Research Articles
Influx and efflux of amphetamine and N‐acetylamphetamine in keratinocytes, pigmented melanocytes, and nonpigmented melanocytes

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Abstract

To establish an in vitro model of drug incorporation into hair and to elucidate the potential roles of hair cell selectivity and hair color in the incorporation of certain drugs into hair, the basic drug amphetamine and its nonbasic analog N‐acetylamphetamine (N‐AcAp) were analyzed for influx and efflux into and out of keratinocytes, pigmented melanocytes (PM), and nonpigmented melanocytes (NPM) as a model for incorporation and efflux of these drugs from hair cells. NPM were of the same melan‐a cell line as PM, but cultured in the presence of the tyrosinase inhibitor phenylthiocarbamide. Results show that PM take up large amounts of the basic drug amphetamine (levels of uptake dependent on melanin content), whereas keratinocytes and NPM take up only small amounts of amphetamine. None of the cells take up N‐AcAp above background levels. Interestingly, whereas keratinocytes and NPM quickly efflux most of the influxed drug, PM are slow to efflux and only efflux ∼65% of influxed drug, if efflux media is not refreshed. (If efflux media is periodically refreshed, PM will eventually redistribute essentially all influxed drug back into the media.) These results demonstrate that pigmented cells take up greater amounts of the basic drug amphetamine, and efflux it more slowly than nonpigmented cells. Also, these results are consistent with previous data for in vivo incorporation of amphetamine in animal hair. In combination with previous data, an overall comparison of the amphetamine and N‐AcAp incorporation data support a non‐diffusion mediated model for drug incorporation into hair cells.

Section snippets

INTRODUCTION

Testing for drugs of abuse in hair has become a widely accepted alternative to traditional urinalysis.1,2 Unfortunately, there are still a few potentially confounding variables involved in hair testing. Such variables include the role of environmental contamination in hair testing and adequacy of laboratory wash procedures designed to remove externally deposited drug, the role(s) of bleaching and/or other chemical treatments on the outcome of a hair test, and the role of hair pigment in drug

Chemicals and Reagents

N‐Acetylamphetamine (N‐AcAp) and N‐acetylamphetamine‐d3 (N‐AcAp‐d3) were synthesized from d‐(+)‐amphetamine sulfate according to the method of Halmekoski and Saarinen29 using acetic anhydride and deuterated acetic anhydride, respectively. The identity of N‐AcAp was verified by melting point, mass spectrometry, and nuclear magnetic resonance spectroscopy (NMR). The identity of N‐AcAp‐d3 was verified by melting point and mass spectrometry. Purity for these synthetic compounds was verified by

RESULTS

Time courses of influx and efflux were carried out so that both initial rates and uptake amounts at equilibrium could be approximated. Initial influx or efflux was always established very quickly (i.e., in a matter of a few min or less). Typical SRM chromatograms obtained from cell uptake/efflux experiments for the purpose of drug quantitation are shown in Figure 2. No smoothing algorithms or special chromatographic processing was performed. Drug‐to‐internal standard peak area ratios were used

DISCUSSION

Various models have been used that assess the potential hair color bias of specific drugs. These include drug–melanin binding studies,11, 12., 13., 14., 15., 16.,34., 35., 36. animal in vivo incorporation studies (specifically with black and white Long‐Evans rats),3,5,7,8,10,37 and human in vivo incorporation studies.6,37., 38., 39 Taken together, these data suggest that basic drugs with pKa values greater than physiologic pH will exhibit a hair color bias, whereas net neutral or acidic drugs

Acknowledgements

The authors express their sincere thanks to Simon Hill and Professor Dorothy Bennett of the Department of Anatomy, St. George's Hospital Medical School, London, for their provision of the melanocytes used in these studies. We also thank Cynthia Jorgensen and Dr. Gerald Krueger, of the Department of Dermatology at the University of Utah, for donating the keratinocytes used in these studies. The authors also thank Linda Schmidt, of the Department of Dermatology at the University of Utah, for her

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    Copyright © 2002 Wiley‐Liss, Inc. and the American Pharmaceutical Association. Published by Elsevier Inc. All rights reserved.