RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Topical Formulations Containing Finasteride. Part I: In Vitro Permeation/Penetration Study and In Vivo Pharmacokinetics in Hairless Rat DANIELA MONTI,1 SILVIA TAMPUCCI,1 SUSI BURGALASSI,1 PATRIZIA CHETONI,1 CARLA LENZI,2 ANDREA PIRONE,2 FEDERICO MAILLAND3 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 25 November 2013; revised 7 May 2014; accepted 7 May 2014 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24028 ABSTRACT: In hair follicle (Hf) cells, the type-2 5-␣-reductase enzyme, implicated in androgenetic alopecia, is selectively inhibited by finasteride (FNS). Because an effective topical formulation to deliver FNS to Hf is currently unavailable, this investigation aimed at evaluating in vitro FNS skin permeation and retention through and into hairless rat and human abdominal skin. Four hydroxypropyl chitosan (HPCH)-based formulations (P-08–012, P-08–016, P-08–063, and P-08–064) and one anhydrous formulation without HPCH (P-10–008) were tested. The pharmacokinetics in plasma and skin after application of P-08–016 or P-10–008 on dorsal rat skin with single and repeated doses was investigated. P-08–016 performed the best in driving FNS to the reticular dermis without producing a high transdermal flux. Neither the in vivo single nor the repeated dose experiments produced plasma levels of FNS and no differences were found between formulations concerning skin retention. No increase in the amount of drug retained in the skin was obtained with the repeated dose experiment. In conclusion, the HPCH-based formulation P-08–016 might represent an alternative to systemic therapy for its ability to C 2014 promote a cutaneous depot of FNS in the region of hair bulbs, minimizing systemic absorption even after repeated treatments.  Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci Keywords: finasteride; hydroxypropyl chitosan; formulation vehicles; permeation; penetration; skin; in vitro models; in vitro/in vivo correlations; pharmacokinetics

INTRODUCTION Androgenetic alopecia, which in men is also known as malepattern baldness, is a common form of scalp hair loss that commences, on average, in men in their mid-twenties and increases directly with age.1 The incidence of androgenetic alopecia varies among ethnic groups, being more common in Caucasian men than in Asians, American natives, and Africans.2 Such a condition may also affect women, even if the pattern of hair loss is different and the prevalence is lower in women than in men.3 On the other hand, it is noteworthy that the hormone testosterone seems to play an important role in male androgenetic alopecia, independent of genetic predisposition.4 Type-2 5"-reductase, which converts testosterone to dihydrotestosterone (DHT) and is expressed in hair follicles (Hf) and other androgen-dependent tissues, appears to be important in male pattern baldness.2 Finasteride (FNS), a synthetic 4-azasteroid compound, has been widely used for benign prostatic hyperplasia at low dose (1 mg/day) and for prostatic cancer at higher dose (5 mg/day). Recently, oral administration of FNS has been used for the treatment of various dermatological and follicular disorders (i.e., acne, seborrhoea, male pattern baldness); in particular,

Abbreviations used: FNS, finasteride; PG, propylene glycol; Ep, epithelium; PD, papillary derma; RD, reticular derma; Hf, hair follicles; Sg, sebaceous glands; A, adipocytes; Mt, muscular tissues. Correspondence to: Monti Daniela (Telephone: +39-0502219662; +390502219659; E-mail: [email protected]) Journal of Pharmaceutical Sciences

 C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association

1 mg/mL oral FNS has been approved for the treatment of androgenetic alopecia.5,6 The mechanism of action is a competitive and selective inhibition of the type-2 5"-reductase isoenzyme.7,8 It has been reported that oral administration of a daily dose of 1 mg reduces concentrations of scalp DHT and serum DHT by 64% and 68%, respectively,9 and inhibits or reverses miniaturization of Hf. Even though continued use does not appear to promote further hair regrowth, the hair density stabilizes with retention of the newly acquired hair.10,11 Even if successful, the treatment should be continued indefinitely because the balding process restarts once treatment ceases.12 Because of several adverse effects observed in the majority of patients13 such as impaired reproductive function, impotence, erectile dysfunction, and gynecomastia,14 topical therapy should be preferable to oral. Notwithstanding some studies on topical FNS having been performed in past years that showed encouraging results,13,15,16 no effective FNS topical formulation is so far commercially available. The aim of the present investigation was, on the one hand, to evaluate in vitro the transdermal permeation and skin retention of FNS of four different hydroxypropyl chitosan (HPCH)based aqueous formulations (P-08–012, P-08–016, P-08–063, and P-08–064) through and into hairless rat skin. On the other hand, the selected best formulation was then compared with an anhydrous formulation devoid of HPCH (P-10–008) by using both hairless rat skin and excided human skin to investigate the appropriateness of these models for the analysis of penetration and storage of topically applied substances in the Hf. Monti et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Table 1.

Composition of the Formulations Under Study

Excipients

Composition (%) P-08–012 P-08–016 P-08–063 P-08–064 P-10–008

FNS Ethyl alcohol 96◦ Propylene glycol Transcutol P Purified water Hydroxypropyl chitosan (HPCH)

0.25 50.00 20.00 – 28.75 1.00

0.25 55.00 5.00 – 38.75 1.00

0.25 55.00 2.50 2.50 38.75 1.00

0.25 55.00 – 5.00 38.75 1.00

0.25 71.25 28.50 – – –

Moreover, the study was aimed at evaluating FNS pharmacokinetics in vivo in plasma samples and in the depths of the skin of hairless rats after single and repeated administration of the formulation that performed best in the in vitro test.

MATERIALS AND METHODS Chemicals The following products were used as received by Polichem S.A. (Lugano Pazzallo, Switzerland): Finasteride (FNS, batch number J7C002F); HPCH-based formulations (P-08–012, P-08–016, P-08–063, and P-08–064) and an anhydrous formulation devoid of HPCH (P-10–008). Details on each formulation composition are reported in Table 1. All other chemicals and solvents were of analytical grade. Animals Experiments were carried out on 5-week-old hairless male rats (HsdHanTM:RNU- Foxn1 rnu; Harlan Italy srl, Correzzana, Italy). 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. The experiments were conducted according to the OECD Test Guideline 427 (in vivo),17 428 (in vitro),18 and the OECD Guidance Document for the Conduct of Skin Absorption Studies.19 The rats were kept individually in cages during the test period with free access to a rodent diet and tap water. A 12h light/12-h dark cycle was maintained throughout the study period. At the end of the study, the animals were sacrificed by cervical dislocation. Temperature and relative humidity in the animal room were monitored and recorded each day of the study. Human Skin Human female abdominal skin was obtained following aesthetic surgery. Full thickness skin was separated from underlying fat and membranes were stored frozen at −20◦ C for a period of up to 6 months. The skin thickness was 1250 ± 420 :m. Histological Evaluation As skin morphology is supposed to influence skin permeability, in order to precisely determine both the thickness of the rat skin used in our experiments and the localization of Hf in the skin depth, some samples were submitted to histological evaluation. The skin samples were fixed in 10% buffered formalin solution, dehydrated, and embedded in JB-4 plastic resin. Coronal sections were cut by a microtome (Reichert-Jung) and mounted Monti et al., JOURNAL OF PHARMACEUTICAL SCIENCES

onto gelatine-coated slides. Sections were stained with methylene blue/toluidine blue for microscopic examination. The specimens were assessed under a then Leitz Diaplan microscope. In particular, the thickness, as the mean of six measurements, of the following regions was evaluated: epithelium (Ep), papillary derma (PD), and reticular derma (RD). In Vitro Permeation/Penetration Studies Permeation tests through excised rat skin or abdominal human skin were carried out as previously described.20 using Gummer-type diffusion cells with an available diffusion area of 1.23 cm2 and the stratum corneum facing the donor compartment. Two-hundred microliters of each formulation was placed on the skin surface. The receiving phase (5 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, 5.0 mL samples of receiving phase were withdrawn for HPLC analysis and replaced with the same volume of fresh fluid. All experiments lasted 24 h. At the end of the permeation experiments, the skin was removed from the cells and rinsed with distilled water. The samples were then frozen and sliced horizontally with a cryomicrotome (MEV Cryostat; Slee-Technik GMBH, Mainz, Germany) after flattening the skin with a 2 kg weight for 1 min. The precision of the microtome, as specified by the manufacturer, gives reproducible sections of 1–60 :m in 1 :m steps up to 10 :m, 2 :m steps up to 20 :m, and 5 :m steps up to 60 :m. The mean thickness of the first incomplete slices, due to the irregular skin surface and skin residues, was calculated from their weight with reference to a standard slice of known weight and thickness.21,22 FNS was extracted from the skin slices by treatment with 2.0 mL of 2% SDS for 24 h. After contact with methanol (4.0 mL) for 1 h, the mixture was centrifuged at 4,000 rpm for 15 min. Two hundred microliters aliquots of supernatant were dried in vacuo and subsequently dissolved in methanol for HPLC analysis. To validate the extraction procedure, series of 20 or 25 :m slices of blank skin were submitted for assay, and the extraction recovery was determined by computing the ratio of the amount of drug extracted from the skin to the amount added. In Vivo Experiments Rats were divided into two groups for topical administration of the two test formulations, namely the anhydrous formulation (P-10–008) and the HPCH-based formulation that performed best in the in vitro tests. A volume of 200 :L of each formulation was applied to a delimited area (20 mm diameter, 3.14 cm2 ) on the dorsal skin site. Twenty-four hours later the animals were either sacrificed (single dose treatment) or further treated every 24 h for 7 days (repeated dose treatment). In both cases, the animals were sacrificed 24 h after the last administration and samples of blood were withdrawn and centrifuged at 10,000 rpm for 10 min in order to separate the plasma fraction. Then, the plasma was treated with a methanol solution containing 6% perchloric acid to precipitate plasma proteins and centrifuged again. Aliquots of the supernatant were dried in vacuo and dissolved in methanol for HPLC analysis. Moreover, the portion of skin submitted to the treatment was excised, carefully rinsed to remove the remaining formulation, sectioned on the cryomicrotome, and processed following the same DOI 10.1002/jps.24028

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

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procedures used for the in vitro skin sections to determine the amount of FNS retained in the skin layers.

Table 2. Results of Histological Examination of Coronal Sections of Hairless Rat Skin (n = 6)

Analytical Methods

Skin Region

The quantitative determination of FNS in receiving fluids and in the biological matrix (skin or plasma) was carried out by HPLC. The apparatus consisted of a Shimadzu LC-20AD system with an UV SPD-10A 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 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 FNS to blank biological matrix. The standard curves were linear in the detection range, and the assay linearity was good. The limit of detection (LOD) and limit of quantitation (LOQ), 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 for receiving fluids, respectively, and 16.75 and 33.5 ng/mL for biological matrixes.

Epidermis Dermis

preparation is intended for the oral route.25 Its mechanism of action is a selective inhibition of the conversion of testosterone to DHT, which mainly happens in the prostate and Hf.16 Androgenetic alopecia is a pathology that if untreated represents a progressive condition; for this reason, the therapy must be continued indefinitely, with consequent patient discomfort and systemic disorders linked to oral drug administration. Topical drug application could be an interesting alternative but, as noted above, presently no company has developed a topical product. The present work focused on developing a formulation for the delivery of FNS on male scalp skin in order to obtain drug concentrations in the dermis, after demonstrating by histological studies that the reticular dermis is the skin region where most of the hair bulbs are located.

Data Analysis

Histological Evaluation

Linear regression analysis of pseudo steady state diffusion plots allowed calculation of the following parameters: steady-state flux (J), given by Q/At, where Q is the amount of permeant diffusing across area A in time t; lag time, indicating the time taken by the drug to saturate the membrane and reach the receiving phase, calculated from the X-axis intercept values of the regression lines; and the percentage of drug permeated at the end of the experiment (Q%24 h ). Moreover, the extraction procedure allowed calculation of the FNS content (FNSskin , mg/g skin) at the end of the permeation studies. All data are the average of five determinations ± standard error (SE). Statistical differences between permeation parameters were assessed by GraphPad Prism software (GraphPad Software Inc., San Diego, California). The evaluation included calculation of means and SEs, and group comparisons using the Student’s two-tailed unpaired t-test. Differences were considered statistically significant at p < 0.05. In order to compare in vitro and in vivo data obtained in the present work with formulation P-08–016, we used the equation reported below23 to calculate the steady-state plasma concentration of FNS estimated on the basis of in vitro permeation data:

The results of the histological examination are reported in Table 2 as mean depth values (±SE) of the Ep, papillary region, and reticular region measured on coronal skin sections from six animals. The measured values of epidermal thickness were about 30 :m, in agreement with Scott et al.,26 whereas other studies have produced different findings.27 In any case, the differences found in these studies could be ascribed to skin morphological characteristics such as the sex or age of the animals used. As shown in Figure 1, Hf are present in both the reticular dermis and the hypodermis region, the latter characterized by the presence of adipocytes (A), again confirming the literature data.27,28

R

 Css = (A × F ) (CLt × BW)

(1)

where A is the skin area available for diffusion (1.23 cm2 ), F is the in vitro permeation rate of FNS (:g/cm2 h), BW is the body weight of the rat (115 g), and CLt is the total body clearance (13.4 mL/min/kg).24

RESULTS AND DISCUSSION Finasteride, a 5"-reductase inhibitor, has been widely studied and used for the treatment of prostatic hyperplasia and androgenetic alopecia, but currently the only commercially available DOI 10.1002/jps.24028

Depth (:m ± SE) Epithelium Papillary region Reticular region

30.81 ± 2.83 108.66 ± 12.98 360.85 ± 36.65

In Vitro Permeation/Penetration Studies In the first part of this research, four HPCH-based aqueous formulations (P-08–012, P-08–016, P-08–063, P-08–064) were evaluated. Chitosans are attracting attention as drug delivery systems due to their excellent biocompatibility, biodegradability, and nontoxicity.29 Some studies have reported permeationenhancing properties of chitosans depending on their molecular weight, degree of deacetylation and pH, together with their concentration in the final formulation.30–32 Furthermore, it has been reported that chitosans present mucoadhesive properties33 and possess film-forming capabilities,34 which could favor an intimate contact of the formulation with the skin, thereby allowing a continuous release of drugs or other actives for many hours after an application. Regarding hair products, Rinaudo et al.35 highlighted that chitosans could remove the sebum coating on the hair shafts due to its hydrophobic character and, when formulated in aqueous solution, they may interact with negatively charged hairs due to electrostatic interactions. For all these reasons, we decided to deliver FNS in HPCH-based aqueous formulations, with the aim of facilitating the contact between formulation Monti et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Table 3. Permeation Parameters of FNS Through Hairless Rat Skin After Application of 200 :L of: (a) the HPCH-Based Aqueous Formulations (P-08–012, P-08–016, P-08–063, and P-08–064); and (b) the Best Performing Formulation Among the HPCH-based Formulations (P-08–016) and the Anhydrous Formulation (P-10–008) (mean ± SE) Vehicle

Flux (J, :g/cm2 h)

Lag time (h)

Q%24 h

(a) P-08–012 P-08–016 P-08–063 P-08–064

2.47 0.77 1.52 1.10

± ± ± ±

0.48 0.27* 0.39 0.54

6.22 2.57 1.49 1.43

± ± ± ±

1.13 1.07 0.68* 0.85*

10.77 3.68 5.83 5.72

± ± ± ±

2.27 1.21* 1.25 1.75

(b) P-08–016 P-10–008 *

0.77 ± 0.27 2.00 ± 0.76

2.57 ± 1.07** 7.01 ± 1.55

3.68 ± 1.21 9.38 ± 4.12

Significantly different from P-08–012, p < 0.05. Significantly different from P-10–008, p < 0.05.

**

Figure 1. Photomicrographs of some of the different samples of skin used. Epithelium (Ep), papillary region (PD), reticular region (RD), hair follicles (Hf), sebaceous glands (Sg), adipocytes (A), and muscular tissue (Mt). Note Hf in the Hypodermis characterized by the presence of A.

and skin, to enhance penetration of the FNS through skin and hair and obtain a larger depot of the drug in the Hf, minimizing transdermal permeation. All formulations contained water to solubilize HPCH and ethanol both to solubilize FNS and to contribute to the formation of an elastic film on skin and/or hair surface, thanks to its rapid evaporation. Moreover, ethanol could dissolve or reorganize the sebum favoring the drug deposition within the follicle.36 Besides, some formulations were added with propylene glycol (PG) and/or Transcutol P, the former being reported either to interact with the lipids in the follicular openings increasing drug accumulation or to improve drug diffusion from the epidermis to outer root sheath once the drug has bypassed the stratum corneum.37 Furthemore, PG is known to act as a drug penetration enhancer with better solubility in alcohols than in water and its action is supposed to be the solvation of keratin within the stratum corneum and the intercalation in the polar head groups of the lipid bilayers.38,39 The latter, namely Transcutol P, has been recognized to increase the skin accumulation of topically applied compounds without a concomitant increase in transdermal permeation, probably by swelling the intercellular lipids of stratum corneum without altering the multiple bilayer structure. These swollen lipids then retain drugs, especially lipophilic compounds, to form the depot with a consequent increased skin accumulation and simultaneous decrease in transdermal permeation. Moreover, when Transcutol P is used in combination with PG, a synergistic enhancement effect can occur and a modulation of the drug permeation enhancement or of its depot in the skin by suitably varying the Transcutol P/PG content is possible.40 Monti et al., JOURNAL OF PHARMACEUTICAL SCIENCES

The results of the present work about the in vitro FNS permeation experiments related to HPCH based formulations are summarized in Table 3a, where the relevant permeation parameters (flux, lag time, and percent FNS permeated after 24 h) are reported. Even though all formulations exhibited transdermal permeation of FNS through hairless rat skin, differences in the permeation parameters could be noticed recorded depending on the composition of the formulations and the nature of the excipients used. In particular, formulation P-08–016 led to a significantly lower flux compared with all the others. The presence of PG seemed to influence the flux in a dose-dependent fashion. Indeed, formulation P-08–012 containing 20% PG produced a flux at a steady state (J) of 2.47 ± 0.48 :g/cm2 h, whereas formulation P-08–016 containing a fourfold lower amount of PG (5%) led to a significant reduction in the transdermal flux of drug (0.77 ± 0.27 :g/cm2 h). Partial or complete substitution of PG with Transcutol P in the case of formulations P-08–063 (Transcutol P 2.5%) and P-08–064 (Transcutol P 5.0%) did not significantly affect any of the permeation parameters except lag time. Neither its enhancing properties nor a hypothetical synergism with PG were found in this study. The in vitro penetration data related to the tested formulations are summarized in Table 4a as mg of FNS per g of skin retained in each skin layer. A large amount of drug was retained in the upper layers of the skin (15–35 :m) with a considerable decrease in the deeper layers, corresponding to dermis (>35 :m as proven by histological analysis), was observed with all formulations. The percentage of PG in formulations P-08–012 and P-08–016 (20% and 5%, respectively) did not influence the amount of drug retained in the skin layers even while it affected the flux of FNS across the skin. In particular, P-08–016 produced an appreciable amount of FNS up to and including the reticular dermis, where most of the hair bulbs are located, while transdermal permeation was sensibly reduced. Replacing PG with 5% Transcutol P (P-08–064), which is reported in the literature to enhance the percutaneous penetration of various active substances,41,42 did not produce an increase in the drug retained in the skin, especially in the DOI 10.1002/jps.24028

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Table 4. Comparison of the Recovery of FNS at Different Skin Depths Following Topical Application on Hairless Rat Skin In Vitro of: (a) the HPCH-Based Aqueous Formulations (P-08–012, P-08–016, P-08–063, and P-08–064); and (b) the Best Performing Formulation Among the HPCH-Based (P-08–016) and an Anhydrous Formulation (P-10–008), (mean mg/g ± SE) (a) Skin Depth (:m)

FNS (mg/g) P-08–012

P-08–016

38.55 ± 11.73 33.92 ± 19.41 23.31 ± 9.32 21.76 ± 10.55 9.87 ± 3.29 12.24 ± 3.42 7.87 ± 2.84 6.07 ± 1.84 9.97 ± 5.11 5.43 ± 1.22 3.77 ± 2.67 5.05 ± 1.20 3.26 ± 1.76 3.60 ± 0.99 2.74 ± 0.90 4.88 ± 1.32 3.85 ± 2.16 1.74 ± 0.25 3.03 ± 1.97 1.87 ± 0.40 3.46 ± 2.30 2.43 ± 0.85 0.82 ± 0.33 0.80 ± 0.51 n.d. 0.72 ± 0.34

15 35 55 75 95 115 135 155 175 195 215 355 455

P-08–063 20.53 15.14 15.22 13.12 8.79 7.97 4.92 5.42 3.89 5.25 3.08 0.65 0.60

± ± ± ± ± ± ± ± ± ± ± ± ±

12.02 4.32 2.79 3.45 3.29 3.03 1.54 1.43 0.96 0.77 0.77 0.23 0.10

P-08–064 24.80 13.87 13.66 13.34 5.14 3.35 2.41 3.11 1.51 1.65 0.76 0.64 0.86

± ± ± ± ± ± ± ± ± ± ± ± ±

9.27 3.76 3.07 1.43 0.91 1.72 1.63 2.52 0.99 0.94 0.25 0.30 0.54

(b) Skin Depth (:m)

FNS (mg/g) P-08–016

155 175 195 215 355 455 *

6.27 1.74 1.87 2.43 0.80 0.72

± ± ± ± ± ±

1.87 0.25 0.40 0.85* 0.51 0.34

P-10–008 2.92 1.28 1.16 0.63 0.65 0.58

± ± ± ± ± ±

0.85 0.30 0.67 0.10 0.10 0.17

Significantly different from P-10–008, p < 0.05.

reticular dermis. Moreover, it is interesting to note how P08–063, containing the PG/Transcutol P combination in the same amount was able to increase FNS retention in the dermis (55–235 :m), producing values in the 15.22 ± 2.79–3.08 ± 0.77 mg/g range. This behavior could depend on the interaction of PG with the polar group regions of the skin lipids which, favoring the absorption of Transcutol P in the skin, was able to create an intracutaneous depot of the drug.40 Anyway, no differences in FNS skin retention were found among formulations P-08–012, P-08–016, and P-08–063, highlighting that PG plays a key role of PG independently from its concentration. On the other hand, the lack of activity of Transcutol P could be attributed to the low concentrations used (2.5%–5%), which were far below those reported in the literature as active (10%–20%).43,44 A low amount of Transcutol P seems not sufficient to carry out its mechanism of action, that is, the swelling of stratum corneum intercellular lipids with consequent drug retention to form an intracutaneous depot.43 Finally, the results obtained from screening the aqueous HPCH-based formulations highlighted that P-08–016 was able to drive FNS down to the reticular dermis without producing DOI 10.1002/jps.24028

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a high transdermal flux of FNS, suggesting a limited systemic absorption. To determine the importance of the technology formulation based on HPCH, the second part of the work aimed at comparing the skin permeation and penetration induced by P-08– 016 with that of P-10–008, an anhydrous formulation devoid of HPCH. For P-10–008, the permeation study on rat skin was performed under the same conditions as for P-08–016, whereas the retention study was focused at a skin depth of 155–455 :m, corresponding to the site of action of FNS. As reported in Table 3b, the flux of FNS from P-10– 008 was higher than that from P-08–016 (2.00 ± 0.76 vs. 0.77 ± 0.27 :g/cm2 h), highlighting that the combination of PG and ethanol in high amounts enhanced FNS permeation as widely reported in literature.45 Skin retention data are reported for both formulations in Table 4b. Those for P-08–016 derive from Table 4a, where the value at 155 :m is calculated as the sum of the data between 15 and 155 :m. P-08–016 appeared to result in greater amounts of drug in all skin layers examined with respect to P-10–008; in particular, the quantity of FNS at a skin depth of 215 :m was about fourfold higher with P-08–016 than with P-10–008 (2.43 vs. 0.63 mg/g), a statistically significant difference. These results may indicate the greater efficacy of the P08–016 formulation to target FNS to its site of action in the RD. Most probably, the film-forming ability of HPCH favors the interaction of the formulation with the skin and the high lipophilicity of the drug (log P = 4.277),14 with consequent good affinity for the stratum corneum, making FNS more readily available for percutaneous absorption when it is formulated in an aqueous medium. On the other hand, the presence of ethanol and PG in large amounts in formulation P-10–008 may produce a synergistic enhancing effect that increases FNS permeation after the lag time.46–48 In addition, HPCH could act as a penetration enhancer by changing the secondary structure of keratin in the stratum corneum and increasing cell membrane fluidity to varying degrees as reported by He et al.,49 contributing to greater amounts of FNS in the dermis. As well as it is noteworthy that rat skin has some limitation as representative of human skin in permeation studies, we performed a preliminary study on excided human skin to validate our results. As expected, we did not find any appreciable amount of FNS in the receiving phase when both formulations were applied. On the other hand, as reported in Table 5, FNS seems to be retained in human skin in lower amounts with respect to rat skin, but with the same trend: formulation P-08–016 produced higher amounts in the region of hair bulbs compared with P10–008 with statistically significant difference at the depths 650 and 750 :m (p = 0.0246 and p = 0.0238, respectively). In Vivo Distribution Studies The pharmacokinetics study on hairless rat performed with the HPCH-based formulation P-08–016 and the anhydrous formulation P-10–008 demonstrated that neither the single nor the repeated dose experiments produced detectable plasma levels of FNS (method sensitivity: LOQ 33.5 ng/mL and LOD 16.75 ng/mL). Although no information about plasma pharmacokinetics after cutaneous administration of FNS is Monti et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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Table 5. Comparison of the Recovery of FNS at Different Skin Depths Following Topical Application on Human Abdominal Skin In Vitro of P-08–016 and P-10–008 (mean mg/g ± SE, n = 3) Skin Depth (:m)

FNS (mg/g) P-08–016

50 150 250 350 450 550 650 750 850 1050 *

13.50 4.87 2.25 0.28 0.25 0.27 0.33 0.35 0.23 0.02

± ± ± ± ± ± ± ± ± ±

3.09 1.66 0.85 0.06 0.002 0.006 0.02* 0.02* 0.003 0.004

P-10–008 17.80 3.06 2.14 0. 35 0.25 0.19 0.22 0.26 0.21 0.03

± ± ± ± ± ± ± ± ± ±

3.88 1.51 0.97 0.10 0.02 0.06 0.02 0.02 0.04 0.01

suggesting quick and complete elimination of the drug from the tissue. Our in vivo study on hairless rats confirmed the absence of systemic absorption, even after repeated administration of the drug for one week. The Css , calculated by using the Eq. (1), was 10.24 ng/mL that we did not reveal because it was below the LOQ of our analytical method. Our findings confirmed the validity of the in vitro preliminary study on rat skin and the usefulness for further investigations. Concerning the FNS retention in the skin obtained with the in vivo study, the behavior of the P-08–016 formulation appeared similar either after single or repeated doses. Data were of the same order of magnitude as the in vitro study, indicating again that rat could be considered a good model for skin penetration studies.

Significantly different from P-10–008, p < 0.05.

CONCLUSIONS

Figure 2. Retention of FNS in hairless rat skin after the “in vivo” (a) single dose experiment and (b) repeated dose experiment (mean ± SE).

available in the literature, our data reflect those regarding oral administration. Indeed, it has been reported that the elimination half-life (t1/2 ) of FNS is 4–7 h and that slow plasma accumulation occurs with multiple doses.50 The in vivo concentration/depth profiles up to the overall thickness of the skin are shown in Figures 2a and 2b for the single and repeated dose experiments, respectively. No differences were found between the two formulations, either in the case of the single dose or that of the repeated dose treatment. Furthermore, it is interesting to note that no increase in the amount of FNS accumulated in the skin was obtained by prolonging the treatment for one week (repeated dose experiment), Monti et al., JOURNAL OF PHARMACEUTICAL SCIENCES

The in vitro study performed on hairless rat skin evidenced that although all formulations were able to produce quantities of FNS in both the receiving fluid and the skin, P-08–016 allowed FNS to reach the reticular dermis while simultaneously producing a low transdermal flux of drug, suggestive of scarce systemic absorption. Even though an isolated in vitro study on rat skin may be of limited regulatory value because it is likely to give an overestimation of absorption, anyway it could provide a rough estimate that could replace a worst case default value. Moreover, studies in literature51 that compared drug permeation from human cadaver skin and rat skin, concluded that rat skin is a good surrogate for human skin in in vitro permeability studies showing positive linear correlation of the in vitro permeation rates. Our preliminary studies on human abdominal skin are in line with literature as we found not an appreciable permeation compared with that obtained with rat skin; however, the retention data are in the same range of magnitude, confirming the acceptability of the rat model; above all, it was highlighted that the formulation P-08–016 is able to produce higher amounts of FNS in deeper dermis, where the follicular bulbs are located. Besides, the pharmacokinetic study was also carried out on rats, the most commonly used specie for animal in vivo studies because it is widely employed in other toxicity and toxicokinetic studies and the results are directly comparable with in vitro studies. In conclusion, the selected formulation P-08–016 might represent a valid alternative to systemic therapy in consideration of its ability to promote a high level cutaneous depot of FNS in the region surrounding the hair bulbs while minimizing systemic absorption, even after repeated treatments. In our opinion, the HPCH-based formulation P-08–016 could become the treatment of choice with respect to P-10–008, which, especially for long-term treatments, could irritate the scalp skin due to the high ethanol/PG concentration and absence of water. Further studies are in progress in our laboratory to verify the exact quantity of drug retained inside the hair bulbs, that is, the active site of FNS. Conflict of interest: Federico Mailland MD is an employee of Polichem S.A. DOI 10.1002/jps.24028

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

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DOI 10.1002/jps.24028