Evaluation of topical bioavailability in human stratum corneum in vivo by tape stripping using a direct sprectroscopic method

The objective of this thesis was to develop a spectrophotometric methodology for
simultaneous determination of a sunscreening agent and relative stratum corneum tissue
density in skin tape strips. This methodology was then employed to investigate the effect of
different vehicles and their application time on the penetration of the sunscreening agent into
the stratum corneum (SC) and the ability of these vehicles to create a reservoir. Tape
stripping of the stratum corneum from the skin is a relatively non-invasive and useful
technique used for bioavailability and dermatopharmacokinetic studies of topically-applied
drugs.
As model compound the sunscreening agent 3-(4-Methylbenzylidene)camphor (4-MBC) was
chosen because of its high absorbance potential (A 930-990 at 299 nm) and its high affinity
to the SC which may be inferred by its low solubility in water (0.00013 g in 100 mL). To be
able to conduct SC measurements directly on a tape by UV/VIS spectroscopy, a Perkin
Elmer UV/VIS spectrophotometer was modified providing a uniform measurement area of
1 cm2 to measure the absorbance through the sample as a result of light scattering by the SC
on the tapes.
A spectroscopic UV/VIS method determining the model compound 4-MBC and SC
simultaneously in skin tape strips was then developed directly on tape strips and validated
according to the International Conference on Harmonization of Technical Requirements for
Registration of Pharmaceuticals for Human Use (ICH) guidelines. For SC measurements this
technique has distinct advantages in terms of facile sample handling procedures when
compared with previously described methods such as tape strip weighing, protein
determination and extraction procedures which are laborious and are susceptible to analytical
artifacts. The validation parameters of the UV/VIS method were compared to those of a
conventional solvent extraction/HPLC method. The data of the two analytical methods were
found to be equally accurate and precise in determining sunscreen abundance in the
corneocyte layers removed by tape stripping. However, the direct spectrophotometric
technique obviates the need for any tape extraction process prior to analysis of 4-MBC and
SC, and is sensitive enough for the accurate determination of low analyte concentrations on
the tape strips since there is no loss of analyte in the sample handling protocol.
The validation was further completed by comparing the data of an in vivo tape stripping
experiment measured by the UV/VIS method and the solvent extraction/HPLC method. For
this purpose the tape stripping procedure has been optimized and standardized using a strict
protocol to minimize the variability of the tape stripping data. The 4-MBC delivery from a 2%
and 4% 4-MBC emulsions was then investigated in vivo using five human volunteers. A dose
of 2 mg/cm2 was applied to the volar forearm for one hour. The tapes were analyzed be the
new UV/VIS method by determining simultaneously the 4-MBC abundance and the relative
density of SC. The tapes were then extracted and the 4-MBC concentrations obtained by
direct spectrophotometric quantification were verified by HPLC. The linear correlation factor
was 0.974 and statistical analysis confirmed that there was no statistical difference (p>0.05)
between the two analytical procedures. The total mass of compound delivered from a vehicle
was calculated by summating the 4-MBC abundance determined per tape and area of tape
strip. Thereby, the first tape was always discarded. The results showed a statistically
significant (p<0.05) influence of the dosing strength (2% vs 4%) of the formulations on the
penetration of 4-MBC.
This investigation revealed that the newly developed spectroscopic method is rapid, precise
and as accurate as a conventional HPLC method for the in vivo determination of
sunscreening agent delivery from topical applied formulations.
Finally, the delivery of 4-MBC from several representative vehicles was compared using the
direct spectrophotometric determination method and according to GCP procedures. To be
able to detect vehicle effects on the penetration of 4-MBC into the SC, all vehicles used in the
study were saturated with 4-MBC.
In the first experiment the influence of propylene glycol and mineral oil and of their application
time on 4-MBC penetration was investigated in five volunteers. Although the solubility of
4-MBC was approximately 3.5 times lower in propylene glycol than in mineral oil, the 4-MBC
delivery into the SC was significantly higher from propylene glycol. This observation can be
explained by the ability of propylene glycol to decrease SC permeability. The number of tape
strips required to completely remove the SC in the presence of the propylene glycol was
significantly lower revealing a loosening of corneocytes and thus a facilitated penetration of
4-MBC into the SC. The application time showed no influence on the 4-MBC penetration.
In a further experiment, PEG 400 vehicles containing 0%, 10% and 50% Transcutol®CG were
evaluated in ten subjects for their ability to create a reservoir of 4-MBC in the skin. This was
evaluated seven hours post vehicle application. The solubility of 4-MBC increased with
increasing Transcutol®CG concentration in the vehicle. The 4-MBC delivery from the three
vehicles was proportional to the 4-MBC concentration in the vehicle. The vehicles containing
0% and 10% Transcutol®CG showed no difference in the 4-MBC amount measured in the SC
seven hours post application compared to the 4-MBC amount measured in the SC one hour
post application. For PEG 400 containing 50% Transcutol®CG the 4-MBC amount remaining
in the SC seven hours post application was smaller than one hour post application.
This study clearly demonstrated that this methodology is, therefore, capable of detecting
differences in the extent of reservoir formation of the sunscreen when applied to the skin in
different delivery vehicles.