Topical formulations containing finasteride. Part II: determination of finasteride penetration into hair follicles using the differential stripping technique.

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Topical Formulations Containing Finasteride. Part II: Determination of Finasteride Penetration into Hair Follicles using the Differential Stripping Technique SILVIA TAMPUCCI,1 SUSI BURGALASSI,1 PATRIZIA CHETONI,1 CARLA LENZI,2 ANDREA PIRONE,2 FEDERICO MAILLAND,3 MAURIZIO CASERINI,3 DANIELA MONTI1 1

Department of Pharmacy, University of Pisa, Pisa I-56126, Italy Department of Veterinary Science, University of Pisa, Pisa I-56124, Italy 3 Scientific Department, Polichem SA, Lugano Pazzallo, Switzerland 2

Received 27 November 2013; revised 19 May 2014; accepted 20 May 2014 Published online 10 June 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24045 ABSTRACT: The differential stripping technique consists of a tape-stripping phase followed by a cyanoacrylate biopsy. This technique not only allows the quantification of drug retained in the stratum corneum (SC) and in the hair follicles but also differentiates transepidermal from transfollicular penetration. Our study aimed at both validating the differential stripping procedure on hairless rat skin and assessing the role of the hair follicle in the cutaneous penetration of finasteride (FNS) after application of two experimental formulations for 6 or 24 h: P-08–016, a hydroxypropyl chitosan (HPCH)-based formulation and P-10–008, an anhydrous formulation devoid of HPCH. Microscopic and histological evaluation showed that after 15 tape strips both the SC and the viable epidermis were completely removed. A subsequent cyanoacrylate skin surface biopsy led to the removal of the infundibula content. The largest amounts of FNS were found in the epidermis and in the appendages after application of P-08–016, regardless of the time from application. In contrast, smaller and statistically significant amounts of FNS were recovered with P-10–008 6 h after application, compared with that at 24 h. In conclusion, the differential stripping technique allowed determination of the amount of FNS localized in different skin districts, focusing particularly on C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:2323–2329, 2014 the follicular contribution. Keywords: finasteride; hydroxypropyl chitosan; formulation; in vitro models; skin; distribution; hair follicles; differential stripping technique; passive diffusion/transport

INTRODUCTION During the last decades, many studies have been performed to determine the penetration pathways of topically applied substances. In particular, the lipid portion of the stratum corneum (SC) was considered as the major pathway, whereas the skin appendages were presumed to play a subordinate role representing not more than 0.1% of the total skin surface area.1 In any case, the contribution of the appendages to the overall penetration process still represents a theme of investigation, especially concerning the hair follicles.2 Studies performed in recent years seem to suggest that follicular drug penetration could be more significant than previously believed.3–5 Suitable techniques to determine follicular penetration and storage should allow differentiation between transepidermal and transfollicular penetration.2 Among the methods used,3,7–10 a noninvasive method that can also be applied in vivo is the cyanoacrylate skin surface biopsy,11–13 which consists in applying superglue on the skin surface and removing it after polymerization, thus entrapping corneocytes and follicular casts. As this method does not allow distinction between the transepidermal and transfollicular routes, an emerging technique is represented by differential stripping, which consists in subjecting the skin to a tape-stripping technique followed by Abbreviations used: FNS, finasteride; HPCH, hydroxypropyl chitosan. Correspondence to: Daniela Monti (Telephone: +39-050-2219661; Fax: +39050-2219659; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 103, 2323–2329 (2014) C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association

cyanoacrylate skin surface biopsy.1,14 The tape-stripping technique is a widely employed method that allows removal of the SC layer by layer, and can be used to determine the amount of a substance retained in the skin surface both in vitro and in vivo. With the differential stripping technique, it is also possible to quantify SC retention after tape stripping and follicular retention by cyanoacrylate casting, making a complete survey of drug retention in the skin. Determining follicular retention could be useful in targeted follicular delivery, which includes the treatment of hair growth abnormalities and hair follicle-associated diseases.15–17 No exhaustive in vitro skin models are currently available to study follicular drug penetration. In fact, human skin, hamster, and pig ear skin, investigated previously,5,6,18–21 revealed several drawbacks such as, the higher hair follicle intensity per cm2 in animal species, which influences the follicular penetration rate. Besides, even though pig ear skin seems to represent a more suitable in vitro model for the analysis of the follicular storage than excised human skin, porcine hair follicles are larger than human hair follicles and this may lead to an overestimation of the penetration process. Moreover, the excised human skin itself is not devoid of limitations as the excision process leads to the contraction of the reticular fibers, reducing the reticular reservoir.19,22 Other studies used rodent skin as a model for targeted follicular delivery.23–26 Despite many differences that can lead to overestimations of permeability, hairless rat continues to be a valid model for dermatological studies. As the permeability of the scalp in patients with alopecia areata or androgenic

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Table 1.

Composition of the Formulations under Study

Batch Ingredients FNS Ethyl alcohol 96% Propylene glycol Purified water HPCH

P-08–016

P-10–008

% (w/w) 0.25 55.00 5.00 38.75 1.00

% (w/w) 0.25 71.25 28.50 – –

alopecia often reveals a reduced epidermal barrier function, hairless rat skin could be especially suitable as an alternative model for the lesional scalp skin. Although hairless rat skin is slightly different from human skin, the pilosebaceous unit and hair density are comparable27 and the sebaceous gland density and size are closely related to those of human forehead skin (100–200 per cm2 ).15 The present investigation aimed at both validating the differential stripping procedure on hairless rat skin as a substrate and at assessing the role of hair follicles in the cutaneous penetration of finasteride (FNS) for the treatment of male baldness after application of one hydroxypropyl chitosan (HPCH)-based formulation (P-08–016) and one anhydrous formulation without HPCH (P-10–008) for either 6 or 24 h. These two formulations previously demonstrated their ability to promote high levels of cutaneous deposition of FNS in the hair bulb region with minimal systemic absorption.28

embedded in JB-4 plastic resin. Coronal sections were cut by a microtome (Reichert-Jung), mounted onto gelatine-coated slides, and stained with methylene blue/toluidine blue for microscopic examination. The specimens were assessed under a Leitz Diaplan microscope. In Vitro Deposition Experiments In vitro permeation tests through excised rat skin were carried out as previously described29 using Gummer-type diffusion cells with an available diffusion area of 1.23 cm2 and the SC facing the donor compartment. Two hundred microliters of each formulation, whose composition is reported in Table 1, was placed on the skin surface. The receiving phase (5.0 mL) was isotonic phosphate buffer saline (66.7 mM, pH 7.4) containing 0.003% (w/v) sodium azide to prevent bacterial growth, maintained at 37◦ C and stirred at 600 rpm. At predetermined time intervals, the receiving phase was completely changed and replaced with fresh fluid to maintain sink conditions. The experiments lasted 6 or 24 h and were replicated five times. At the end of the in vitro permeation experiments, the skin was removed from the diffusion cells, rinsed with distilled water to remove excess formulation from the skin surface and gently wiped with cotton-wool tampons. The skin specimens were then positioned on a homemade specific apparatus delimiting the drug-exposed surface. Afterward, the skin was subjected first to tape stripping and then to cyanoacrylate skin surface biopsy. Adhesive Tape-Stripping Technique

MATERIALS AND METHODS Chemicals Two experimental liquid formulations, P-08–016 and P-10–008 (Polichem S.A., Lugano Pazzallo, Switzerland; composition reported in Table 1) were used as received. FNS (batch number J7C002F) was supplied by Polichem S.A. All other chemicals and solvents were of analytical grade.

The tape-stripping procedure was performed as described by Weigmann et al.30 The skin was stripped using an adhesive tape (Tesa film N. 5529; Beiersdorf, Hamburg, Germany) and the tape strips were pressed on the skin by applying uniform pressure in order to obtain intimate contact between the film and the skin. The first tape strip was discarded, as this represents unabsorbed material only. Then, the subsequent strips were carefully removed and the entire procedure was repeated 15 times (tape strips no. 1–15).

Animals Rat skin was obtained from 5-week-old hairless male animals (HsdHanTM:RNU-Foxn1 rnu; Harlan Italy srl, Correzzana, Italy). The animals were killed by cervical dislocation immediately before the experiments, the skin was carefully excised, and the adhering fat and subcutaneous tissue were removed. The study was approved by the Ethics Committee of the University of Pisa and the protocol was compliant with European Union Directive 86/609/EEC for the use of experimental animals. Tuning and Validation of the Differential Stripping Technique

Cyanoacrylate Skin Surface Biopsy Following the removal of 15 tape strips, a drop of Superglue ¨ (UHU GmbH & Co. KG, Buhl/Baden, Germany) was placed on the skin stripped area and the glue was covered with the adhesive tape under slight pressure. After 10 min, the cyanoacrylate polymerized and the strip was removed with a single quick motion, entrapping the casts of the hair follicles. Each tape-strip sample removed from the treated skin area and each sample derived from cyanoacrylate skin surface biopsy was cut to a size of 1.9 × 3.0 cm2 and placed in a glass vial containing 5 mL ethanol, sonicated for 10 min, and submitted to centrifugation (15 min, 4,000 rpm). The supernatant was collected for HPLC analysis.

After positioning the rat skin on a support and delimiting a precise area with a Teflon mask, the skin was subjected to a certain number of tape strips (from 1 to 20). In order to determine how many strips were necessary to completely remove the SC and the viable epidermis, each strip was observed by light microscopy. After that, a cyanoacrylate biopsy of the stripped skin was performed and the obtained sample was observed microscopically. Moreover, skin residuals after different numbers of tape strips and after the complete differential stripping procedure were fixed in 10% buffered formalin solution, dehydrated, and

The quantitative determination of FNS recovered in skin samples was carried out by HPLC. The apparatus consisted of a Shimadzu (Kyoto, Japan) LC-20AD system with an UV SPD10A detector equipped of autosampler SIL-10AD VP and a computer integrating system. The injection valve was a Rheodyne with a capacity of 20 :L. A Luna C8 (10 :m; 250 × 4.6 mm) column was employed. The mobile phase consisted of a mixture of acetonitrile:methanol:water (20:40:40). The detection


DOI 10.1002/jps.24045

Analytical Methods

R


wavelength was 210 nm, the flux was 1.0 mL/min, and the retention time was 12.35 min. The amount of FNS in the samples was determined by comparison with appropriate standard curves. In the case of biological materials, standard curves were obtained by adding increasing amounts of drug to blank biological samples. The standard curve was linear in the detection range and the assay linearity was good (r2 = 0.9983). The limit of detection and limit of quantization, calculated on the basis of the response and slope of the regression equation and signal-to-noise ratio, were 3.81 and 27.5 ng/mL, respectively. Experimental Strategy and Data Analysis The tape strips were weighed and the cumulative amount and thickness of epidermis removed were calculated, assuming a density of 1 g cm−3 and uniform coverage of tissue on the tape strip. The absolute amount of drug on each pool of tape strips was determined by HPLC, and the weight of skin removed on a tape strip divided by the density of the tissue yielded a value of the volume, thereby permitting the drug concentration to be expressed in molar units. The values on the abscissa (x/L) are calculated from the ratio j i = 1 Mi /n i = 1 Mi where Mi is the epidermis mass removed by the ith tape strip and n i = 1 Mi = Mt , the total epidermis mass removed by all n tape strips combined.31 The epidermis concentration (Cx ) versus normalized depth (x/L) profiles of FNS were fitted to the following solution of Fick’s second law of diffusion: Cx = KCv

∞ nBx x 2 1 −Dn2 B2 t 1− − sin exp (1) L B n=1 n L L2

The applicable boundary conditions are as follows: the applied drug concentration (Cv ) remains constant during the treatment period (t), the dermis acts as a perfect sink for the drug and the SC contains no drug at t = 0. The fitting generates values of K and D/L2 (R Core Team, 2013. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria). The former is the epidermis/vehicle partition coefficient, a thermodynamic parameter reflecting the relative affinity of the drug for the epidermis over the vehicle. The second parameter has units of (time)−1 and can be considered as a first-order kinetic constant describing drug diffusion across the epidermis. Integration of Eq. (1) across the epidermis thickness (i.e., from x/L = 0 to x/L = 1) provides an expression for the area under the epidermis concentration versus relative depth profile (area under the curve), which equals the total amount of drug present in the membrane divided by volume of this compartment.32,33 Statistical analysis of the data was performed with GraphPad Prism software (GraphPad Software Inc., San Diego, California). The evaluation included calculating the means and standard errors (SEs) of the recovered FNS concentration in the SC, epidermis, and follicular infundibula after different permeation times. Group comparisons were carried out using the Student’s two-tailed unpaired t-test. Differences were considered statistically significant at p < 0.05. All data are the average of five determinations ± SE. DOI 10.1002/jps.24045

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RESULTS AND DISCUSSION Adhesive tape stripping is a widely accepted method to measure the localization and distribution of substances in the SC. Literature studies report that 15 tape strips are necessary to remove completely the SC from the abdominal area of hairless rat skin, data experimentally confirmed by transepidermal water loss measurements.34 In any case, several factors, such as skin hydration, vehicle composition, cohesion among the corneocytes, and interindividual differences in total SC thickness, can influence the amount of SC that is removed by a single tape strip.35 Moreover, for correct determination of the amount of a substance accumulated in the hair follicles using the differential stripping technique, it is extremely important to completely remove the infundibular content of the hair follicle without interference from skin corneocytes and epidermal cells. In a previous, study Monti et al.28 selected two formulations, P-08–016 and P-10–008, which exhibited relatively low FNS transdermal permeation and good drug retention in the skin layers in vitro. In particular, P-08–016 allowed FNS to reach the reticular dermis without producing a high transdermal flux. In vivo experiments revealed that FNS was not found in plasma and any differences existing between formulations concerned skin retention after administration of single versus repeated doses of drug. Moreover, any increase in the amount of drug accumulated in the skin was obtained with the repeated dose experiment. Our present work, besides validating the differential stripping procedure using hairless rat skin as substrate, aimed at evaluating the amount of FNS localized in the different skin districts after in vitro topical application of the formulations under study, focusing particularly on the follicular contribution. Selective FNS targeting to hair follicles could represent a valid alternative to systemic therapy for the treatment of male pattern baldness, bypassing the several adverse effects observed in high percentages of patients.36 Tuning and Validation of the Differential Stripping Technique Microscopic evaluation of the tape strips removed from hairless rat skin was performed. The most relevant microscopic images are shown in Figure 1. After the removal of 15 tape strips, both the SC and the viable epidermis were completely removed (f). Vellum hair and corneocytes represented the content of the first tape strip (a), whereas epidermal cells appeared only from the seventh tape strip onward (c). Increasing the number of tape strips from 15 to 20 did not show any change, as every epidermal cell was already completely removed from the skin. Application of the cyanoacrylate skin surface biopsy after the 15 tape strips led to removal of the infundibula content, as shown in Figure 2. Furthermore, histological evaluation on paraffin-embedded samples of the stripped skin confirmed that 15 tape strips were necessary to completely remove the epidermal layer from the skin (Fig. 3). Magnified images of intact and empty hair follicles are illustrated in Figures 4a and 4b, respectively. Skin Deposition Studies Data regarding recovery of FNS in the tape strips and follicular casts are reported in Table 2. Greater amounts of FNS were found in both the epidermis and the appendages following application of P-08–016 with Tampucci et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:2323–2329, 2014

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Table 2. Comparison of the Recovery of FNS in the Epidermis and Follicular Infundibulum Following Topical Application of the Formulations under Study to Hairless Rat Skin “in vitro” (mean mg ± SE) FNS (mg) Skin Layers

Stratum corneum Epidermis

Follicular cast

6h

Strip 1 Strip 2–4 Strip 5–8 Strip 9–12 Strip 13–15 Cyanoacrylate biopsy

24 h

P-08–016

P-10–008

P-08–016

P-10–008

0.0132 ± 0.0023 0.0266 ± 0.0076 0.0390 ± 0.0139 0.0227 ± 0.0122 0.0218 ± 0.0114 0.0118 ± 0.0034

0.0051 ± 0.0010 0.0102 ± 0.0018 0.0066 ± 0.0009 0.0052 ± 0.0005 0.0051 ± 0.0005 0.0080 ± 0.0018

0.0397 ± 0.0107 0.0667 ± 0.0135 0.0560 ± 0.0275 0.0520 ± 0.0178 0.0220 ± 0.0042 0.0166 ± 0.0041

0.033 ± 0.0093 0.0343 ± 0.0075 0.0393 ± 0.0114 0.0110 ± 0.0033 0.0115 ± 0.0040 0.0128 ± 0.0034

Figure 2. Microscopic image of a cyanoacrylate skin surface biopsy with infundibular casts (IC) and vellus hair (VH). The follicle infundibulum is completely embedded in the cyanoacrylic matrix.

casts, respectively, whereas only 1.9- and 1.4-fold was obtained with P-08–016. The 4-fold increase in epidermis retention obtained with P10–008, notwithstanding quantitatively lower than that obtained with P-08–016, may be indicative of an effect of the Figure 1. Microscopic images of (a) tape strip no. 1 with vellum hair and corneocytes, (b) tape strip no. 4 with few vellum hair and corneocytes, (c) tape strip no. 7 with some epidermal cells between corneocytes, (d) tape strip no. 8 with epidermal cells and some follicular infundibula, (e) tape strip no.11 with epidermal cells, and (f) tape strip no. 15 completely free from epidermal cells.

respect to P-10–008, the differences being statistically significant in the 6 h experiment (p = 0.0033). The combination of HPCH, water, and ethanol and the lower content of propylene glycol (5-fold less than in P-10–008) in formulation P-08–016 appeared to lead to greater recovery of FNS in the tape strips and follicular casts with respect to the anhydrous formulation P-10–008 without HPCH. It is important to notice that a higher FNS accumulation in the epidermis with respect to follicular retention over longer exposure was obtained for both formulations, particularly emphasized with formulation P-10–008. Indeed, when the experiments lasted 24 h, an increase in FNS retention of 4-fold and 1.6-fold was found for P-10–008 in epidermis and follicular

Figure 3. Histological section of hairless rat stripped skin (15 tape strips).


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Figure 4. Hystological slice of (a) intact follicles before differential stripping and (b) empty hair follicles after 15 tape strips and cyanoacrylate skin surface biopsy.

formulation on the interfollicular pathway that could lead over time to a higher skin permeation as previously observed.28 Probably, the high amounts of propylene glycol and ethanol in formulation P-10–008 require longer time to affect the barrier properties of SC, opening ways for the passage of drugs.37 As far as the aim of our work was to target the pilosebaceous unit as depot of FNS for localized therapy without systemic effects, formulation P-08–016 seems to reach the goal. A limiting step for drug accumulation in the follicular apparatus could be diffusion through the sebum, which covers the follicular opening.26 Published data indicated that chitosanderived molecules are able to hinder the sebum coating of the hair shaft.38 In this context, HPCH might be able to hinder the sebum and enhance FNS accumulation in the follicular cast.

The presence of HPCH seems to enhance FNS recovery in hair follicles, increasing by 1.5- and 1.3-fold the amount of drug at the site of action in the experiment at 6 and 24 h, respectively. Besides, ethanol is useful for partitioning FNS into lipid-rich skin compartments, including follicles filled with sebum. On the other hand, the presence of water could be important for FNS distribution both in the viable epidermis and hair follicles, once the SC and sebum barriers are overcome, as evidenced from the larger FNS depot produced by the P-08–016 formulation with respect to P-10–008. A further observation was that P-10–008 requires 24 h to accumulate the same amount of FNS in the hair follicles obtained in 6 h with formulation P-08–016: 0.0128 mg for P-10–008 at 24 h versus 0.0118 mg for P-08–016 at 6 h (p = 0.8516). This phenomenon could depend on the fact that HPCH, which has a great affinity to the skin, penetrates into the hair follicles carrying FNS with. Such a result could suggest reduced times of therapy using formulation P-08–016, which could be an advantage for compliance and above all, safety reasons. In fact, female partners of childbearing potential must avoid contact with FNS and this formulation may, for example, be applied on the morning and washed out in the evening, before going to bed, avoiding the need of a nightcap. Notwithstanding the percentage retained in the follicular infundibulum was fairly low (about 3%) with respect to the applied dose, it is important to consider that follicular drug recovery could be influenced by several factors such as hair follicle morphology, follicular density per cm2 , and functional status.22 Anyway, the efficacy of a formulation containing 0.25% FNS and HPCH was demonstrated in preliminary studies on men after topical administration on scalp, the desired site of action in men with male pattern hair loss. Data showed that 1-week treatment was able to reduce DHT scalp-levels up to 70% with respect to 50% after 1 mg oral tablet once daily.39

Mechanistic Analysis A more objective evaluation of this conclusion was facilitated by fitting the individual profiles to Eq. (1) and by a comparison of the resulting partitioning and diffusivity parameters (K and D/L2 , respectively). Table 3 presents the average results for K and D/L2 derived from fitting Eq. (1) to the individual profiles. The table includes also the values of AUC given as mean ± SD. From the values of K and D/L2 , together with the corresponding epidermis thickness (L) determined for each tissue, it is possible to deduce the permeability coefficient (Kp ) of FNS across the epidermis from each of the formulations tested using

Table 3. Transport Parameters of FNS Across Epidermis Following Topical Drug Delivery from P-08–016 and P-10–008 after 6 and 24 h (mean ± ES) Formulation P-08–016 (6 h) P-10–008 (6 h) P-08–016 (24 h) P-10–008 (24 h)

k × 102 8.64 3.77 28.15 34.46

± ± ± ±

3.10*, ** 0.71**, * 4.90 9.83

D/L2 (h−1 ) 0.015 0.027 0.0084 0.014

± ± ± ±

0.002 0.007* 0.0038 0.0098

Kp (cm/h) 260.1 145.5 350.7 551.5

± 101.10 ± 12.35 ± 153.80 ± 270.95

Jss (mg/cm2 h) 1.70 0.95 2.30 3.61

± ± ± ±

0.66 0.08 1.01 1.77

AUC (M) 3.12 0.93 6.25 3.79

± ± ± ±

0.93*** 0.12 0.76 0.76

*Significantly different from P-08–016 (24 h; p < 0.05, unpaired t-test). **Significantly different from P-10–008 (24 h; p < 0.05, unpaired t-test). ***Significantly different from P-10–008 (6 h; p < 0.05, unpaired t-test). DOI 10.1002/jps.24045


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Eq. (2):

1. Teichmann A, Jacobi U, Ossadnik M, Richter H, Koch S, Sterry W, Lademann J. 2005. Differential stripping: Determination of the amount of topically applied substances penetrated into the hair follicle. J Invest Dermatol 125:264–269.

2. Jacobi U, Tassopoulos T, Surber C, Lademann J. 2006. Cutaneous distribution and localization of dyes affected by vehicles all with different lipophilicity. Arch Dermatol Res 297:303–310. 3. Meidan VM, Bonner MC, Michniak BB. 2005. Transfollicular drug delivery—Is it a reality? Int J Pharm 306:1–14. 4. Knorr F, Lademann J, Patzelt A, Sterry W, Blume-Peytavi U, Vogt A. 2009. Follicular transport route-research progress and future perspectives. Eur J Pharm Biopharm 71:173–180. 5. Grice J, Ciotti S, Weiner N, Lockwood P, Cross SE, Roberts MS. 2010. Relative uptake of minoxidil into appendages and stratum corneum and permeation through human skin in vitro. J Pharm Sci 99(2):712– 718. 6. Lademann J, Richter H, Meinke M, Sterry W, Patzelt A. 2010. Which skin model is the most appropriate for the investigation of topically applied substances into the hair follicle? Skin Pharmacol Physiol 23:47– 52. 7. Otberg N, Richter H, Schaefer H, Blume-Peytavi U, Sterry W, Lademann J. 2004. Variations of hair follicle size and distribution in different body sites. J Invest Dermatol 122:14–19. 8. Otberg N, Patzelt A, Rasulev U, Hagemeister T, Linscheid M, Sinkgraven R, Sterry W, Lademann J. 2008. The role of hair follicles in the percutaneous absorption of caffeine. Br J Clin Pharmacol 65(4):488–92. 9. Grams YY, Bouwstra JA. 2002. Penetration and distribution of three lipophilic probes in vitro in human skin focusing on the hair follicle. J Contr Release 83:253–262. 10. Teichmann A, Otberg N, Jacobi U, Sterry W, Lademann J. 2006. Follicular penetration: Development of a method to block the follicles selectively against the penetration of topically applied substances. Skin Pharmacol Physiol 19:216–223. 11. Finlay A, Marks R. 1982. Determination of corticoid concentration profiles in stratum corneum using the skin surface biopsy technique. Br J Dermatol 107:33. 12. Bojar, R, Cutcliffe, A, Graupe, K, Cunliffe W, Holland K. 1993. Follicular concentrations of azelaic acid after a single topical application. Br J Dermatol 129:399–402. 13. Pagnoni A, Kligman AM, el Gammal S, Stoudemayer T. 1994. Determination of density of follicles on various regions of the face by cyanoacrylate biopsy: Correlation with sebum output. Br J Dermatol 131:862–865. 14. Wosika H, Cal K. 2010. Targeting to hair follicles: Current status and potential. J Dermatol Sci 57:83–89. 15. Valiveti S, Lou GW. 2007. Diffusion properties of model compounds in artificial sebum. Int J Pharm 345:88–94. 16. Bernard E, Dubois J, Wepierre J. 1997. Importance of sebaceous glands in cutaneous penetration of an antiandrogen: Target effect of liposomes. J Pharm Sci 56(5):573–578. 17. Vogt A, Blume-Peytavi U. 2014. Selective hair therapy? Bringing science to the fiction. Exp Dermatol 10.1111(exd.1231823:83–86. 18. Lademann J, Otberg N, Jacobi U, Hoffman RM, Blume-Peytavi U. 2005. Follicular penetration and targeting. J Invest Dermatol Symp Proc 10:301–305. 19. Lademann J, Patzelt A, Richter H, Schanzer S, Sterry W, Filbry A, Bohnsack K, Rippke F, Meinke M. 2009. Comparison of two in vitro models for the analysis of follicular penetration and its prevention by barrier emulsions. Eur J Pharm Biopharm 72(3):600–4. 20. Tabbakhian M, Tavakoli N, Jaafari MR, Daneshamouz S. 2006. Enhancement of follicular delivery of finasteride by liposomes and niosomes 1. In vitro permeation and in vivo deposition studies using hamster flank and ear models. Int J Pharm 323:1–10. 21. Patzelt A, Richter H, Buettemeyer R, Huber HJR, Blume-Peytavi U, Sterry W, Lademann J. 2008. Differential stripping demonstrates significant reduction of the hair follicle reservoir in vitro compared to in vivo. Eur J Pharm Biopharm 70:234–238. 22. Patzelt A, Lademann J. 2013. Drug delivery to hair follicles. Expert Opin Drug Deliv 10(6):797–797. 23. Mordon S, Sumian C, Devoiselle JM. 2003. Site-specific methylene blue delivery to pilosebaceous structures using highly porous nylon


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Kp = K

D KD L= L2 L

(2)

Moreover, knowing Kp and the concentration of drug in the vehicle (Cv ), the steady-state flux (Jss ) across the epidermis can be estimated: Jss = K p Cv =

KD Cv L

(3)

Data obtained show that the diffusion kinetic (D/L2 ) is independent from the formulation, whereas the values of K seem to be influenced by the nature of the formulation and appear to depend from the length of the experiment. P-08–016 interacts better with the skin in the first 6 h (8.64 × 102 vs. 3.77 × 102 ), whereas P-10–008 requires longer times, as previously discussed. Moreover, the values of Kp and Jss move in the same direction. Indeed, P-08–016 produced a higher flux in the epidermis than P-10–008 thanks to the action of chitosan previously explained. When the experiment lasted 24 h, P-08–016 did not enhance FNS permeation differently from P-10–008, which produced a 3.8-fold increment for Kp and Jss . On the other hand, it is immediately striking that to obtain the same molar concentration of FNS (expressed by the AUC) obtained in 6 h with P-08–016, are necessary 24 h with P-10– 008. In addition, P-08–016 showed an AUC value at 24 h 1.65fold higher than P-10–008, indicative of a better attitude for P-08–16 to retain FNS inside the epidermis (p = 0.051).

CONCLUSIONS The results obtained in this study demonstrated that the differential stripping technique is able to quantify the amount of FNS recovered in the follicular apparatus, making a valuable contribution toward determining an optimal treatment protocol. Furthermore, the results obtained with P-08–016 and P10–008 demonstrated distinct differences in the localization of FNS within the SC, viable epidermis, and hair follicles depending both on the formulation used and the length of the application. Considering that, when the experiments are performed in vitro, the elastic fibers are contracted due to the cutting process, thus causing a reduction in the follicular reservoir of about 10%,40 the quantities of FNS recovered under these experimental conditions are likely to be underestimations of the amounts that can reach the follicles in an in vivo system.

ACKNOWLEDGMENT We gratefully acknowledge Prof. Renato Urso for scientific contribution.

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DOI 10.1002/jps.24045


penetration study and in vivo pharmacokinetics in hairless rat.

Topical minoxidil fortified with finasteride: An account of maintenance of hair density after replacing oral finasteride.

Enhanced topical delivery of finasteride using glyceryl monooleate-based liquid crystalline nanoparticles stabilized by cremophor surfactants.

Finasteride drug safety alert.

Finasteride and the Uncertainties of Establishing Harms.

Determination of arylsulfatase C in hair follicles.

Chitosan-decorated polystyrene-b-poly(acrylic acid) polymersomes as novel carriers for topical delivery of finasteride.

High-performance liquid chromatographic method for the determination of finasteride in human plasma at therapeutic doses.

Finasteride-mediated hair regrowth and reversal of atrophy in a patient with frontal fibrosing alopecia.

Finasteride and dutasteride may reduce melanoma risk.

Hair growth effects of oral administration of finasteride, a steroid 5 alpha-reductase inhibitor, alone and in combination with topical minoxidil in the balding stumptail macaque.

The influence of low dose finasteride, a type II 5α-reductase inhibitor, on circulating neuroactive steroids.

Finasteride use and acute pancreatitis in Taiwan.

Finasteride is effective for the treatment of central serous chorioretinopathy.

The clinical development of a 5 alpha-reductase inhibitor, finasteride.

Penetration of topical chloramphenicol into the anterior chamber: response.

Penetration of topical chloramphenicol into the anterior chamber.

Increasing utility of finasteride for frontal fibrosing alopecia.

Delivery and targeting of nanoparticles into hair follicles.

Stability determination of the formulations containing hyaluronic acid.

Effects of flutamide and finasteride on rat testicular descent.

Grade-dependent Response to Finasteride in Early Prostate Cancer.

A novel finasteride 0.25% topical solution for androgenetic alopecia: pharmacokinetics and effects on plasma androgen levels in healthy male volunteers.

Combined treatment with oral finasteride and topical minoxidil in male androgenetic alopecia: a randomized and comparative study in Chinese patients.