Usefulness in dermatopharmacology
This technique consists of sequentially re-moving microscopic layers (typically, 0.5-1 µm) of stratum corneum. It is usually per-formed by placing an adhesive tape-strip onto
the skin surface, followed by gentle pressing to ensure a good contact, and subsequently removal by a sharp upward movement. The procedure is relatively painless and non-invasive, given that only dead cells (corneo-cytes) embedded in their lipid matrix are re-moved. It is widely used in various fields of cutaneous biology, namely for the study of stratum corneum barrier recovery [14,15], barrier function [16,17], morphology [18] and other related investigations [19]. In the field of dermatopharmacology, tape stripping is used either to evaluate the effect of stratum corneum-depleted skin on percutaneous ab-sorption [20], or to assess cutaneous drug lev-els after topical treatment (either in the re-moved strips or directly in the tape-stripped skin, as discussed in the next two sections). The latter assessment allows the local drug bioavailability in the stratum corneum (the target tissue) to be determined, e.g., after an antifungal treatment [21,22].
However, it is also applicable to other drugs,
Table I: Skin compartments and most current sampling techniques for the local bioavailability assessment on human subjects. TS, tape stripping; SSB, skin surface biopsy; SHB, shave biopsy; PB, punch biopsy; SB, suction blister; MD, microdialysis; FR, follicle removal.
Target skin layer Disease Drug TS SSB SHB PB SB MD FR
Stratum corneum Mycosis Antimycotics × × × ×
Viable epidermis Psoriasis Corticosteroids × × × ×
Herpes Antivirals × × × ×
Dermis Dermatitis Corticosteroids × × ×
Subcutis Arthritis NSAID × ×
Contusions NSAID × ×
Follicles Acne Retnoids,antibiotics ×
not necessarily targeted to the stratum corneum, since due to the proximity, it is rea-sonable to assume that the drug concentra-tions in the underlying skin layers (viable epidermis, dermis or subcutis) are directly related to the concentration in the stratum corneum, just as drug concentrations in this layer have been found to be directly related to systemic levels by Rougier and coworkers [23]. This group showed that the human stra-tum corneum reservoir after a 30-minute topi-cal application of radiolabeled benzoic acid correlated nicely with the absorbed amount after 96 hours, as determined from urinary excretion. This approach is attractive, but cur-rently intense effort is being devoted in order to validate the relevance of the stratum corneum drug reservoir levels to other more pertinent parameters, such as local clini-cal/pharmacological activity [24-27].
This makes the stratum corneum an excel-lent candidate for bioavailability/ bioequiva-lence assessment of any topical dermatologi-cal formulation, since drugs may be quanti-fied in a single compartment, just as the sys-temic compartment is used for assessing the pharmacokinetics of all kind of drugs de-signed to exert their action systemically. Fur-thermore, given that the barrier acts as a res-ervoir for the penetrating drugs, assessment of their levels is feasible with conventional ana-lytical techniques, without having recourse to more sensitive techniques requiring the use of radiolabeled drugs.
Local drug bioavailability from the removed tape-strips
Sequential tape stripping of the stratum corneum allows horizontal fraction layers to be obtained. The tape-strips carrying the re-moved tissues must subsequently be specifi-cally extracted in order to recover and quan-tify the absorbed drug. Local bioavailability may therefore be assessed either from the combined or the individual tape strips.
Quantification of the amount of drug in the combined tape-strips enables the total amount of drugs in the stratum corneum reservoir to be determined. By sampling different skin sites at progressively application times (Fig-ure 1A), it is possible to derive kinetic uptake metrics for local bioavailability evaluation, providing a means for assessing bioequiva-lence of two dermatological drug products. In the same way, a fixed treatment time for all skin sites followed by sequential tape strip-ping at increasing times post-treatment re-moval allows the clearance of the drug from the stratum corneum to be assessed (Figure 1B). This in vivo approach is also known as
"dermatopharmacokinetics" (DPK), and is attracting growing interest. The U.S. FDA is seriously considering this method for regula-tory purposes, and after much discussion [3,4,28-30] a draft guidance reviewing this procedure has been published [31]. However, further validation is necessary, in order to assess the feasibility of the approach, and
many groups (ours is one of those) have de-voted much effort in performing preliminary studies on human subjects.
In particular, Pershing and coworkers have recently shown that the tape stripping meth-odology was feasible and sufficiently sensi-tive even using a conventional HPLC tech-nique for marketed 0.05% betamethasone dipropionate formulations [24], and further that local bioavailability differences between various formulations were detected by the method [25], as confirmed by the skin blanch-ing assay. Other studies were conducted usblanch-ing a 5% commercial acyclovir cream [32], 2%
miconazole and 2% ketoconazole creams [22]. Schaefer et al. also compared two com-mercially available 2.5% hydrocortisone creams on human subjects by the tape strip-ping technique and the skin blanching re-sponse [27]. The drug levels in the removed strips were determined by a specific radioim-munoassay technique, which was validated by HPLC. There was a clear difference in stra-tum corneum drug levels between the two formulations, which reflected differences in the skin blanching response (generated in the underlying dermis) and also in the in vitro release of the drug from the formulations. The same group also assessed the local bioavail-ability of another corticosteroid, 0.05% clobe-tasol propionate, in a USP cream and a com-mercial cream with or without emollient [33].
For the creams without emollient, no signifi-cant differences were observed in the stratum
Figure 1: Experimental setup and profiles of a typical bioequivalence dermatopharmacokinetic assessment of two hypothetical test and reference formulations contain-ing the same drug but in different vehicles. In (A) the up-take kinetics of the drug is compared by tape-stripping the stratum corneum immediately after each treatment time, whereas in (B) the clearance profile of the drug is obtained by tape-stripping the stratum corneum after a post-treatment delay. CTR refers to a control formulation, not containing any drug.
0 Test treatment time (min)
Reference treatment time (min) 180 Test post-treatment time (h)
Reference post-treatment time (h) 24
corneum drug levels, as confirmed by the skin blanching assay, whereas for the emollient-containing cream a lower content was meas-ured, related to a lower blanching of the treated site (in this case, the results were at-tributed to a lateral diffusion of the drug). The methodology was also validated by other au-thors for sunscreening agents, where sun pro-tection factor measurements were concomi-tantly performed [34], for caffeine [35] meas-ured by surface recovery and HPLC, and for radiolabelled bifonazole and clotrimazole [21]
assessed by liquid scintillation counting.
In contrast to the "pooled" approach, indi-vidual analysis of the tape-strips allows the stratum corneum distribution of the molecule to be displayed as a concentration/tape-strip number or concentration/depth profile, which is an instantaneous and static picture of the drug penetration at a given time, and which could serve as the basis of a DPK study if several skin sites are treated. Given the sig-nificant reduction of drug amount recovered in each single tape-strip, analysis of the pene-trant sometimes requires very sensitive tech-niques, but this is not always the case.
Original investigations on human subjects using a concentration/tape-strip number methodology have been reported by many authors. Among those having used radio-labeled molecules, Hui et al. studied the stra-tum corneum clearance of peldesine after a 24-hour topical application, in ten individual tape-strips, in a set up also comparing other
parameters [36]. Bucks et al.tested the stra-tum corneum uptake of betaine-derived sur-factants after an exposure of 30 minutes and stripping of 15 stratum corneum layers [37].
Other sensitive techniques such as fluores-cence spectroscopy have been used to study the stratum corneum uptake of liposome-encapsulated fluorescein after 30 minutes contact and 15 strippings (first, three tapes quantified individually, subsequently com-bined in groups of three) [38,39]. More con-ventional techniques such as HPLC have shown this approach to be feasible, since it was also implemented for the analysis of a 40 mg/ml erythromycin lotion after 7 hours treatment and 15 strippings, yielding values above the detection limit [40].
Although profiles of drug levels versus tape-strip number give an estimate of the local stratum corneum bioavailability, they are based upon the assumption that each tape-strip removes a constant and reproducible amount of stratum corneum if the procedure is standardized (e.g. by applying a constant pressure over a given time). This is question-able, since the physiological and normal un-even cohesion of the corneocytes has been well established by Marks and coworkers: the dead cells are looser at the exterior surface and more compact in the stratum corneum interior, resulting in removal of a greater amount of tissue in the upper third of the stra-tum corneum during tape stripping [41,42].
This outcome was confirmed in a study
car-ried out by Alberti et al. (data not published), showing essentially the same pattern (Figure 2). Furthermore, during the stripping proce-dure clusters of corneocytes are removed, rather than perfect layers, and furrows are obviously not evenly removed with the super-ficial tape-strips [43]. Swelling by skin hydra-tion provoked by emollient or occlusive for-mulations may also result in removal of non-reproducible amounts of stratum corneum [44], this may be especially true at long appli-cation times [45].
In order to overcome these problems, the removed stratum corneum must be quantified by weighing the tape-strips before and after the procedure, thus yielding absolute amounts of tissue which can be converted to a depth, assuming a constant stratum corneum density and a constant tape-stripped area. One can
thus write x = m/A∗d, where x is the stratum corneum absolute depth, m is the amount re-moved, A is the stripped area, and d is the stratum corneum density. This normalization ensures that differences in concentra-tion/depth profiles result from differences in drug penetration, and not merely to artifacts due to removal of variable amounts of tissue (Figure 3), thus allowing proper interpretation of the profiles. A further normalization as relative instead of absolute stratum corneum depth was proposed by Kalia and coworkers [46,47], who measured transepidermal water loss during sequential tape stripping. Theo-retical barrier function at the bottom of stra-tum corneum can be deduced by this method-ology, and total thickness (L) of the layer may be extrapolated, thus permitting the relative x/L depth to be obtained.
Figure 2: Amount of stratum corneum removed by each individual tape-strip (mean of 5 volunteers) after 2 hours topical treatment on the ventral forearm with a 500 mg/ml solution of terbinafine in 3 different vehicles: (A) 50%
isopropyl myristate in ethanol, (B) A + 5% oleic acid, and (C) A + 1% urea. It should be noted that the first few tape-strips remove considerably more tissue, thus reflecting the increased cohesion in the deeper layers of the stratum corneum (Alberti et al., data not published).
Drug local bioavailability from the tape-stripped skin
With the emergence of more sensitive ana-lytical techniques over the last two decades, it has been made possible to analyze drugs di-rectly in the skin, in vivo. Attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy is probably the most promising technique, albeit only semi-quantitative. The instrument consists of an infrared source emitting a beam through an infrared-transparent crystal which is in direct contact with the treated skin (Figure 4). The beam is reflected internally through the crys-tal, but a certain amount of radiation (reflect-ing at a critical angle) escapes out of it, pene-trates very superficially into the skin (no more than a few microns), and finally scatters and possibly returns into the crystal and to the
detector. Multiple reflections amplify the sig-nal. Any molecule absorbing infrared radia-tion in the range of 2.5-15 µm is detected, and may therefore be analyzed if skin constituents do not interfere. Preliminary studies of feasi-bility of the method were performed in the early seventies on dodecylbenzene sulphonate [48] and later on polydimethylsiloxanes [49].
Figure 3: Stratum corneum concentration profiles after application of 500 mg/ml terbinafine in isopropyl myristate:ethanol 50:50, after 2 hours topical treatment on the ventral forearm of one volunteer. The two representa-tions (as a function of tape-strip number and of stratum corneum relative depth) exhibit significant differences, dem-onstrating that, obviously, each tape-strip does not remove a constant amount of tissue (Alberti et al., data not pub-lished).
Figure 4: Schematic diagram of an attenuated total reflectance (ATR) device mounted in a Fourier trans-form infrared (FTIR) spectrometer. The sampling area of the crystal (here a ZnSe model) must be in contact with the treated skin site. IR radiation from the spec-trometer is directed by a first mirror up to the input face of the ATR crystal. It then reflects through the latter, penetrating superficially into the skin and scat-tering or reflecting again into the crystal. At the out-put end, the beam is finally directed to the detector.
------ Tape strip number
0.6
Stratum corneum relative depth
TBF level in SC (mol/l)
20
When coupled to sequential tape stripping, this technique allows drug uptake to be moni-tored as a function of stratum corneum depth [45], thus permitting the local drug bioavail-ability to be assessed.
Limitations and unsolved issues of the Tape stripping technique
Despite the promising future of the tape stripping technique for local bioavailabil-ity/bioequivalence assessments, many issues remain to be resolved in order to allow rou-tine studies to be performed.
One essential limitation of the tape strip-ping technique is that the stratum corneum cannot be completely removed. Total removal would logically give a more accurate measure of the drug levels in the reservoir, but such a procedure is, of course, ethically unacceptable when performed on human subjects, since it locally disrupts the barrier function, and re-sults in skin glistening (due to the exposure of the aqueous viable epidermis), erythema and, possibly, bleeding scars leaving an unaes-thetic brown pigmentation for several months [50].
Another limitation is the weighing of the tape-strips, which is a tedious and time-consuming procedure necessitating a micro- or semi-micro balance. Artifacts are some-times observed, due essentially to the electri-cal properties of the backing layer of the tapes, creating electrostatic charges over the
balance pan, especially in a dry environment.
A special and expensive deionizer is then necessary, but more simple precautions may be taken, such as humidification of the weigh-ing room with a saturated solution of a mois-ture-releasing salt. Some more rapid and less tedious alternatives to weighing have been proposed, including light scattering [51], pro-tein quantification [52], optical absorbance [53], and image analysis [54,55].
Extraction of the drug from the tape-strips is often complicated by the adhesive coating:
the polymeric matrix could precipitate or en-trap the drug, thus reducing the recovery rate.
The adhesive matrix should not necessarily need to be dissolved, since the drug is only superficially stuck to it, and the extraction solvent would probably not need to penetrate deeper than the removed tissue. If the adhe-sive does dissolve in the solvent, problems can arise during an HPLC analysis, since pre-cipitation (due to interaction with the mobile phase) may occur in the tubings or in the col-umn. To overcome this problem, it is advis-able to extract the tapes in the mobile phase, and/or to add a pre-column.
Related techniques
The stratum corneum can also be removed by "glass slide stripping". Actually, this tech-nique consists in placing a drop of cyanoacry-late adhesive onto the skin surface, covering it with a glass slide, pressing lightly and finally
removing it after 30-40 seconds by a quick movement. A thick layer of stratum corneum is thus removed with the adhesive which re-mains attached to the glass slide. The proce-dure was formerly used for histological ex-amination of the skin surface [56], but was limited to flat skin surfaces. It was later adapted for irregular surfaces, such as the space between the toes, using a polyester film instead of a glass slide [57]. This technique appears useless, since self-adhesive tapes re-quire less manipulation in order to strip the stratum corneum. However, the amounts of tissue harvested by a single operation are far higher than after a single tape-strip (up to five slides are enough), but in turn this obviously increases the risk of total depletion and sub-sequent damage to the barrier.
Local drug uptake was assessed by Marks and coworkers [10] by this technique and HPLC for a piroxicam gel on a 40×25 mm surface of the knee, after 0.5, 1, 2, and 4 hours treatment and five serial strippings. Local drug levels were high, but concomitant plasma levels were almost under the detection limit, showing an insignificant systemic expo-sure of the anti-inflammatory drug. The same group also used the technique for the determi-nation of corticosteroid levels [58].