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A rapid method for measuring drug enrichment in epidermis

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Summary

A rapid and simple method is described for measuring the enrichment of small molecules in epidermal tissue. To measure such an enrichment, a small tissue sample (2–10 mg) is allowed to equilibrate with a buffered solution of a labelled substance for periods of 12–36 h. The concentration of the radioactive molecule in the tissue is measured as a decrease of radioactivity in the solution. Concentration measurements in the tissue itself can be performed, but are not required to detect enrichment in the tissue or to assess its magnitude.

The specific density of appendage free human epidermis has been determined and was found to be 1.20 g/cm3. Using this value, tissue weight can be translated into volume and concentration changes in the solution can be recalculated to yield the concentration of the substance in the tissue itself. Close agreement was found between the calculated tissue concentration and the values actually measured, following digestion of the epidermis with NaOH and measuring the activity in the tissue digest.

The enrichment of five substances in human epidermis was measured: α-estradiol, thiopyronine, 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP), and theophylline. Of these substances, the first four are concentrated by human epidermis and the concentrations reached within the tissue are 10–500 times higher than the concentration of the same substance in the surrounding buffer. The enrichment data has been analysed in an attempt to distinguish between reversible affinity binding to specific tissue sites and partitioning of the substances between buffer and tissue components (lipids, membranes, etc.). In the case of thiopyronine and 8-MOP, reversible binding is indicated with dissociation constants of 10-7 M and 10-5 M, respectively, while partitioning distribution could account for the behavior of 5-MOP and α-estradiol. The method can be used either as a rapid screening method or as a quantitative analysis for the characterization of tissue enrichment with specific drugs.

Zusammenfassung

In dieser Arbeit wird eine einfache Methode zur Messung der Anreicherung von verschiedenen Substanzen im epidermalen Gewebe beschrieben. Die Gewebeprobe (2–10 mg) wird in einer physiologischen Lösung mit der radioaktivmarkierten Substanz bis zum Gleichgewicht inkubiert (12–36 h). Die Radioaktivität der Lösung wird vor und nach der Inkubation gemenssen. Die Differenz zwischen den beiden Messungen wird als Maß für die Anreicherung der Substanz im Gewebe genommen.

Unter Berücksichtigung der spezifischen dichte der Epidermis, die bei Vorversuchen an isolierter menschlicher Epidermis als 1,2 g/cm3 bestimmt wurde, wird die Konzentration der Substanz in der Epidermis berechnet. Die nach dieser indirekten Methode berechnete Konzentration stimmt mit der tatsächlich gefundenen Konzentration der Substanz in der Epidermis nach alkalischer Hydrolyse überein.

Die Anreicherung im epidermalen Gewebe wurde an alpha-Östradiol, Thiopyronin, 5-MOP, 8-MOP und Theophyllin untersucht. Außer bei Theophyllin wurde bei allen anderen untersuchten Substanzen eine 10–500 fach höhere Konzentration im Gewebe gefunden.

Die Analyse der Ergebnisse, um eine reversible Bindung von einer Verteilung der Substanz zwischen den Zellkomponenten zu unterscheiden, zeigte sich im Falle von Thiopyronin und 8-MOP eine reversible Bindung mit einer Dissoziationskostanten von 10-7 und 10-5, hinsichtlich von 5-MOP und alpha-Östradiol eine partielle Verteilung.

Diese Methode kann als eine schnelle Screening-Methode oder als eine quantitative Analyse zur Charkterisierung der Anreicherung von Substanzen im Gewebe angewendet werden.

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This work was supported by the Volkswagen Foundation, Hannover, Federal Republic of Germany

This work is part of the Ph. D. thesis to be submitted by A. M. to the Technical University of Berlin

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Artuc, M., Reinhold, C., Stüttgen, G. et al. A rapid method for measuring drug enrichment in epidermis. Arch Dermatol Res 268, 129–140 (1980). https://doi.org/10.1007/BF00403797

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  • DOI: https://doi.org/10.1007/BF00403797

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