Relative Uptake of Minoxidil into Appendages and Stratum Corneum and Permeation through Human Skin In Vitro JEFFREY E. GRICE,1 SUSAN CIOTTI,2 NORMAN WEINER,3 PETER LOCKWOOD,2 SHEREE E. CROSS,1 MICHAEL S. ROBERTS1 1Therapeutics Research Unit, School of Medicine, The University of Queensland, Princess Alexandra Hospital, Brisbane, Woolloongabba, Qld 4102, Australia 2Formerly Pfizer Global Research and Development, Ann Arbor, Michigan 3College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109 Received 25 March 2009; revised 8 May 2009; accepted 19 May 2009 Published online 18 June 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21856 ABSTRACT: We examined uptake of the model therapeutic agent, minoxidil, into appendages, stratum corneum (SC), and through human skin, under the influence of different vehicles. Quantitative estimation of therapeutic drug deposition into all three areas has not previously been reported. Finite doses of minoxidil (2%, w/v) in formula- tions containing varying amounts of ethanol, propylene glycol (PG), and water (60:20:20, 80:20:0, and 0:80:20 by volume, respectively) were used. Minoxidil in SC (by tape stripping), appendages (by cyanoacrylate casting), and receptor fluid was determined by liquid scintillation counting. At early times (30 min, 2 h), ethanol-containing for- mulations (60:20:20 and 80:20:0) caused significantly greater minoxidil retention in SC and appendages, compared to the formulation lacking ethanol (0:80:20). A significant increase in minoxidil receptor penetration occurred with the PG-rich 0:80:20 formula- tion after 12 h. We showed that deposition of minoxidil into appendages, SC, and skin penetration into receptor fluid were similar in magnitude. Transport by the appendageal route is likely to be a key determinant of hair growth promotion by minoxidil. ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:712–718, 2010 Keywords: absorption; formulation vehicle; membrane transport; passive diffusion; skin; transdermal drug delivery INTRODUCTION there have been a number of studies examining appendageal deposition, particularly of nanopar- The differential targeting of topically applied com- ticles,1 and other work aiming to determine the pounds into appendages, stratum corneum (SC) contribution of appendageal transport to skin and through skin is an important goal. While penetration,2 to our knowledge there are no reports examining the deposition of therapeutic compounds to all three areas. The aim of this work Susan Ciotti’s present address is NanoBio Corporation, was to study uptake, through a quantitative esti- 2311 Green Road, Suite A, Ann Arbor, MI 48105. Peter Lockwood’s present address is Pfizer Global Research mate of deposition of the model therapeutic agent, and Development, Groton, CT 06340. minoxidil, into appendages, SC, and through Correspondence to: Michael S. Roberts (Telephone: þ61-7- human skin, under the influence of different 3240-5803; Fax: þ61-7-3240-5806; E-mail: [email protected]) vehicles. Journal of Pharmaceutical Sciences, Vol. 99, 712–718 (2010) There is increasing recognition of the contribu- ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association tions to transdermal delivery made by ‘‘shunt 712 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010 RELATIVE UPTAKE OF MINOXIDIL INTO APPENDAGES AND STRATUM CORNEUM 713 pathways’’ or ‘‘appendageal transport,’’1,3 parti- within the follicle. Three prototype formulations cularly at early times.4 This contribution has been with differing ethanol, PG, and water content evaluated, with varying degrees of acceptance, by were chosen; in proportion by volume of 60:20:20, indirect methods, for example, by a comparison 80:20:0 (no water) and 0:80:20 (no ethanol), between areas of different follicular density5 or respectively. Finite doses of 2% minoxidil (5 mL/ between intact and scarred skin,6 by physical cm2) were applied to excised full thickness human blocking of follicular openings,7 or by the use of skin. This dose is similar to that applied to the the in vitro skin sandwich technique.8 In addition, human scalp under ‘‘in use’’ conditions (3–5 mL/ rather than regarding follicles as shunts for trans- cm2). The results were interpreted in terms of dermal delivery, some attention has been given to solvent evaporation and individual vehicle effects. direct targeting of hair follicles by therapeutic agents, such as for acne and alopecia. To this end, attempts have been made to quantitate material retained in follicles after topical application of EXPERIMENTAL model penetrants such as dyes9 and nanoparti- cles,1 but little has been done with therapeutic Chemicals compounds and the area remains difficult and 3H-minoxidil (Minoxidil, [piperyl-3,4-3H], ART- incompletely validated.10 A useful approach is 0711-250mCi) was purchased from American that of differential stripping.11 Introduced by Radiolabeled Chemicals, Inc. (St. Louis, MO). Marks and Dawber,12 this technique allows Minoxidil was supplied by the Therapeutics quantitation of SC retention by tape stripping Research Unit. Ethanol and PG were purchased and of follicular retention by cyanoacrylate from Sigma–Aldrich Pty. Ltd. (Sydney, Australia). casting.1 The scintillation cocktail used was Emulsifier- The targeting of topically applied substances Safe (PerkinElmer Pty Ltd, Melbourne, Austra- to particular skin sites can be approached by lia). For cyanoacrylate casting, we used ‘‘Quick manipulating the components of the formulation. Fix Supa Glue’’ (Selleys Pty Ltd, Padstow, In this work, we chose to prepare formulations Australia). Formulations containing 2% (g/mL) with different proportions of three common minoxidil and a trace amount of 3H-minoxidil vehicles, ethanol, propylene glycol (PG), and (approx. 1 mCi/7 mL) were prepared in three water. Ethanol and PG rapidly diffuse into different vehicle mixtures. These contained (by the SC13 and may act as penetration enhancers volume) 60% ethanol, 20% PG, and 20% water by a variety of mechanisms.13–15 Of interest here (formulation 1); 80% ethanol and 20% PG are the effects on membrane properties to alter (formulation 2); and 80% PG and 20% water drug permeation, following alcohol diffusion into (formulation 3). In the following, these formula- the membrane. As well, ethanol could potentially tions will be designated 60:20:20, 80:20:0, and extract or solubilize sebum to promote follicular 0:80:20, respectively. delivery in some cases. The use of water in formulations aids skin hydration, thus enhancing transdermal delivery of many drugs. This may be Human Skin Membranes particularly important when vehicles which tend to desiccate the skin are present, such as ethanol Human abdominal skin was obtained from volun- and PG. teers undergoing elective surgery. The collection While the mechanism of action of the model of skin was approved by the Princess Alexandra compound, minoxidil remains unclear, the hair Hospital Research Committee (Approval no. 097/ follicle has been regarded as the site of action16 090, administered by the University of Queens- and a recent report identified K(ATP) channels land Human Ethics Committee) and donors gave responsive to minoxidil located in human follicu- informed written consent. Full thickness skin, lar dermal papillae.17 The uptake of minoxidil was which was used in the majority of experiments, studied by measuring penetration into the recep- was separated from underlying fat and epidermal tor of Franz diffusion cells and by differential and dermal membranes were prepared from full stripping, consisting of tape stripping (20 times) to thickness skin by floating the skin in water at sample SC content, which reflects partitioning of 608C for 1 min and peeling off the epidermal layer. minoxidil into the skin barrier layer and cyanoa- Both full thickness skin and epidermal mem- crylate casting to sample minoxidil deposited branes were stored frozen at À208C until use. DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 2, FEBUARY 2010 714 GRICE ET AL. In Vitro Permeation Studies adhesive tape to the exposed skin surface and careful removal, up to a maximum of 20 times. The experiments were performed using full Individual tapes were placed into separate scin- thickness skin as described above, from three tillation vials and soaked overnight with 2 mL different donors (five replicates per skin) in glass scintillation fluid (Emulsifier-Safe) before vortex- horizontal static Franz-type diffusion cells, using ing and quantitation by liquid scintillation phosphate buffered saline, pH 7.4 (PBS) as the counting (LSC). The total amount of minoxidil receptor phase. The exposed skin surface area was deposited within the SC was obtained by summa- 1.33 cm2 in all cases. The diffusion cells were tion of the amounts measured on the individual placed in a water bath, to maintain the receptor tapes. Results for the first two tapes were not phase temperature at 358C, over a magnetic included, as this represents unabsorbed surface 15 point stirring plate. Magnetic stirrer bars in material only. the bottom of each receptor chamber maintained Following tape stripping, follicular casts were mixing of the receptor fluid. The prepared Franz prepared. One drop of superglue was placed on a cells remained in the water bath for a minimum of glass microscope slide, which was then pressed 30 min before dosing, to ensure the skin was onto the surface of the stripped skin and held in adequately hydrated. After hydration, resistance