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1 AAC Accepts, published online ahead of print on 29 December 2014 Antimicrob. Agents Chemother. doi: /aac Copyright 2014, American Society for Microbiology. All Rights Reserved Ability of Hydroxypropyl Chitosan (HPCH) Nail Solution to Protect Against Dermatophyte Nail Infection Running Title: Hydroxypropyl Chitosan (HPCH) Nail Solution MA Ghannoum 1 #, L Long 1, N Isham 1, A Bulgheroni 2, M Setaro 3, M Caserini 2, R Palmieri 2 and F Mailland 2 1 Center for Medical Mycology, Case Western Reserve University, Cleveland, OH, 2 Scientific Dept, Polichem S.A., Lugano, Switzerland, 3 Tecnolab, Verbania Fondotoce, Italy Corresponding Author: Mahmoud Ghannoum Center for Medical Mycology Case Western Reserve University Euclid Ave Cleveland, OH mahmoud.ghannoum@case.edu Downloaded from on October 2, 2018 by guest 1

2 ABSTRACT Background: Development of a topical agent that would strengthen the nail, improve the natural barrier, and provide better drug penetration to the nail bed is needed. In this study, we report the effects of a hydroxypropyl chitosan (HPCH)-based nail solution using a bovine hoof model. Methods: Following nail solution application, changes in the hardness of the hoof samples were measured using the Vickers method. Tensile and flexural strength were tested by stretching or punching the samples, respectively. Ultrastructure was examined using scanning electron microscopy, and samples stained with periodic acid-schiff were used to determine fungal penetration depth. Comparators included 40% urea and 70% isopropyl alcohol solutions. Results: HPCH nail solution increased hoof sample hardness in comparison to the untreated control sample (mean 22.3 vs HV). Similarly, the HPCH solution increased tensile strength (mean vs MPa) and flexural strength (mean vs MPa) as compared to the untreated control. By contrast, the comparators had adverse effects on hardness and strength. SEM showed that the HPCH solution reduced the area of sample crumbling following abrasion compared to the untreated control (7418 vs pixels), and the PAS-stained images showed that the HPCH solution reduced penetration of dermatophyte hyphae (e.g. penetration by T. mentagrophytes was <25 μm at Day 9, compared to 275 μm in the untreated control). Conclusion: In contrast to chemicals normally used in cosmetic treatments, repeated application of HPCH nail solution may help prevent the establishment of new or recurring fungal nail infection. 2

3 INTRODUCTION The treatment of onychomycosis has improved recently with the addition of novel topical agents, used either alone or in combination with systemic oral drugs. However, recurrent or persistent infection occurs in up to 25% of patients. [1] Several predisposing factors may contribute to a patient's susceptibility to fungal nail infection, including age, impaired blood circulation, diabetes, physical trauma, occupation, and lifestyle. [1-5] The health of the nail itself may have an important bearing on the ability of the dermatophyte fungi to establish an infection, as most cases of onychomycosis are secondary to a previously established skin infection and/or nail trauma, either derived from mechanical insults or from exposure to chemical agents which can alter the natural nail barrier function. In this regard, the use of urea and/or isopropyl alcohol, chemicals often used in nail care to dissolve the nail keratin or to remove solutions from nail surface, can undermine the nail structure and make it more prone to fungal infections. Therefore, a topical agent that would strengthen the nail, improving the natural barrier to infection, might represent a valuable option to prevent new or recurrent fungal infections. The HPCH-based nail solution (Genadur ), developed by Polichem, is a hydroxypropyl chitosan solution based on aminosaccharide chitin extracted from crab carapace. The other two components, methyl sulfonyl methane and Equisetum arvense (an herbaceous perennial plant commonly known as horsetail), provide sulfur and silica, which contribute to strengthening the nail and which provide minerals to form the collagen that cements nail cells. This water-based HPCH solution forms a protective film that remineralizes and restructures nails, protects keratin, and maintains hydration. In clinical 3

4 studies, it has been shown to reduce the signs of fragility and roughness in nails of patients with psoriatic nail dystrophy. [7-9] HPCH-based nail solution is not intended as a cure for onychomycosis, as it does not exhibit antifungal activity, but instead is able to form a film on the nail surface that may prevent fungal invasion. Moreover, the solution can be used indefinitely, being devoid of any pharmacological or toxicological effect. In this paper, we describe the effects of an HPCH-based nail solution, compared to urea and isopropyl alcohol, on hardness, ultrastructure, tensile and flexural strength, using bovine hooves, which have been validated as equivalent in structure to human nails. [10] We used bovine hooves in this study because they are less expensive and more easily obtained than human cadaver nails. In addition, though the bovine keratin is chemically slightly different from that of humans, these differences were not believed to affect the results of the investigation. On the other hand, the great inter-individual variability of human nails, e.g. due to sex, age, thickness and length, may have affected the results. In our investigation, the bovine samples used were cut to a standardized size and thickness for the various assays. MATERIALS AND METHODS Test items HPCH-based nail solution, containing hydroxypropyl chitosan, Equisetum arvense glycolic extract, methylsulfonyl methane, diethylene glycole monoethylether, water, and 4

5 97 98 ethanol, was the primary test article in this study. Comparators included 70% isopropyl alcohol and 40% urea water solutions. 99 Fungal strains Two strains obtained from the American Type Culture Collection (ATCC), Trichophyton mentagrophytes ATCC and T. rubrum MYA 4438, were used in the penetration studies. Preparation of Hoof Samples In order to ensure uniform test product application, hoof samples were polished with abrasive paper, wiped with a wet cloth to remove excess material, and cut into slices of various thicknesses depending on the test assay. All samples for all assays were processed in the same manner, and were subsequently stored at 20±2ºC at 50±2% relative humidity throughout the testing procedures. Each test product was applied as a thin layer to one surface of the sample using a special brush and allowed to air dry. After 24 hrs, the sample was rinsed with water, air dried, and re-treated with product. This procedure was repeated daily for a total of 6 days to mimic a repeat-dose regimen. Test groups included HPCH-based nail solution, 40% urea solution, 70% isopropyl alcohol solution, and untreated control. Hardness Test Hoof slices (3 mm thick) (n=20 per test article) were tested for hardness using the Vickers method that measures the footprint left by an indenter on the surface of the 5

6 sample. The unit of hardness given by the test is known as the Vickers Pyramid Number (HV) and represents the test material's ability to resist deformation from a standard source, which in this case is a diamond in the form of a square-based pyramid. [11] Tensile Strength To determine the relative tensile strength, uniaxial samples (n=20 per test article) were prepared at a thickness of mm with widened shoulders to facilitate testing with an RS 232 SAUTER FH 500 instrument. The sample was pulled with a controlled, gradually increasing force until the sample changed shape or was ruptured. Tensile strength was measured as force per unit area, and expressed in megapascals (MPa). [12] Flexural Strength The RS 232 SAUTER FH 500 instrument was also used to test flexural strength of the hoof samples. Samples (n=20 per test article) were prepared at a thickness of mm and were processed to achieve a constant rectangular section throughout the entire length of the sample. The load was gradually increased to obtain the rupture of the tested specimens. The flexural or bend strength, defined as a material's ability to resist deformation under load, was also measured in megapascals (MPa). [13] Ultrastructure Ultrastructure images of the samples were obtained using a Scanning Electron Microscope (SEM) Hitachi S-2400 from 0.5 cm 2 samples, 2-5 mm thick (n=20 per test article). Images were taken before and after abrasion of the hoof surface with abrasive 6

7 paper applied in 2 directions for 15 seconds each. The "index of crumbling", defined as the difference between the percentage area of shades of gray before and after the abrasion, is an indicator of the protective effect of the applied test product Dermatophyte penetration In order to study the effects of test articles on the ability of dermatophytes to penetrate the hoof samples, disposable biopsy punches were used to cut 4 mm disks from autoclaved samples measuring mm thick (n=5 per test article). Treatments were applied to the disks once daily for six days prior to inoculation with dermatophytes. Mature colonies of Trichophyton mentagrophytes ATCC and T. rubrum MYA 4438 were harvested in Phosphate buffered saline (PBS) to a concentration of CFU/mL. This inoculum was used to seed Potato dextrose agar (PDA) plates upon which the disks were placed. Disks were evaluated after 1, 3, 5, 7, 9, 11, 13, and 15 days of incubation at 30º C, which were chosen as optimal time points for demonstration of the various rates of penetration. The ability of the fungi to invade the hoof disk was evaluated using two methodologies: determination of the fungal colony forming units (CFUs) infecting the hoof samples and histopathological examination. For determining CFUs, additional hoof disks were wiped to remove any residual inoculum from the surface, weighed, and then ground to powder. Ground hooves were cultured on Potato dextrose agar (PDA), incubated for 4 days at 30 C and then analyzed for colony count. Since determination of CFUs by this method is only semi-quantitative and is not standardized, the depth of penetration by fungal hyphae 7

8 was also determined by histological evaluation. Disks were stained with periodic acid- Schiff (PAS) for visualization of fungal elements in order to monitor the penetration of the fungal infection Statistical Analyses The results for hardness and ultrastructure were analyzed using the Kruskal-Wallis test, followed by the Wilcoxon-Mann-Whitney test. For tensile strength and flexural strength an ANOVA was performed, followed by the t-test. RESULTS Hardness Test The results of the hardness test are summarized in Table 1. As can be seen, application of the HPCH-based nail solution to the hooves significantly increased their hardness in comparison to the untreated control sample (mean ±2.01 HV compared to ± 1.23 HV, P=<0.001). By contrast, both urea and isopropyl alcohol had a significantly adverse effect on the hardness of the bovine hoof (17.98 ±1.39 HV and ±1.22 HV, respectively, P=<0.001). The effect of the HPCH-based test product was significant also vs. urea and isopropyl alcohol (both P<0.001). Tensile Strength As reported in Figure 2, the application of the HPCH-based nail solution increased the tensile strength of the hoof samples compared to the untreated control (mean ±5.46 compared to ±5.39 MPa P=<0.05). On the contrary the application of urea or 8

9 isopropyl alcohol rendered the samples more fragile (mean ±4.02 and ±7.48 MPa, respectively). The effect of the HPCH-based test product was significant also vs. urea and isopropyl alcohol (P<0.001) Flexural Strength Results obtained on the flexural strength suggested that the application of the HPCHbased nail solution appeared to increase the resistance of the hoof samples to flexing or bending as compared to the untreated control (mean ±44.75 compared to ±49.71 MPa). In contrast, urea and isopropyl alcohol made the samples more easily flexed ( ±61.89 and ±35.71 MPa, respectively), though there were no significant differences between treatments. Ultrastructure A very high "index of crumbling", measured HPCH-based nail solution as the difference in the areas of gray shading on SEM images taken before and after sample abrasion, was recorded in the untreated hooves. In contrast, the application of HPCH-based nail solution resulted in significantly smaller shaded areas in the images captured before and after abrasion as compared to the control (P= <0.05 and P= <0.001 respectively), indicating that the HPCH-based nail solution rendered the samples less brittle. Similarly, urea and isopropyl alcohol significantly reduced the index of crumbling compared to the untreated control after abrasion (P= <0.001). 209 Dermatophyte penetration 9

10 Figure 3 shows the fungal burden (CFUs) per gram of hooves pre-treated with either the HPCH-based nail solution or isopropyl alcohol or urea and infected with either T. mentagrophytes or T. rubrum. As can be seen, the fungal burden of dermatophytes was consistently lower in hooves treated with the HPCH-based nail solution as compared to the untreated control. Additionally, the fungal burden of hooves treated with the HPCHbased nail solution was generally lower than that of hooves pre-treated with urea or isopropyl alcohol, at all time points. Further, there was a notable delay in the proliferation of T. rubrum growth on the HPCHbased nail solution-treated hooves. There were no colonies of T. rubrum isolated from the HPCH-based nail solution-treated hooves evaluated at Days 3 and 5 as compared to a 3.30 Log CFU/g colony count in the untreated hooves at Day 5. As can be seen in Table 1, the depth of fungal penetration by both dermatophyte species was much lower in the HPCH-based nail solution -treated hooves than that achieved in the untreated control or hooves treated with urea or isopropyl alcohol. In fact, the penetration depth in the HPCH-based nail solution -treated hooves by T. mentagrophytes was <25 μm at Day 9, compared to 275 μm in the untreated control. The penetration depth in the HPCH-based nail solution-treated hooves by T. rubrum at Day 5 was <25 μm as compared to 125 μm in the untreated control. Examples of PAS-stained hoof slices inoculated with T. mentagrophytes and T. rubrum at Day 7 are shown in Figures 4 and 5, respectively. As can be seen in both figures, the 10

11 hooves treated with the HPCH-based nail solution demonstrated superficial fungal elements with limited hoof penetration in contrast to the hooves treated with isopropyl alcohol or urea or the untreated control. Moreover, images taken following inoculation with either dermatophyte strain showed marked degradation of the hooves treated with isopropyl alcohol or urea by Day 15, as demonstrated in Figure 6. DISCUSSION The population at risk for developing fungal nail infection includes those with nail dystrophy, brittle or psoriatic nails, and mechanical or chemical nail injury. These various nail conditions highlight the importance of nail integrity in preventing the ability of dermatophytes and non-dermatophyte nail pathogens from establishing an infection. Although it is common awareness that aggressive chemicals, normally used for cosmetic or therapeutic purposes, can result in weakening the nails, to our knowledge this is the first time it is experimentally demonstrated. The use of isopropyl alcohol to remove previously applied solution layers of some nail formulations or the use of urea as nail pretreatment to improve penetration of antifungals is not supported by our data. In fact, adverse changes in the physical characteristics of the hoof translated into a higher susceptibility to fungal nail permeation. This may account for failure to treat onychomycosis by topical products, which contain penetration enhancers [6]. On the contrary, strengthening the nail may improve the natural barrier to infection, as shown by our investigation with HPCH-based nail solution. 11

12 The composite results of this study indicate that the application of the HPCH-based nail solution may improve the nail barrier characteristics as demonstrated by the enhanced hoof hardness and tensile strength and by protection against nail abrasion. Taken together, these findings may translate into a more effective barrier against nail fungal infection. In conclusion, the results obtained in this study suggest that repeated application of the HPCH-based nail solution may reinforce the nail structure and thus prevent nail fungal colonization and infection. ACKNOWLEDGEMENTS This work was supported by Polichem SA, Lugano, Switzerland. REFERENCES 1. Scher RK, Baran R Onychomycosis in clinical practice: factors contributing to recurrence. Br J Dermatol. 149 Suppl 65: Haneke E Fungal infections of the nail. Semin Dermatol. 10(1): Jesudanam TM, Rao GR, Laksmi DJ, Kumari, GR Onychomycosis: a significant medical problem. Ind J Dermatol Venerol Leprol. 68(6): Boonchai W, Kulthanan K, Maungprasat C, Suthipinittham P Clinical characteristics and mycology of onychomycosis in autoimmune patients. J Med Assoc Thai. 86(11): Ghannoum MA, Hajjeh RA, Scher R, Konnikov N, Gupta A K, Summerbell R, Sullivan S, Daniel R, Krusinski P, Fleckman P, Rich P, Odom R, Aly R, Pariser D, Zaiac M, Rebell G, Lesher J, Gerlach B, Ponce-de-Leon GF, 12

13 Ghannoum A, Warner J, Isham N, Elewski B A large-scale North American study of fungal isolates from nails: The frequency of onychomycosis, fungal distribution, and antifungal. J Am Acad Dermatol. 43: Elewski BE, Rich P, Tosti A, Pariser DM, Scher, R, Daniel RC, Gupta AK Onychomycosis: an overview. J Drugs Dermatol. 12(7):s Tasic S, Stojanovic S, Poljacki M Etiopathogenesis, clinical picture and diagnosis of onychomycosis. Med Pregl. 54(1-2): Cantoresi F, Sorgi P, Arcese A, Bidoli A, Bruni F, Carnevale C, Calvieri S Improvement of psoriatic onychodystrophy by a water-soluble nail solution. J Eur Acad Dermatol Venereol. 23(7): Cantoresi F, Caserini M, Bidoli A, Maggio F, Marino R, Carnevale C, Sorgi P, Palmieri R Randomised, controlled trial of a water-soluble nail solution based on hydroxypropyl-chitosan (HPCH), in the management of nail psoriasis. Clin Cosm Inv Dermatol. 7: Monti D, Saccomani L, Chetoni P, Burgalassi S, Tampucci S, Mailland F Validation of bovine hoof slices as a model for infected human toenails: in vitro ciclopirox transungual permeation. Br J Dermatol. 165(1): Smith RL, Sandland GE "An Accurate Method of Determining the Hardness of Metals, with Particular Reference to Those of a High Degree of Hardness," In Proceedings of the Institution of Mechanical Engineers, Vol. I, Czichos H In Springer Handbook of Materials Measurement Methods. Berlin: Springer. p ISBN

14 Hodgkinson JM In Mechanical Testing of Advanced Fibre Composites, Cambridge: Woodhead Publishing, Ltd., p

15 Fig. 1. Comparison of the effects of HPCH-based nail solution and comparators on the hardness and tensile strength of hoof samples in HV (Vickers Pyramid number) and megapascals (MPa), respectively) *P values <0.001 HPCH-based nail solution vs. comparators for both hardness and tensile strength. Downloaded from on October 2, 2018 by guest 15

16 Fig. 2. Comparison of the effects of HPCH-based nail solution and comparators on the "crumbling index" following sample abrasion in pixels

17 Figure 3. Fungal burden of hooves pre-treated with HPCH-based nail solution, urea, or isopropanol, and untreated control after exposure to dermatophytes

18 Table 1. Tissue penetration (in μm) of hooves inoculated with dermatophytes. Days of fungal growth HPCH-based nail solution 40% Urea 70% Isopropyl alcohol Untreated TM TR TM TR TM TR TM TR < <25 < < < *NS NS *NS NS *NS - Sample damaged during processing; TM: T. mentagrophytes; TR: T. rubrum Downloaded from on October 2, 2018 by guest 18

19 Figure 4. Microscopy of hooves at Day 7 post-exposure to T. mentagrophytes and stained with PAS at 20. A. Untreated Control B. HPCH-based nail solution C. Isopropyl alcohol D. Urea Downloaded from on October 2, 2018 by guest 19

20 Figure 5. Microscopy of hooves at Day 7 post-exposure to T. rubrum and stained with PAS at 20. A. Untreated Control B. HPCH-based nail solution C. Isopropyl alcohol D. Urea Downloaded from on October 2, 2018 by guest 20

21 Figure 6. Microscopy of hooves at Day 15 post exposure to T. rubrum and stained with PAS at 20. A. Untreated Control B. HPCH-based nail solution C. Isopropyl alcohol D. Urea Downloaded from on October 2, 2018 by guest 21

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