Measuring skin delivery

Unfortunately, this is not always the case predominantly due to the lack of skin delivery of the active ingredient. In order for such an ingredient to exert its activity, it will need to be delivered to the right site in the substrate (most often the skin) at the right concentration and for the correct period of time. Moreover, attention will need to be paid to the active ingredient itself to ensure that it is not metabolised during skin passage.

But awareness of these facts alone is not enough. In order to assess whether sufficient delivery has been achieved, one needs to be able to measure skin delivery. In this short article, some basic principles of measuring skin absorption will be described, ranging from in vitro and in vivo methods to the differences between animal and human skin for skin penetration. It ends with some thoughts for the requirements of new techniques for the assessment of dermal delivery.

In vitro methods

Most skin penetration work is done in in vitro set-ups, using either the static Franz diffusion cell system1 (schematically depicted in Fig. 1) or the Bronaugh flowthrough cell system2 (shown in Fig. 2).

In both these techniques, a piece of skin is placed in the skin penetration cell and a receptor fluid is placed underneath the skin in contact with the tissue. Either all receptor fluid is collected continuously (Bronaugh flow-through cells) or samples thereof are taken periodically (Franz diffusion cells). Bronaugh flow-through cells have a much smaller receptor compartment, so that the time delay between the moment that an active ingredient reaches the receptor compartment and when it is being sampled is as short as possible. It has been shown experimentally that the volume needs to be replaced at least roughly 7 times per hour for this not to create a problem.3 As receptor fluid flowrates are typically 1 ml/h, the volume of the receptor compartment should therefore be 143 µl or smaller. Increasing the flow or inserting small glass beads or magnetic stirrers may help to ensure that a sufficient replacement factor (i.e. flow rate/receptor compartment volume) is achieved.

In Franz diffusion cells, however, the receptor volumes are deliberately large in order to avoid the build-up of a significant concentration of the active ingredient in the receptor compartment. This would reduce transdermal skin penetration because it reduces the concentration gradient over the skin.4

It is often asked whether the use of Franz or Bronaugh diffusion cells makes any difference at all. Having worked with both of them, I do not really favour one above the other. But analytical capabilities for the determination of the active ingredient may constitute a good argument for selecting the Bronaugh cell over the Franz cell because the latter tends to have lower concentrations due to the larger receptor compartment. Moreover, because the Bronaugh flow-through cell system is used in combination with a fraction collector, samples can be collected throughout the night. More recent models of the Franz diffusion cell system also allow for automatic sampling.

One of the mistakes often made in in vitro skin penetration studies is to express the results as a percentage of the dose applied. This is wrong because skin penetration increases with the concentration of the active ingredient in the formulation until a certain concentration is reached after which skin penetration remains constant. Above this level, percentage penetration goes down whereas absolute penetration remains constant. Therefore, transdermal flux results should always be expressed as µg/cm2.h and dermal delivery results as µg/cm2 or – if the exact volume is known – as a concentration in µg/cm3.

In vivo methods

In in vivo skin absorption studies, an active ingredient is applied onto the skin of volunteers and the extent of skin penetration is assessed via determination of the active ingredient in various samples. Under very strict conditions, the use of 14C- or 3H-labelled tracer molecules is allowed which facilitates the analysis of penetrated and non-penetrated material. Skin penetration is the sum of the amounts of active ingredient retrieved in tape strips (levels in the superficial layers of the stratum corneum), urine, faeces, sweat and/or expired air. The applied dose and the non-absorbed dose should also be determined as this allows the calculation of the so-called total recovery (the sum of all the samples relative to the applied dose).5 Such values should be between 95-102% of the dose in order to be seen as reliable.

The main problems of in vivo studies are the high costs and the low concentrations of active ingredients due to large sample volumes, but the answer is – of course – of much more relevance than that of in vitro studies.

Animal skin versus human skin

Of course, results obtained on human skin will be of more relevance than those obtained on animal skin. In cosmetics, most products are applied on healthy skin but care should be taken to use parts of the human body on which the product will ultimately be applied. This precaution is necessary because skin permeability varies with body site, the more permeable sites being the scrotum and the ear lobe.4 The face is a part of the human body to which cosmetic products are frequently applied but it should be realised that skin permeability even varies within the face as recently indirectly described by Distante et al who reported the sitevariance of TEWL values on the face.6

If given no other choice than to use animal skin, pigskin is strongly recommended. The structure and characteristics of pig ear skin are most similar to that of human skin.7 Particularly in the past, skin penetration experiments predominantly used mouse, rat or rabbit skin. When using such historic data it should be realised that rodent skin is about a factor 4 more permeable than human skin, but also that this may change considerably between chemicals.

Need for new nondestructive methods

The use of in vitro cells allows a determination of transdermal delivery in a non-destructive way and this can therefore be continuous. Determining dermal delivery per se is not more difficult but, unfortunately, it is a destructive method. After all, the skin needs to be extracted or dissolved to allow the determination of the amount of active ingredient within the dermal layers. In order to study this more extensively as a function of formulation, time and concentration, new innovative techniques to assess dermal delivery should be developed that are nondestructive.

If they could be performed in vivo, it would be even better. Whereas the previously proposed methods of confocal fluorescence spectroscopy8 and of cutaneous microdialysis9 were in fact still invasive, the recently described confocal Raman microspectroscopy technique10 may truly be a great step in the right direction to the design of new skin delivery systems that allow continuous quantification of dermal delivery. Now the resolution of this technique has been improved, it can be used as a screening method for cosmetic formulations in vivo on the relevant application sites under realistic conditions. This method will greatly help in the development of new, truly active cosmetic products.

A much less expensive, but more cumbersome, technique to measure dermal delivery is the use of tape-strips in combination with transepidermal water loss measurements to predict dermal concentrations, as developed by Herkenne et al,11 although it can still be argued that tape-stripping is invasive and destructive.

Conclusions

As cosmetic science is transforming its products from containing active ingredients to being active products, it will be increasingly important for the cosmetic scientist to understand how to measure transdermal and dermal delivery. Some of the more important aspects have been discussed here, focusing on the differences between in vitro and in vivo skin penetration techniques, animal and human skin, as well as the need for more non-destructive methods to assess dermal delivery. Without these techniques routinely being used in product development, we will not necessarily progress to truly active cosmetic products.

References

1 Franz T.J. Percutaneous absorption. On the relevance of in vitro data. J. Invest. Dermatol., 54 (1975) 399-404.

2 Bronaugh R.L., and Stewart R.F. Methods for percutaneous absorption studies. IV. The flowthrough diffusion cell. J. Pharm. Sci., 74 (1985) 64-67.

3 Wiechers J.W. Unpublished data.

4 Wiechers J.W. The barrier function of the skin in relation to percutaneous absorption of drugs. Pharm. Wkbl. Sci. Ed., 11 (1989) 185-198.

5 Bucks D.A.W., McMaster J.R., Maibach H.I., and Guy R.H. Bioavailability of topically administered steroids: A ‘mass balance’ technique. J. Invest. Dermatol., 91 (1988) 29-33.

6 Distante F., Rigano L., D’Agostino R., Bonfigli A., and Berardesca E. Intra- and inter-individual differences in sensitive skin. Cosmet. Toilet., 117 (2002) (7) 39-46.

7 Jacobi U., Kaiser M., Toll R., Mangelsdorf S., Audring H., Otberg N., Wolfram S., and Lademann, J. Porcine ear skin: an in vitro model for human skin. Skin Res. Technol., 13 (2007) 19-24.

8 Cullander C., and Guy R.H. Visualising the pathways of iontophoretic current flow in real time with laser-scanning confocal microscopy and the vibrating probe electrode, in: Scott R.C., Guy R.H., Hadgraft J., and Boddé H.E. (Eds.). Prediction of percutaneous penetration, Volume 2, IBC Technical Services Ltd, London (1991) 229-237.

9 Anderson C. Cutaneous microdialysis, in: Brain K.R., and Walters K.A. (Eds.). Prediction of percutaneous penetration, Volume 6a, STS Publishing, Cardiff (1998) 16.

10 Caspers P.J., Lucassen G.W., Carter E.A., Bruining H.A., and Puppels G.J. In vivo confocal Raman microspectroscopy of the skin: Noninvasive determination of molecular concentration profiles. J. Invest. Dermatol., 116 (2001) 434-442.

11 Herkenne C., Naik A., Kalia Y.N., Hadgraft J., and Guy R.H. Ibuprofen transport into and through skin from topical applied formulations: In vitro/in vivo comparison. J. Invest. Dermatol., 127 (2007) 135-142.

SOURCE OF ARTICLE
This article is based on Chapter 2 “Measuring Skin Delivery” of the book Science and Applications of Skin Delivery Systems, edited by Johann W. Wiechers, Allured Publishing Corp. (Carol Stream, Illinois, USA), 2008, 552 pages, ISBN-10: 1-932633-37-5, ISBN-13: 978-1-932633-37-5. This book encompasses 28 chapters subdivided in sections on measuring skin delivery, skin delivery from emulsions, encapsulation techniques, electrical ways to enhance skin delivery, special delivery routes and future perspectives. The book is available via the publisher’s website: http://allured.stores.yahoo.net/scandapofskd.html Copies will be available for purchase during the Personal Care and Household Ingredients show being held in Shanghai in March 2008 and in-cosmetics Europe in Amsterdam, The Netherlands, in April 2008.            

PROFESSOR DR JOHANN W. WIECHERS
After earning his Ph.D. in skin penetration enhancement from the University of Groningen, The Netherlands, Johann Wiechers has spent the past 18 years establishing himself in the field of cosmetic science. He has worked for companies including Unilever Research and Uniqema as well as become a visiting professor at the University of London, School of Pharmacy. On 1 July 2007, Johann became technical advisor for Allured Publishing Corporation, serving as advisor for Cosmetics & Toiletries magazine, as well as the book division. In July 2007 he opened an independent consultancy business in cosmetic science and in September 2007 he became the president of IFSCC.