Transungual Delivery, Anti-Inflammatory Activity, and In Vivo Assessment of a Cyclodextrin Polypseudorotaxanes Nail Lacquer

pharmaceutics Article Transungual Delivery, Anti-Inflammatory Activity, and In Vivo Assessment of a Cyclodextrin Polypseudorotaxanes Nail Lacquer Francisco Fernández-Campos 1,* , Francesc Navarro 1, Adrian Corrales 1, Jordi Picas 1, Eloy Pena 1, Jordi González 1and Francisco J. Otero-Espinar 2,3 1R & D Department. Reig Jofre Laboratories, Avda dels Flors, s/n, 08970 Sant Joan Despi, Spain; [email protected] (F.N.); [email protected] (A.C.); [email protected] (J.P.); [email protected] (E.P.); [email protected] (J.G.) 2 Department of Pharmacology, Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Santiago de Compostela (USC), 15782 Santiago de Compostela, Spain; [email protected] 3Paraquasil Group, Health Research Institute of Santiago de Compostela (FIDIS), 15782 Santiago de Compostela, Spain *Correspondence: [email protected]; Tel.: +34-93-480-6710 Received: 27 June 2020; Accepted: 31 July 2020; Published: 4 August 2020   Abstract: A new cyclodextrin polypseudorotaxanes nail lacquer (Regenail ® ) containing biotin, methyl sulphonyl methane (MSM), and dimethylsilanediol salicylate was developed and evaluated in vitro and in vivo . The product was developed to improve nail status and diminish signs of pathological nail alterations. A reference product (Betalfatrus ® ) was used for comparative purposes. An in vitro permeation experiment in hooves showed high MSM and biotin absorption. The content of sulfur and silicon in hooves was also found to be higher compared with the reference product. MSM was tested in human keratinocytes, exhibiting a good cytotoxicity profile and anti-inflammatory activity by the reduction in IL-8 and TNFα under LPS stimuli. A clinical study was performed to check product safety and efficacy against nail brittleness and alterations such as Beau’s lines and onychorrhexis. A reduction in both alterations and in surface roughness without alteration of nail structure was observed, with a good level of patient acceptance and satisfaction. Keywords: nail lacquers; cyclodextrins; methylsulfonylmethane; active penetration; biotin 1. Introduction Nails are a complex structure composed of a nail bed (the deeper layer), nail matrix, nail folds, and the outer layer, nail plate. The nail bed is formed of a noncornified living epithelium tissue underlying the papilla dermis and contains blood vessels, nerves, and lymphatic fluid [ 1 ]. The nail plate contains between 80 and 90 layers of dense keratinized (mainly α -keratin) dead cells, with desmosome unions. This structure protects the nail bed from external damage. Nail folds are attached to the nail matrix and are a continuation of the skin, developing the cuticle, which seals the nail surface, preventing the entrance of chemicals and microorganisms. Finally, the nail plate is the outermost layer, formed by translucent dead keratinocytes and with a high keratin content, rich in cysteine, glycine, and tyrosine proteins. Lunula is the most characteristic distal semicircular white section of the nail plate [ 2 – 4 ]. Unlike the skin, nails have a low lipid content (around 1%), mainly composed of cholesterol, and the water content is higher than in the stratum corneum at around 10–30% (as a protein solvate) in a normal state [ 4 ]. These facts mean nails have different permeability properties from skin, which should be considered in formula development. Pharmaceutics 2020,12, 730; doi:10.3390/pharmaceutics12080730 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2020,12, 730 2 of 18 Different factors could affect nail status, such as environmental conditions, external factors, and several diseases. Environmental factors (e.g., low relative humidity, winter, etc.) could reduce the water content, leading to nail brittleness. Constant use of nail cosmetic products (nail polish) based on a high amount of organic solvents (e.g., butyl acetate, ethyl acetate) could extract nail water and lipids, leading to progressive dehydration. These products also contain chemical compounds (plasticizers, i.e., dibutyl phthalate and dioctyl phthalate; pearlizers, i.e., bismuth oxychloride; resins, i.e., toluene sulphonamide formaldehyde, shellac; etc.), that could induce contact dermatitis and nail plate coloration [ 5 ]. Nutritional deficiencies (biotin deficiencies [ 6 ]) and diseases (i.e., microbial infections, psoriasis, lichen planus, alopecia areata, Darier’s disease, etc.) could affect the nail structure [7,8]. Fungal infections (onychomycosis) are one of the most common nail diseases and are highly recurrent [ 9 , 10 ]. The prevalence ranged between 8% and 14% in North America, increasing with age up to 50% in the elderly [ 11 , 12 ]. They could be caused by dermatophytes, with Trichophyton rubrum being the main microorganism involved in around 71% of fungal infections, and the second most common cause being Trichophyton mentagrophyte (20%). Nondermatophyte microorganisms are less common, with Candida albicans responsible for 5.6% of nail infections [ 12 ]. Several clinical presentations could be present—hyperkeratosis, onycholysis, dyschromia (melanonychia), longitudinal striates, and inflammation—leading to nail structure alteration and function impairment. Topical antifungals are usually employed, and an oral alternative is reserved to treat more invasive and/or extensive manifestations [11]. Onychomycosis is present in 18% of patients, with nail psoriasis being an aggravating factor of the disease [ 13 ]. Psoriasis is a chronic immune disease affecting the skin. Keratinocyte hyperproliferation and inflammation are observed in patients and the involvement of the nail apparatus happens in around 50% of cases [ 14 ]. Nail alterations in psoriasis usually involve the nail matrix (pitting, leukonychia or white spots, and crumbling) and the nail bed (onycholysis, hyperkeratosis, and discoloration). In addition, longitudinal (onychorrhexis) and transversal ridges (also known as Beau’s lines) are usually present as a clinical manifestation. Topical drug delivery systems to treat nail alterations are usually preferred to avoid the adverse effects of systemic medications. In addition, they have good patient acceptance and are cost-effective. The main drawback of topical therapy is low drug absorption due to the limited permeability of the nail structure, reducing the access of the drug to the nail bed. In addition, the nail bed turnover increases the reduction in drug concentrations [ 15 ]. To maintain an effective drug flux, several strategies (physical and chemical) are reported in the literature. Physical agents such as iontophoresis have been reported to increase antifungal and corticoid concentrations in the nail structure, improving therapeutic outcomes. Ultrasounds, photodynamic and laser therapy were also described as promising tools to increase drug effectiveness [ 12 ]. Physical abrasion with sandpaper aimed to reduce nail thickness and thereby increase drug diffusion across the ungual structure. This has been reported to increase terbinafine effectiveness in onychomycosis [16]. Transient chemical modification of the nail structure by permeation enhancers to increase drug diffusion is widely described in the literature. Considering that the main constituent of the nail is keratin, an alteration in protein packing is one mechanism of enhancement. Sulfhydryl compounds, such as cysteine, acetyl cysteine, and mercaptoethanol [ 17 ], and papaine, an endopeptidase with reactive sulfhydryl residues, reduce disulfide bounds and destabilize keratin. Softening compounds (urea and salicylic acid) have keratolytic properties and increase water uptake and swell keratin, making it less dense and compact and increasing the pore size [ 4 ]. Surfactants such as sodium lauryl sulfate could also alter the protein packing by electrostatic repulsion, induce micelle formation and absorption promotion, and reduce the contact angle between the formulation and nail surface to ensure better water access, increasing swelling [ 18 ]. Cyclodextrins are cyclic oligosaccharides and had the ability to form complexes with various active ingredients, involving the active ingredient in the lipophilic center. Their complex formation allows us to change the physical and chemical properties of Pharmaceutics 2020,12, 730 3 of 18 the active ingredients [ 19 , 20 ]. Cyclodextrins had been reported to increase drug absorption across nails due to the solubilization of hydrophobic molecules—increasing the hydration of the nail plate, making pores broaden, and interacting with aromatic amino acids, limiting the protein folding. It was also demonstrated that the water itself could act as an ungual enhancer by the hydration and swelling of keratin fibers [ 21 – 23 ]. In addition, the hydroalcoholic-based formulation causes a higher concentration of the drug in the film than from the originally applied formulations after the evaporation of the solvent, leading to thermodynamic activity increase, which favors drug diffusion [24]. Nogueiras-Nieto et al. developed in situ gelling formulations of Poloxamer 407 and hydroxypropyl β -cyclodextrin (HPβ -CD), obtaining a polypseudorotaxane supramolecular structure that increases the delivery of antifungals (ciclopirox) and corticoid (triamcinolone) across human nails and hooves [ 25 ]. Later, the same group used methylβ -cyclodextrin and Poloxamer 407 in a hydroalcoholic solution to deliver ciclopirox olamine and compared the results with other marketed ciclopirox olamine formulations. The proposed polypseudorotaxane lacquer increased the permeation and accumulation of the drug into the nail structure [ 22 ]. Chouhan et al. also demonstrated in vitro the enhancing effect of HPβ -CD for terbinafine ungual delivery [ 21 ]. Cutrin-Gomez et al., in 2018, showed the capacity of the soluble cyclodextrin derivatives (methylβ -cyclodextrin and HPβ -CD) to modify the microporous characteristics of the nail plate via interaction with nail components. These modifications produce a significant increase in the drug permeability and drug accumulation in the nail [23]. There are a limited number of products on the market to treat nail alterations topically, based on hydroalcoholic solutions. Most of them are antimycotic drugs, formulated in a variety of nail lacquers bases. There are few nonmycotic agents to treat other nail alterations or protect from external aggressions. Methyl sulfonyl methane (MSM), an important volatile component in the sulfur cycle, has long been thought of as a sulfur donor for sulfur-containing compounds such as methionine, cysteine, homocysteine, taurine, and many others [ 26 – 28 ]. MSM is present in keratin, where it is responsible for making nails hard and is a crucial component of collagen production [ 26 ]. MSM is structurally related to DMSO but differs in the oxidation state; it is a small (MW 94.14 Da) and hydrophilic compound (logP −1.41). It is nonionizable in ambient conditions. The presence of soft and brittle nails can also indicate a systemic deficiency of silicon [ 29 , 30 ]. Silicon also helps with the synthesis of glycosaminoglycans, along with collagen [ 30 ]. Barel et al. [ 31 ] evaluated the effect of the intake of supplements containing choline-stabilized orthosilicic acid (ch-OSA) on the skin, hair, and nails. After treatment, they observed a significant improvement in the fragility of nails and hair in the group using the ch-OSA. Dimethylsilanediol salicylate is an organic form of water-soluble silicon (MW 212.3 Da, log P 3.13, Log D (pH 5.5) 2.59). A salicylic radical could enhance properties for nail penetration. Biotin has an important role in protein synthesis, especially in keratins, and has been claimed to contribute to healthy nails and hair. Oral biotin led to improved hardness and thickness in nails [ 6 , 32 , 33 ]. In vitro studies of keratinocytes showed the stimulation of cell differentiation and the production of cytokeratins [ 34 ]. They were also shown to stimulate lipid synthesis, responsible for the binding of keratinocytes in the nail plate [ 33 ]. Biotin is a small molecule of 244.3 Da, slightly soluble in water in its nonsalt form (logP 0.39, pKa 4.4) and leading to good permeability across nails. We hypothesized that the topical administration of MSM, dimethylsilanediol salicylate, and biotin in polypseudorotaxane hydroalcoholic nail lacquer will effectively deliver these compounds across the nail barrier and thereby improve nail status and diminish signs of nail pathological alterations. Hence, the aim of this study was to further investigate the transungual permeation/penetration profiles of MSM and biotin, as well as the amounts of sulfur and silicon in the nail, after the application of an experimental nail lacquer (Regenail ® : REG) and a commercial nail lacquer (Betalfatrus ® : BET). Secondary objectives included the evaluation of the clinical efficacy and safety of the experimental lacquer and of the user-friendliness, as gauged by the subject’s self-assessment and a medical examination and the effect in terms of the improvement of alterations in nails achieved by Regenail ® . Pharmaceutics 2020,12, 730 4 of 18 2. Materials and Methods 2.1. Materials MSM (10% w/w) and bamboo glycolic extract (Pracofar S.A., Barcelona, Spain), biotin (0.2% w/w) (Siemgluss Iberica S.A., Barcelona, Spain), HPβ -CD (8.7% w/w) (molar substitution degree of 0.65 and molecular weight of 1399 Da, HPB Kleptose, Roquette, Lestrem, France), dimethylsilanediol salicylate (0.246% w/w, Exsymol, Monaco), sodium lauryl sulfate (0.87% w/w, BASF, Barcelona, Spain), ethanol 96 ◦ (34.7%, Alcoholes Oliva, Barcelona, Spain), and purified water were used for the experimental nail lacquer (REG) production. Components were added one by one, from the least to the most concentrated, to the ethanol-water mixture. The commercial nail lacquer (BET) chosen as a reference had the following composition: Equisetum arvense, methyl-sulfonyl-methane (~5.55% w/w), hydroxypropyl-chitosan, ethanol, water, and diethylenglycol monoethylether. 2.2. Methods 2.2.1. Regenail Formulation Characterization The produced formulation, Regenail, was characterized in terms of appearance (visual observation) and HPLC quantification of biotin and MSM. These determinations assessed the stability according to ICH long-term conditions 25 ◦ C/60% HR, 30 ◦ C/75% HR and in accelerated conditions 40 ◦ C/75% HR. The HPLC quantification was as described in Section 2.2.2. Finally, a release experiment was performed on Franz cells (VidraFoc, Barcelona, Spain; 100 mL volume and an effective diffusional area of 2 cm 2 ) using a dialysis membrane (10–12 kDa). One milliliter of REG was placed in a donor compartment (n=6) and 0.3 µ L samples were taken at 1, 2, 4, 20, 22, and 24 h and replenished with the same volume of fresh receptor medium (5% HPβ -CD in phosphate buffered saline (PBS, pH 7.4, to maintain sink conditions). The system was continuously stirred at 500 rpm and maintained at 37 ◦ C throughout the experiment. Biotin and MSM release were quantified according to the methods described in Section 2.2.2. 2.2.2. In Vitro Penetration of the Nail Plate We created in vitro permeation profiles of MSM and biotin (only in the case of REG), and recorded the amounts of sulfur and silicon in the nails after treatment with the experimental nail lacquer or the commercial nail lacquer. Permeation Study The bovine hooves were obtained from freshly slaughtered cattle, stripped of adhering cartilaginous and connective tissue. Hooves were cut into flat sections of approximately 300–700 µ m in thickness and frozen until use. The prepared hoof membrane was used in a Franz diffusion cell (n=6 per formulation) with an effective diffusion area of 0.196 cm 2 . For this, hoof samples were cut into small discs using an 8-mm punch and placed between two PTFE adapters with an O-shaped ring of 5 mm in diameter. The ratio between the area of the O-shaped ring and the bovine hoof samples was 0.392 and, therefore, in accordance with Palliyil et al. [ 35 ], the lateral diffusion caused by the edge effect is not expected to have a significant influence on our results. The receptor compartment was filled with 5 mL of phosphate buffered saline (PBS, pH 7.4) and with 5% of HPβ -CD, and the cells were equilibrated at 37 ◦ C in a water bath with magnetic stirring. Two milliliters of the nail lacquers or PBS (blank) were placed in the donor compartment to avoid depletion during the 11 days of the experiment. Once a day, a 1 mL sample of receptor medium was sampled and replenished with the same volume of fresh medium. The receptor fluid was sampled for the determination of MSM by gas chromatography (GC) and biotin by HPLC. In order to determine Pharmaceutics 2020,12, 730 5 of 18 the amounts of biotin, silicon and sulfur that remained in the hoof after the permeation experiment, hooves were cleaned with distilled water and dried using cellulose paper for further quantification. Biotin Quantification in Receptor Medium and Hooves The biotin content of the samples (from receptor medium, n=6; from hoof, n=6) was directly analyzed by a validated HPLC (Water Corporation, Barcelona, Spain) method (mobile phase: Acetonitrile/buffer (1 g sodium perchlorate, 10 mL phosphoric acid up to 1 L of purified water) (10:90), flow 1.0 mL/min, column Symmetry 200 C18 3.5 µ m, 150 mm × 4.6 mm). Biotin was extracted from hoof samples by incubation with 5 mL of 5% methanol in a PBS solution for five days (at 25 ◦ C). To quantify biotin content, a calibration line in the range 0.7–506 µ g/mL was prepared. The cumulative amounts permeated were normalized versus the permeation surface. The slope of cumulative amounts (normalized by surface) vs. time (h) was calculated. MSM Quantification in Receptor Medium The MSM content of the receptor medium samples (n=6 per formulation) was analyzed by CG (Agilent Technologies, Barcelona, Spain; mobile phase helium, flow 8.0 mL/min, column DB-1 50 m, 0.53 mm internal diameter, 5 µ m). A calibration curve in the range of 3 to 13,300 µ g/mL was prepared in a methanol solvent. The cumulative amounts released were normalized versus the permeation surface. Sulfur and Silicon Quantification in Hooves after Permeation In order to determine the amount of sulfur and silicon in the hooves, they were treated by digestion for 120 min with 2 mL of a solution of 2.5% TMAH (tetramethylammonium hydroxide, Sigma-Aldrich, Barcelona, Spain) in water at 90 ◦ C. The intensity value obtained for the blank was subtracted from the values obtained with each one of the samples. The sulfur and silicon content in hooves was analyzed by ICP-OES (Inductively Coupled Plasma-Optical Emission Spectrometry). Sulfur was determined at 182.0 nm and silicon was determined at 288.2 nm. 2.2.3. In vitro Cytotoxicity Assay The cytotoxic effect of MSM was evaluated in vitro (n=6) by direct contact with the cell, following the ISO 10993-5:2009 recommendation guidelines [36]. The HaCaT cells were seeded in a 96-well plate at a density of 10,000 cells/well, in a fresh culture medium supplemented with 10% fetal bovine serum, penicillin (100 IU/mL), and streptomycin (100 µ g/mL) (Fisher Scientific, Barcelona, Spain). The plates were incubated for 24 h in a humidified atmosphere of 5% CO 2 at 37 ◦ C and treated with different concentrations of MSM from 10 µ M to 0.003 µ M, diluted in growth medium. For cell proliferation quantification, the general cell viability endpoint MTT (Sigma-Aldrich, Barcelona, Spain) reduction (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) was used [ 37 ]. Accordingly, the previous culture medium was removed and replaced with a fresh medium containing MTT (1:10). The cells were incubated at 37 ◦ C for 3 h; after this time, the medium was removed and the intracellular formazan crystals were solubilized and extracted with dimethylsulfoxide (Panreac, Barcelona, Spain). After 15 min at room temperature, the absorbance was measured at 550 nm in a microplate reader (Perkin Elmer, Barcelona, Spain). 2.2.4. TNFαand IL8 RNA Isolation and RT-PCR RNA was isolated from cell samples (n=6) using the method described by Chomczynski [ 38 ], using the RNeasy kit (Qiagen) and treating with DNAse-I to remove any contamination from genomic DNA. The purity of the RNA was estimated by 1.5% agarose gel electrophoresis, and the RNA concentration was determined by nano-drop spectrophotometry (Thermo Fisher Scientific, Barcelona, Spain). Pharmaceutics 2020,12, 730 6 of 18 Reverse transcription of 1 µ g RNA for complementary DNA (cDNA) synthesis was performed using a First-strand Synthesis kit (Takara-Clontech, Saint-Germain-en-Laye, France). This cDNA from treated or untreated cells (control) was used to determine the relative gene expression of TNF α and IL-8 through RT-qPCR. Real-time (RT)-qPCR was performed in a QuantStudio 5, Applied BioSystem (Barcelona, Spain) apparatus. For the normalization of cDNA loading in the PCR reaction, the amplification of ACT for every sample was used. Relative changes in gene expression were calculated using the Pfafflmethod, following standard procedures [39]. 2.2.5. Clinical Study Design The main objective of the study was to evaluate the clinical efficacy and subjective perception of the improvement in nail alterations achieved by the experimental nail lacquer (REG) or the commercial nail lacquer (BET). The safety of both products was also evaluated as a secondary endpoint. Thirty volunteers (between 18 and 65 years old) from both sexes were recruited to participate in the study. The inclusion criteria were: Both genders, general good health (physical and psychological), alterations on the surface of the nails of both hands, adequate understanding of the clinical study, agreement to participate, and availability to attend the visits. No other topical nail product could be used during the study and any other treatment that could affect the evaluation of the study endpoints was forbidden. Each subject received oral and written information concerning the studied product. Written consent was obtained before any study-specific procedures, in accordance with the Helsinki Declaration. Eligible subjects were randomly assigned to an experimental group. Patients were treated for a period of 28 days. Both products were applied once daily, preferably at night (to ensure a contact time of 6 h), with a brush, covering the complete (cleaned) nail. After application of the nail lacquer, patients were instructed not to clean their hands, because the obtained film in nails is hydrophilic. The following day, patients could do their routine daily cleaning (shower, hand washing, etc.). Nails’ evolution was evaluated at day 28, compared with D 0 (the baseline). On day 14, volunteers came to the study site for a control visit. On day 0 and day 28, the nails were examined by a dermatologist to evaluate the nail alterations’ progression (Beau’s lines and onychorrhexis). A categorical scale ranging from 0 (alteration absence) to 4 (severe alteration) was used by the dermatologist for evaluation. The nail roughness was evaluated with PRIMOS-CR ® (Canfield Scientific, NY, USA). The transonychial water loss (TOWL) was also evaluated with the Tewameter ® TM 300 (Courage +Khazaka electronic GmbH, Köln Germany). Participants had washed their hands prior to the topographical evaluation. Measurements of camera distance and ambient illumination were standardized. A subjective questionnaire was included at the end of the study to evaluate product usability and efficacy. A Likert scale (from 0—totally disagree, to 5—totally agree) was employed for this evaluation. The compatibility of the product with nails and any undesirable effects were assessed at every visit by a dermatologist. 2.2.6. Data Analysis The slope of the linear portion of the cumulative amounts vs. the time of permeation profile was estimated as the pseudo steady-state flux (Js) of penetrant permeation. The lag times (Tlag) for each active compound were derived from the x-intercept of the slope at pseudo steady-state. The permeability coefficients (Kp) of each active compound were calculated as compound flux/compound concentration in the donor chamber. The diffusion parameter P 1 and the portioning parameter P 2 were calculated according to Equations (1) and (2), respectively. The results were expressed as mean ± standard deviation (SD) and were statistically analyzed with Minitab software (Coventry, United Kingdom). Kp =P1·P2(1) Pharmaceutics 2020,12, 730 7 of 18 Tlag =1/(6 P2) (2) The significance of the effect of the treatment over time on the response variables was evaluated using an ANOVA test. A significance value of 0.05 was established for all statistical tests used in the data analysis. 3. Results 3.1. Formulation Characterization Figure 1shows the results of biotin and MSM under different climatic conditions for 12 months. The data show low variation (less than 5%) compared with the initial time point for both active compounds in all conditions. The results at 30 ◦ C/75% HR almost overlap with those at 25 ◦ C/60% HR for both MSM and biotin, with no differences shown between these stability conditions. According to ICH Q1E [ 40 ], if no significant variation (less than 5%) is seen in the accelerated stability conditions, then an extrapolation of long-term conditions up to two years could be stated, based on the obtained results. A long-term study will continue until the proposed extrapolated shelf-life (two years) to confirm the obtained results. The regression equations at 25 ◦ C/60% HR (long-term conditions at climate zone II) were for biotin y =102.57 − 0.067*Time and for MSM y =103.9 +0.25*Time. The regression equations at 30 ◦ C/75% HR (long-term conditions at climate zone IVb) were for biotin y =102.43 − 0.117*Time and for MSM y=104.02 +0.275*Time . To evaluate whether there are differences in the regression lines, ANCOVA analysis (covariance analysis) was performed. In both biotin and MSM, there were no differences in interaction factor Time x Climate condition (p<0.05), which means that the regression line slopes are considered parallel and the factor climate conditions (which represents the y-intercept) is not significant (p<0.05). The overall biotin and MSM regression lines could be considered coincident at both climate conditions (25 ◦C/60% HR and 30 ◦C/75%HR). Pharmaceutics 2020, 12, x FOR PEER REVIEW 7 of 18 Kp = P1∙P2 (1) Tlag = 1⁄(6 P2) (2) The significance of the effect of the treatment over time on the response variables was evaluated using an ANOVA test. A significance value of 0.05 was established for all statistical tests used in the data analysis. 3. Results 3.1. Formulation Characterization Figure 1 shows the results of biotin and MSM under different climatic conditions for 12 months. The data show low variation (less than 5%) compared with the initial time point for both active compounds in all conditions. The results at 30 °C/75% HR almost overlap with those at 25 °C/60% HR for both MSM and biotin, with no differences shown between these stability conditions. According to ICH Q1E [40], if no significant variation (less than 5%) is seen in the accelerated stability conditions, then an extrapolation of long‐term conditions up to two years could be stated, based on the obtained results. A long‐term study will continue until the proposed extrapolated shelf‐life (two years) to confirm the obtained results. The regression equations at 25 °C/60% HR (long‐term conditions at climate zone II) were for biotin y = 102.57 − 0.067*Time and for MSM y = 103.9 + 0.25*Time. The regression equations at 30 °C/75% HR (long‐term conditions at climate zone IVb) were for biotin y = 102.43 − 0.117*Time and for MSM y = 104.02 + 0.275*Time. To evaluate whether there are differences in the regression lines, ANCOVA analysis (covariance analysis) was performed. In both biotin and MSM, there were no differences in interaction factor Time x Climate condition (p < 0.05), which means that the regression line slopes are considered parallel and the factor climate conditions (which represents the y‐intercept) is not significant (p < 0.05). The overall biotin and MSM regression lines could be considered coincident at both climate conditions (25 °C/60% HR and 30 °C/75%HR). Figure 1. Biotin (upper panel) and methyl sulphonyl methane (MSM) (lower panel) content of Regenail® (REG) in stability studies under different climate conditions. Figure 1. Biotin ( upper panel ) and methyl sulphonyl methane (MSM) ( lower panel ) content of Regenail®(REG) in stability studies under different climate conditions. Release experiments (Figure 2) showed a slow release of both compounds, probably due to the dialysis membrane being the rate-limiting step (the active compounds are not expected to be Pharmaceutics 2020,12, 730 8 of 18 encapsulated, because the high solubility in the hydroalcoholic vehicle). This membrane was chosen to prevent the HPβ -CD polypseudorotaxane complex from passing through to the receptor medium. In addition, the small pore of these membranes could be more similar to hoof pores than the standard membranes of 0.22 or 0.45 µ m usually employed in release experiments. A limitation of release experiments is that artificial membranes, such as cellulose dialysis membranes, are not sensitive to permeation enhancers. For this reason, in vitro permeation with hooves was performed. Pharmaceutics 2020, 12, x FOR PEER REVIEW 8 of 18 Release experiments (Figure 2) showed a slow release of both compounds, probably due to the dialysis membrane being the rate‐limiting step (the active compounds are not expected to be encapsulated, because the high solubility in the hydroalcoholic vehicle). This membrane was chosen to prevent the HP‐β‐CD polypseudorotaxane complex from passing through to the receptor medium. In addition, the small pore of these membranes could be more similar to hoof pores than the standard membranes of 0.22 or 0.45 µm usually employed in release experiments. A limitation of release experiments is that artificial membranes, such as cellulose dialysis membranes, are not sensitive to permeation enhancers. For this reason, in vitro permeation with hooves was performed. Figure 2. MSM (upper panel) and biotin (lower panel) release over time from REG nail lacquer. 3.2. In Vitro Permeation Study Nail penetration of active compounds was investigated in Franz diffusion cells after topical application of experimental nail lacquer (REG) or commercial nail lacquer (BET) to hooves for 11 days. Infinite dose conditions were set up to assess formulation differences in terms of the permeation profiles. Both tested products permeated the bovine hoof plate, but differences in permeation parameters were noticed, related to the active compounds and the formulation. The permeation profiles of MSM across the hoof are shown in Figure 3. The transungual permeation parameters of active compounds containing the formulations are presented in Table 1. The calculated permeation steady state flux of MSM through REG formulation, across the hoof plate, was observed to be 10,839.695 µg/h/cm2, more than double that of the reference product. The lag times (Tlag) were statistically different: 105.12 h for REG vs. 98.77 h for BET (p < 0.05). The mean values for the P1 (diffusion‐related parameter) and permeability coefficients (Kp) of MSM were also more than double the corresponding values for the reference. However, P2 (the partitioning‐related parameter) was similar for the two nail lacquers, thus indicating that the partitioning coefficient of the MSM between the formulation and the nail is similar, so both formulations are similar in terms of the relative polarity of the MSM. Figure 2. MSM (upper panel) and biotin (lower panel) release over time from REG nail lacquer. 3.2. In Vitro Permeation Study Nail penetration of active compounds was investigated in Franz diffusion cells after topical application of experimental nail lacquer (REG) or commercial nail lacquer (BET) to hooves for 11 days. Infinite dose conditions were set up to assess formulation differences in terms of the permeation profiles. Both tested products permeated the bovine hoof plate, but differences in permeation parameters were noticed, related to the active compounds and the formulation. The permeation profiles of MSM across the hoof are shown in Figure 3. The transungual permeation parameters of active compounds containing the formulations are presented in Table 1. The calculated permeation steady state flux of MSM through REG formulation, across the hoof plate, was observed to be 10,839.695 µ g/h/cm 2 , more than double that of the reference product. The lag times (Tlag) were statistically different: 105.12 h for REG vs. 98.77 h for BET (p<0.05). The mean values for the P 1 (diffusion-related parameter) and permeability coefficients (Kp) of MSM were also more than double the corresponding values for the reference. However, P 2 (the partitioning-related parameter) was similar for the two nail lacquers, thus indicating that the partitioning coefficient of the MSM between the formulation and the nail is similar, so both formulations are similar in terms of the relative polarity of the MSM. These results clearly indicate that REG allows higher permeation levels of MSM than those obtained with the reference product BET. Pharmaceutics 2020,12, 730 9 of 18 Pharmaceutics 2020, 12, x FOR PEER REVIEW 9 of 18 These results clearly indicate that REG allows higher permeation levels of MSM than those obtained with the reference product BET. Figure 3. Permeation profiles of MSM through the bovine hoof plate as a model of human nail permeation. Additionally, these data were normalized by the amount of MSM applied in each test (the MSM concentration between both products is different: 10% w/w for REG and around 5.5% w/w for BET). When these normalized values are considered, the profiles of both formulations get closer. The mean values for the P1, P2, and Kp of MSM that permeated through hooves showed negligible differences between the two formulations. The permeation steady state flux values, however, were significantly different: 1083.97 µg/h/cm2 for REG vs. 647.89 µg/h/cm2 for BET. Although most of the differences between permeation parameters were caused by different MSM concentrations, the increased flux after dose normalization indicates the superiority of the test vehicle over the commercial formulation. According to the molecular structure of MSM, it is not expanded to encapsulate or interact with HP‐β‐CD, so the increase in transungual flux could be caused by the shelf promoting effect of the formulation. It was demonstrated previously [22,23,25] that cyclodextrin‐soluble polypseudorotaxanes increase the size of nail channels and interact with keratin residues, reducing protein folding. Sodium lauryl sulfate could also increase the diffusivity of MSM by contribution to protein interaction. MSM is not expected to reduce disulfide bonds. In addition, the proposed formulation can increase the absorption of hydrophilic active compounds despite the well‐known solubilization capacity of lipophilic molecules, which is the main promotion mechanism in these compounds. When permeated concentrations of MSM at 24 h (around 21,700 µg/cm2) are compared with release concentrations at the same time point (around 1800 µg/cm2), we see the enhancing effect of REG caused by the interaction of the system with the hoof structure, compared with the pure diffusion seen in release experiments across dialysis membranes. The amount of sulfur obtained through the MSM at the end of experiments after application of REG formulation was 948.66 ppm, 2.3‐fold higher than that obtained with the BET solution (412.78 ppm). Biotin profiles from REG could not be compared with the reference product because it does not contain biotin. The permeation profiles of biotin across a hoof are shown in Figure 4. The transungual biotin parameters are shown in Table 1. As P1 is higher than P2, diffusion is the main driving force that allows for active penetration. There are no previous investigations that have described the topical absorption of biotin, so this is the first time that transungual delivery of biotin has been described. It is shown that this route of administration could lead to topical absorption across the nail plate, leading to pharmacological action at the nail bed. 0.0 50,000.0 100,000.0 150,000.0 200,000.0 250,000.0 300,000.0 350,000.0 0 50 100 150 200 250 300 MSM (µg/cm2) Time (h) Regenail Betalfatru s Figure 3. Permeation profiles of MSM through the bovine hoof plate as a model of human nail permeation. Additionally, these data were normalized by the amount of MSM applied in each test (the MSM concentration between both products is different: 10% w/wfor REG and around 5.5% w/wfor BET). When these normalized values are considered, the profiles of both formulations get closer. The mean values for the P 1 , P 2 , and Kp of MSM that permeated through hooves showed negligible differences between the two formulations. The permeation steady state flux values, however, were significantly different: 1083.97 µ g/h/cm 2 for REG vs. 647.89 µ g/h/cm 2 for BET. Although most of the differences between permeation parameters were caused by different MSM concentrations, the increased flux after dose normalization indicates the superiority of the test vehicle over the commercial formulation. According to the molecular structure of MSM, it is not expanded to encapsulate or interact with HPβ -CD, so the increase in transungual flux could be caused by the shelf promoting effect of the formulation. It was demonstrated previously [ 22 , 23 , 25 ] that cyclodextrin-soluble polypseudorotaxanes increase the size of nail channels and interact with keratin residues, reducing protein folding. Sodium lauryl sulfate could also increase the diffusivity of MSM by contribution to protein interaction. MSM is not expected to reduce disulfide bonds. In addition, the proposed formulation can increase the absorption of hydrophilic active compounds despite the well-known solubilization capacity of lipophilic molecules, which is the main promotion mechanism in these compounds. When permeated concentrations of MSM at 24 h (around 21,700 µ g/cm 2 ) are compared with release concentrations at the same time point (around 1800 µ g/cm 2 ), we see the enhancing effect of REG caused by the interaction of the system with the hoof structure, compared with the pure diffusion seen in release experiments across dialysis membranes. Table 1. Transungual permeation parameters of test and reference formulation. ( * )=statistical differences between formulations with a p-value <0.05. Formulation Active Compounds Js (µg/h/cm2)Kp (cm/h) P1(cm) P2(h−1)Tlag (h) Active Content (µg/mg) Regenail Biotin 74.98 ±35.34 0.0187 ±0.0102 6.7163 ±4.5610 0.00279 ±0.00118 59.72 ±5.34 0.161 ±0.098 MSM 10,839.695 * ±5786.239 0.054 * ±0.014 34.185 * ±8.661 0.00159 ±0.00098 105.12 * ±3.56 – Betalfatrus MSM 3563.382 * ±963.993 0.0324 * ±0.011 19.198 * ±4.821 0.00170 ±0.00125 98.77 * ±3.01 – The amount of sulfur obtained through the MSM at the end of experiments after application of REG formulation was 948.66 ppm, 2.3-fold higher than that obtained with the BET solution (412.78 ppm). Biotin profiles from REG could not be compared with the reference product because it does not contain biotin. The permeation profiles of biotin across a hoof are shown in Figure 4. The transungual biotin parameters are shown in Table 1. 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