dermatologists. J. Eur. Acad. Dermatol. Venereol. 12(Suppl. 1):S6 S9, S Seebacher, C., J. P. Bouchara, and B. Mignon Updates on the epidemiology of dermatophyte infections. Mycopathologia 166: Sigurgeirsson, B., et al Long-term effectiveness of treatment with terbinafine vs itraconazole in onychomycosis: a 5-year blinded prospective follow-up study. Arch. Dermatol. 138: Tosti, A., R. Hay, and R. Arenas-Guzmán Patients at risk of onychomycosis-risk factor identification and active prevention. J. Eur. Acad. Dermatol. Venereol. 19(Suppl. 1): Tosti, A., B. M. Piraccini, C. Stinchi, and M. D. Colombo Relapses of onychomycosis after successful treatment with systemic antifungals: a threeyear follow-up. Dermatology 197: Welsh, O., et al Dermatophytoses in Monterrey, Mexico. Mycoses 49: Yenişehirli, G., et al Onychomycosis infections in the Middle Black Sea Region, Turkey. Int. J. Dermatol. 48: