Investigation of piroctone olamine delivery to the skin from single, binary and ternary solvent systems

Disruption of the protective stratum corneum barrier increases the skin's vulnerability to microorganisms and facilitates conditions such as dandruff. Dandruff is a disorder of the scalp that causes increased scaling of the SC and is associated with Malassezia fungus. Consequently, many anti‐dandruff commercial products use anti‐fungal active ingredients such as piroctone olamine also known as Octopirox (OPX). OPX is an active ingredient used in a number of topical preparations for the management of dandruff. The characterization of the physicochemical properties of OPX was previously reported. The aim of the present work was to investigate a range of solvent systems for their effects on OPX interaction with human skin.


Conclusion:
To our knowledge, this is the first study to examine the permeation behaviour of OPX for a range of single, binary and ternary solvent systems.

K E Y W O R D S
dandruff, finite dose, formulation, in vitro, piroctone olamine (OPX), skin delivery

INTRODUCTION
The scalp is a unique skin area of the body consisting of five layers: the skin, connective tissue, galea aponeurotica, loose areolar connective tissue and the pericranium [1].This part of the body is characterized by thick skin and a high follicular density with a large number of sebaceous glands [2].The presence of these characteristics in combination with a dark and warm environment, makes the scalp more prone to fungal infections such as dandruff [3].Dandruff is defined as a non-inflammatory, condition characterized by increased scaling of the scalp [4] that also causes itching.Almost 50% of the world's population is affected with dandruff at some point, and thus it is one of the most common dermatological skin conditions [5].
Dandruff and seborrheic dermatitis are thought to be caused by Malassezia yeast spp, sebaceous secretions and the individual's susceptibility [6].Malassezia are a type of basidiomycetous fungi and are part of the normal skin flora of a variety of animals [7].Most species are lipophilic, and owing to their lipophilicity they tend to colonize sites of the body that are seborrheic such as the scalp [8].Certain skin diseases are primarily associated with Malassezia yeasts and 17 different species of Malassezia have so far been identified [9].Among the identified species, Malassezia restricta is the most dominant on human skin [10][11][12][13].Studies have proposed that several Malassezia species including M. restricta require lipids and that the breakdown of sebaceous triglycerides releases irritating free fatty acids that result in inflammation and scalp flaking [14,15].Some Malassezia species including M. restricta utilizes the carbon in lipid-rich areas for growth, for the colonization of Malassezia spp, sebum would be ideal [16].
Piroctone olamine or Octopirox® (OPX) is a cytostatic, anti-fungal agent widely used in cosmetic products such as anti-dandruff rinse-off shampoos (Figure 1) [17].The physicochemical properties of OPX are described in Table 1 [18].The exact mechanism of action of OPX is not fully understood [17].One theory proposes that OPX enters the cell wall of the fungal cells and chelates with iron (III) ions [19].The creation of these complexes leads to the inhibition of energy metabolism in the mitochondria resulting in fungicidal effects [20].In addition, OPX was shown to exhibit inhibitory action on collagenolytic activity [21] with other authors noting that the compound may have the potential to protect against skin ageing [22].
In a study involving 21 subjects with severe dandruff, Piérard-Franchimont et al. showed that a shampoo formulation containing 1% OPX induced significant reductions in the colonization of Malassezia yeast and scalp scaliness [23].Lodén and Wessman investigated the use of shampoos containing 0.75% OPX/2% salicylic acid compared with 1% zinc pyrithione in a double-blind, randomized study with 19 subjects.The combination of OPX and salicylic acid was noted to be more effective in reducing the area and severity of scaling than zinc pyrithione [24].
To effectively target superficial fungal colonization such as Malassezia spp, the anti-fungal agent must be delivered to the SC, hair follicles or nails with limited permeation into the systemic circulation [25].Although OPX is widely used in commercial formulations, there is little reported information about the skin uptake of OPX from such products and there are no studies that have examined OPX permeation in human skin.The aim of the present study was therefore to investigate the interaction of OPX with the skin in vitro following application in a range of solvents including single, binary and ternary systems.Solvents were selected based on their use in commercial skin and shampoo formulations and included propylene glycol (PG), diethylene glycol monoethyl ether as Transcutol® P (TC), PG caprylate as Capryol® 90 (CAP), PG monolaurate (PGML), caprylic/capric triglycerides as Labrafac™ lipophile WL 1349 (LAB), polyglyceryl-3 oleate as Plurol® Oleique CC 497 (PIOI), isostearyl isostearate (ISIS) and isopropyl myristate (IPM).

T A B L E 1
The physicochemical properties of Octopirox (OPX) [18].

Solubility studies and solubility parameter calculation
Solubility was determined as reported previously [16].
Briefly, OPX was added to saturation to 0.5 mL of solvent(s) in sample tubes and kept at 32 ± 1°C for 48 h in a JB Nova thermostatically controlled water bath (Grant, UK) equipped with an HP 15 stirring system (Variomag®, USA).Samples were centrifuged (5415 R centrifuge Eppendorf®, UK) at 13 200 rpm for 30 min at 32°C, and the supernatant was withdrawn and suitably diluted to lie within the range of the calibration curve.Samples were analysed by HPLC.All measurements were conducted in triplicate.The solubility parameter was calculated with Molecular Modelling Pro® (Version 6.3.3).

In vitro permeation experiments
Piroctone olamine has been approved for use in cosmetic products at a maximum concentration of 1% (w/v) in rinseoff products [26].This concentration of active was used for all in vitro permeation studies in the present work.The excised human epidermis used in in vitro permeation experiments was prepared by the heat separation method as reported previously [27].The experiments were performed as finite dose studies with an application volume of 10 μL.The receptor fluid used in the Franz cells was PBS with the addition of 6% (w/v) Brij® O20 to maintain sink conditions.The temperature was controlled with a JB Nova water bath (Grant, UK) and the receptor fluid in the Franz cells was stirred continuously with a HP 15 stirring system (Variomag®, USA).The Franz cells were placed in the water bath until the skin surface reached 32 ± 1°C.Solutions of 1% w/v OPX in the selected solvent system were subsequently applied to each cell.Permeation experiments were conducted for 24 h, followed by mass balance studies.The time points at which 200 μL samples were collected from the receptor compartment were 0, 0.5, 1, 2, 4, 8, 12, 24 h; all samples were analysed by high-performance liquid chromatography (HPLC).

Mass balance studies
After 24 h, each cell was washed five times with 1 mL of the appropriate solvent.Each cell was swabbed with a cotton bud and then cut to fit 2 mL Eppendorf microcentrifuge tubes (Eppendorf®, UK).After swabbing, the cells were disassembled, and the skin was cut into small pieces and inserted into separate microcentrifuge tubes.All sample tubes were shaken for 24 h at 32 ± 1°C.After 24 h, all extraction samples were subjected to two cycles of 15 min of ultrasonication (Ultrasonic Cleaner USC-THD VWR International, USA) followed by 15 min of centrifugation (5415 R centrifuge Eppendorf®, UK).

High-performance liquid chromatography analysis
HPLC analysis was performed using a 1100 series HPLC (Agilent, USA) with a reverse phase C 18 column (Poroshell 120 EC-C18, 100 × 4.6 mm × 4 μm; Agilent, USA).The mobile phase was a gradient mixture of mobile phase A consisting of 95:5 v/v water:ACN with 0.1% TFA and mobile phase B consisting of 95:5 v/v ACN:water with 0.1% TFA as described in Table 2.The column temperature, mobile phase flow rate and injection volume were set at 32°C, 1 mL min −1 and 20 μL, respectively.The detection wavelength was set to 305 nm.Calibration curves in the range of 0.5-100 μg ml −1 were prepared in ACN:water (50:50 v/v) and the HPLC method was validated as per ICH guidelines [28].

Statistical analysis
Statistical analysis of the data collected was conducted using GraphPad Prism Statistics software (GraphPad Software LLC, San Diego, CA, USA).The assessment of normality was conducted with the Shapiro-Wilk test.
Data were evaluated using one-way analysis of variance (one-way ANOVA) and grouped data was evaluated using two-way analysis of variance (two-way ANOVA).A post hoc multiple comparisons test was used to compare the means between groups using the Tukey test.

Solubility parameter of single, binary and ternary solvent systems
Figure 2 shows a plot of the calculated solubility parameter of the solvents investigated against the saturated solubility of OPX.The range of OPX solubility in the solvents used for this study ranged from 5.2 ± 0.07 to 624.96 ± 25.49 mg ml −1 .The solvent system with the highest OPX solubility was found to be PG:PGML:LAB (60:30:10).In solvent systems that contain PG, the solubility of OPX was found to increase with higher amounts of PG.The solubility parameter values determined ranged from 8.1 to 14.07 (cal/cm 3 ) 1/2 .

Single solvent systems
The skin permeation of OPX was investigated for six single solvents and mass balance results are also reported (Figure 3).OPX did not permeate the skin and was not detected in the receptor compartment.As OPX is a salt it is possible that limited or slow diffusion across the SC may account for the absence of permeation [29].The three highest skin extraction values for OPX were obtained for PGML, TC and PG, with values of 10.09% ± 0.97%, 6.92% ± 1.47% and 5.12% ± 0.55% of the applied dose respectively.The results for PGML were significantly higher compared with the other solvent systems, (p < 0.05).
The amount of OPX recovered from the skin surface for all single solvent systems ranged from 81.69% to 91.54% of the dose applied (Figure 3).With the exception of PG, significantly higher amounts of OPX were recovered from the skin surface for CAP compared with the other solvents (p < 0.05).The highest amount of OPX recovered was for PG with a total recovery value of 93.95% ± 1.96%.The recovery percentage of OPX in PG was significantly higher compared with TC and ISIS with total recovery percentage values of 89.70% ± 2.56% and 89.66% ± 2.81% respectively (p < 0.05).When the recovery percentage of OPX in PG was compared with other solvent systems, the values were statistically similar (p > 0.05).
PGML is a fatty acid ester used as a solubilizer for active ingredients with poor solubility and it is also used as a cosurfactant.Previous reports have also demonstrated the potential of PGML to function as a skin penetration enhancer [30][31][32].In the present study, the highest amount of OPX extracted from the skin was from PGML as shown in Figure 3 hexamidine diisethionate and its dihydrochloride form compared to the neat solvents alone [32].The authors also suggested that the free fatty acid fraction of the ester may play a role in promoting skin uptake of these actives.
TC is a monoethyl ether of diethylene glycol that has been reported to increase drug solubility and promote skin penetration [33].TC is thought to penetrate the stratum corneum and strongly interact with water along the intercellular path.This may then lead to a decrease in skin barrier function due to the modification of proteins and lipids and therefore more active permeation [34].Iliopoulos et al. investigated the influence of neat solvents on the permeation of niacinamide.The researchers noted that TC promoted the greatest skin penetration of niacinamide but did not promote skin retention compared with other solvents studied [35].As we have noted previously the results reported here confirm that solvents such as TC may not always act as skin penetration enhancers and in some cases might actually retard skin delivery.

PG:TC
OPX permeation was further investigated using binary solvent systems of PG and TC.It is interesting to note that while there was no permeation of OPX from single solvent systems, OPX did permeate through the skin for all binary solvent systems (Figure 4).may be the driver for the higher OPX skin penetration.Notably the amount of OPX that permeated for PG:TC (50:50) was 7.09 ± 0.43 μg/cm 2 compared with a value of 6.68 ± 0.88 μg/cm 2 for PG:TC (25:75).However, these values are not significantly different (p > 0.05).
The inclusion of additional solvents in the case of binary and ternary solvent systems has been suggested to produce a synergistic effect on the permeation of various compounds [30,32,36,37].Zhang et al. investigated the permeation of niacinamide from various binary and ternary formulations in porcine and human skin and an artificial membrane model.Higher amounts of niacinamide permeated in human skin for binary solvent systems of PG with a fatty acid compared with neat solvents [36].Kung et al. explored single, binary and ternary solvent systems for the delivery of methadone.The researchers reported that PG:TC (50:50) delivered significantly more methadone through human skin after 24 h compared with d-limonene and combinations of d-limonene with ethyl oleate, TC and octyl salicylate (p < 0.05) [37].It was suggested that the promotion of permeation from the use of the binary solvent system PG:TC might bone attributed to PG increasing the permeation of both the active compound and TC.The higher uptake of TC may also lead to additional permeation of the active compound [37].

PG:PGML:Lab
Building on the results reported in the previous section, the solvents PG, PGML and LAB were selected as a ternary solvent system.The specific ratios were identified based on miscibility evaluation (data not shown).Permeation of OPX was observed for all ternary solvent systems.Permeation was not observed until the 8 h timepoint (data not shown).At the 24 h timepoint, significantly more OPX permeated for PG:PGML:LAB The most frequently utilized glycol in topical and transdermal products is PG.Although the mechanism of action of PG is not fully understood, it is commonly used as a co-solvent and is reported to be a permeation enhancer [33].The mass balance study in this work indicates that increasing amounts of PG results in increased permeation and skin retention of OPX.Similar findings were reported by Kung et  The results for the mass balance study of OPX for PG:TC:LAB vehicles are summarized in Figure 6.For PG:TC:LAB (20:60:20) the percentage of the dose applied recovered from the skin surface was 69.32% ± 1.61%.However, there are no significant differences for the three systems with reference to deposition of OPX on the skin (p > 0.05).The highest OPX extraction from within the skin was observed for PG:TC:LAB (10:55:35) with a value of 17.03% ± 2.10% of the dose applied.Similar to the results from the skin surface, the results were not significantly different for the three vehicles (p > 0.05).The highest permeation of OPX was from PG:TC:LAB (25:65:10) with a value of 9.19% ± 1.07% of the dose applied.OPX permeated significantly more for this system compared with PG:TC:LAB (10:55:35) (p < 0.05) but permeation was not significantly higher (p > 0.05) when compared with PG:TC:LAB (20:60:20).

Solvent system
Percentage of applied dose of OPX (%) Table 3 shows the results from the mass balance studies from all the solvent systems tested.The amount of OPX that remained on the skin surface ranged from 55.50% ± 4.64% to 91.55% ± 2.48% of the dose applied.For OPX skin extraction, values ranged from 1.25% ± 0.23% to 22.52% ± 3.44% of the dose applied.As noted, no permeation was observed for single solvents.For binary and ternary solvent systems, the permeated values of OPX range from 4.10% ± 0.41% to 11.75% ± 1.81% dose applied.As well as enhancing skin permeation it is evident that most of the binary and ternary systems also promote OPX skin retention.

CONCLUSION
Only one report of the skin permeation of OPX in vivo in rodents has been published to date [39].To our knowledge, this is the first comprehensive evaluation of OPX penetration and distribution in human skin in vitro.In the present work, while no permeation was observed for simple vehicles, more complex binary and ternary systems generally promoted both skin penetration as well as skin retention.This may reflect changes in the solubility and partition behaviour of OPX in the SC or structural changes within the SC itself with increasing vehicle complexity.As differing amounts of retention and penetration were observed in different systems the potential for vehicle manipulation to preferentially promote skin retention rather than skin penetration should be explored further.This will require monitoring of the vehicle as well as the active.Appropriate techniques would be HPLC to monitor OPX and gas chromatography for the vehicle.

T A B L E 2
Mobile phase gradient for high-performance liquid chromatography analysis of Octopirox.
. Parisi et al. investigated the topical delivery of hexamidine in a range of single and binary solvent systems.The researchers concluded that the addition of PGML clearly improved the topical delivery of both F I G U R E 2 Plot of solvent solubility parameter against saturated solubility of OPX in the solvents and mixtures used at 32 ± 1°C (n = 3, mean ± SD).OPX, Octopirox.