Measurement of Bioavailability: Measurement of Absorption Through Skin In Vitro

Sharon A.M. Hotchkiss1

1 Imperial College School of Medicine, London
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 5.1
DOI:  10.1002/0471140856.tx0501s00
Online Posting Date:  May, 2001
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Abstract

New therapeutic compounds intended for use on the skin or for delivery through application to the skin and agrochemicals, whose use may result in skin exposure, must be tested for bioavailability as the result of absorption. This unit contains a protocol for measuring skin absorption in vitro using the diffusion cell skin absorption method (SAM), which can be used to measure percutaneous absorption after topical application. Usually a radiolabeled compound is used, but if a suitable specific assay is available, nonradioactive compounds may be tested. The procedure is applicable to skin from a variety of species.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • Receptor fluid: HHBSS (see recipe) or tissue culture medium
  • Skin sample (human or animal)
  • 70% and 100% (v/v) ethanol
  • Enzyme solution (e.g., Dispase, Sigma; optional)
  • Test compound (usually radiolabeled; >98% pure)
  • Vehicle: e.g., dimethyl sulfoxide (DMSO), ethanol, or water
  • Scintillation fluid (e.g., Ecoscint, National Diagnostics)
  • 2% (v/v) aqueous soap solution (liquid hand soap, e.g., Labguard microbial hand soap, Day Impex; optional)
  • Decontamination liquid (e.g., Decon from Decon Laboratories)
  • Skin digest solution (see recipe)
  • 4.4 M nitric acid (HNO 3)
  • Skin absorption model (SAM) system (Fig. ), including:
    •  Fraction collector (Crown Glass)
    •  Flow‐through diffusion cells (Crown Glass)
    •  Receptor fluid reservoir
    •  Heated water circulator set at 36°C (Churchill)
    •  Peristaltic pump (Watson‐Marlow)
    •  Teflon septa and caps (for occluded skin experiments; Crown Glass)
  • 20‐ml plastic and glass scintillation vials
  • Heated mantle blocks (Crown Glass)
  • Vacuum filtration system with 0.45‐µm filter
  • Dermatome (e.g., Padgett electric model; optional)
  • Surgical tools (scissors, scalpel, forceps)
  • Rubber mat or cork board
  • Circular steel cutter with 1.7‐cm‐diameter circular cutting edge (custom made at the Imperial College School of Medicine)
  • Hammer
  • 0.63‐µm‐bore orange‐white marprene or polyvinyl chloride (PVC) tubing (Watson‐Marlow)
  • 10‐µl Hamilton syringe and blunt‐ended needle
  • Gauze swabs cut into ∼1.5‐cm2 pieces
  • 70°C shaking water bath
  • Spreadsheet software (e.g., Microsoft Excel)
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Figures

Videos

Literature Cited

Literature Cited
   Bartek, M.J., LaBudde, J.A., and Maibach, H.I. 1972. Skin permeability in vivo:Comparison in rat, rabbit, pig and man. J. Investig. Dermatol. 58:114‐123.
   Beckley‐Kartey, S.A.J., Hotchkiss, S.A.M., and Capel, M. 1997. Comparative in vitro skin absorption and metabolism of coumarin (1,2‐benzopyrone) in human, rat and mouse. Toxicol. Appl. Pharmacol. 145:34‐42.
   Blank, I.H., Scheuplein, R.J., and Macfarlane, D.J. 1967. Mechanisms of percutaneous absorption. iii. The effect of temperature on the transport of non electrolytes across the skin. J. Investig. Dermatol. 49:582‐588.
   Bronaugh, R.L. and Stewart, R.F. 1985. Methods for in vitro percutaneous absorption studies. IV. The flow‐through diffusion cell. J. Pharm. Sci. 74:64‐67.
   Bronaugh, R.L., Stewart, R.F., Congdon, E.R., and Giles, A.L. Jr. 1982. Methods for in vitro percutaneous absorption studies. I. Comparison with in vivo results. Toxicol. Appl. Pharmacol. 62:474‐480.
   Bucks, D.A.W., Maibach, H.I., and Guy, R. 1989. Occlusion does not uniformly enhance absorption in vivo. In Percutaneous Absorption. Mechanisms, Methodology, Drug Delivery. (R.L. Bronaugh and H.I. Maibach, eds.) pp. 13‐27. Marcel Dekker, New York.
   de Lange, J., van Eck, G.R., Bruijnzeal, P.L.B., and Elliott, G.R. 1994. The rate of percutaneous permeation of xylene, measured using the perfused pig ear model, is dependent on the effective protein concentration of the perfusing medium. Toxicol. Appl. Pharmacol. 127:298‐305.
   Frantz, S.W., Dittenber, D.A., Eisenbrandt, D.L., and Watanabe, P.G. 1990. Evaluation of a flow through diffusion in vitro skin penetration chamber method using acetone‐deposited organic solids. J. Cut. Oc. Toxicol. 9:277‐299.
   Franz, T.J. 1975. Percutaneous absorption. On the relevance of in vitro data. J. Investig. Dermatol. 64:190‐195.
   Hotchkiss, S.A.M. 1995. Skin absorption of occupational chemicals. In Handbook of Occupational Hygiene, installment 46, pp.1‐38. Croner, Surrey, U.K.
   Hotchkiss, S.A.M. 1998. Dermal metabolism. In Dermal absorption & toxicity assessment (M.S. Roberts and K.A. Walters, eds.) pp.43‐101. Marcel Dekker, New York.
   Hotchkiss, S.A., Chidgey, M.A.J., Rose, S., and Caldwell, J. 1990. Percutaneous absorption of benzyl acetate through rat skin in vitro. 1. Validation of an in vitro model against in vivo data. Food Chem. Toxicol. 28:443‐447.
   Hotchkiss, S.A.M., Hewitt, P., Caldwell, J., Chen, W.L., and Rowe, R.R. 1992a. Percutaneous absorption of nicotinic acid, phenol, benzoic acid and triclopyr butoxyethyl ester through rat and human skin in vitro. Further validation of an in vitro model by comparison with in vivo data. Food Chem. Toxicol. 30:891‐899.
   Hotchkiss, S.A.M., Miller, J.M., and Caldwell, J. 1992b. Percutaneous absorption of benzyl acetate through rat skin in vitro. 2. Effect of vehicle and occlusion. Food Chem. Toxicol. 30:145‐153.
   Hotchkiss, S.A.M., Hewitt, P., and Caldwell, J. 1993. Percutaneous absorption of 4,4′‐methylene‐bis‐(2‐chloroaniline) and 4,4′‐methylene dianiline through rat and human skin in vitro. Toxic. In Vitro 7:141‐148.
   Kao, J. and Hall, J. 1987. Skin absorption and cutaneous first‐pass metabolism of topical steroids: In vitro studies with mouse skin in organ culture. J. Pharmacol. Exp. Ther. 241:482‐487.
   Kao, J., Hall, J., and Helman, G. 1988. In vitro percutaneous absorption in mouse skin: Influence of skin appendages. Toxicol. Appl. Pharmacol. 94:93‐103.
   Kreidstein, M.L., Pang, C.Y., Levine, R.H., and Knowlton, R.J. 1991. The isolated perfused human skin flap:Design, perfusion technique, metabolism and vascular reactivity. Plast. Reconstr. Surg. 87:741‐749.
   Maibach, H.I., Feldmann, R.J., Milby, T.H., and Serat, W.F. 1971. Regional variation in percutaneous penetration in man. Arch. Environ. Health 23:208‐211.
   Mint, A., Hotchkiss, S.A.M., and Caldwell, J. 1994. Percutaneous absorption of diethyl phthalate through rat and human skin in vitro. Toxicol. In Vitro 8:251‐256.
   Potts, R.O., McNeil, S.C., Desbonnet, C.R., and Wakshull, E. 1989. Transdermal drug transport and metabolism. II. The role of competing kinetics events. Pharm. Res. 6:119‐124.
   Riviere, J.E., Bowman, K.F., and Monterio‐Riviere, N.A. 1986. The isolated perfused porcine skin flap (IPPSF). 1. A novel in vitro model for percutaneous absorption and cutaneous toxicity studies. Fundam. Appl. Toxicol. 7:444‐453.
   Rougier, A., Dupuis, D., Lotte, C., and Roguet, R. 1985. The measurement of the stratum corneum reservoir. A predictive model for in vivo percutaneous absorption studies:Influence of application time. J. Investig. Dermatol. 84:660‐666.
   Scheuplein, R.J. and Bronaugh, R.L. 1983. Percutaneous absorption. In Biochemistry and Physiology of the Skin (L.A. Goldsmith, ed.) pp. 1255‐1295. Oxford University Press, Oxford.
   Scott, R.C., Corrigan, M.A., Smith, F., and Mason, H. 1991. The influence of skin structure on permeability: An intersite and interspecies comparison with hydrophobic penetrants. J. Investig. Dermatol. 96:921‐925.
   Storm, J.E., Collier, S.W., Stewart, R.F., and Bronaugh, R.L. 1990. Metabolism of xenobiotics during percutaneous absorption: Role of absorption rate and cutaneous enzyme activity. Fundam. Appl. Toxicol. 15:132‐141.
   Suber, C., Wilhelm, K.P., and Maibach, H.I. 1991. In vitro skin pharmacokinetics of acitretin:Percutaneous absorption studies in intact and modified skin from three different species using different receptor solutions. J. Pharm. Pharmacol. 43:836‐840.
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