Plucked human hair as a tissue in which to assess pharmacodynamic end points during drug development studies

Abstract

We have demonstrated the feasibility of detecting and quantifying six cell-cycle-related nuclear markers (Ki67, pRb, p27, phosphop27 (phosphorylated p27), phospho-pRb (phosphorylated pRb), phospho-HH3 (phosphorylated histone H3)) in plucked human scalp and eyebrow hair. Estimates of the proportion of plucked hairs that are lost or damaged during processing plus the intra-and intersubject variability of each nuclear marker with these techniques are provided to inform sizing decisions for intervention studies with drugs potentially impacting on these markers in the future. (Moll, 1996;Gho et al, 2004); they are therefore potentially attractive as an easily accessible tissue in which to assess the pharmacodynamic (PD) effects of drugs that interfere with cell proliferation. Skin biopsies have already been used to similar ends during the development of a number of agents targeted against the epidermal growth factor receptor (Albanell et al, 2002;Malik et al, 2003;Salazar et al, 2004;Vanhoefer et al, 2004). Plucked hairs would offer certain advantages over skin biopsies for these purposes including improved tolerability, a greater potential for multiple time-point analyses and the possibility of a higher signal within hair follicles for certain key biomarkers compared to the cells of the epidermis (Albanell et al, 2002). We have developed a technique for quantifying cell-cycle-related proteins within the sheath cells of plucked human hair. MATERIALS AND METHODS Trial designIn all, 12 healthy Caucasian males, within the age range 18 -45 years, and with scalp hair greater than or equal to 5 mm in length, were recruited. Following thorough combing to remove any loose hairs, five suitable hairs with visible bulbs were plucked from each of the left eyebrow, right eyebrow and four different scalp sites using a pair of blunt-nosed forceps. Each set of five hairs was trimmed with scissors to a workable length (approximately 1 cm), retaining the bulb end of each hair, and then immersed in prelabelled 1.5 ml microcentrifuge tubes containing 1 ml 100% acetone at 41C. Plucked hairs without visible bulbs were discarded and not processed. Immunohistochemistry (IHC) and signal quantificationHair processing Following acetone fixation for 10 min, each hair was air-dried for 10 min and then placed, in batches of five from each sampling site, in 1 ml of 0.5 M, pH 7.6, Tris-buffered saline.Hair IHC In our hands, it was not possible to paraffin embed and then section the hairs for later staining without producing significant loss or damage of the fragile material; we therefore developed techniques based on staining intact hairs followed by rigid epoxy resin embedding and then sectioning.Briefly, a peroxidase block (5 min at room temperature), followed by a nonspecific protein block (30 min at room temperature), was employed on the intact hairs. A primary antibody, or buffer for negative controls, was then applied (antiKi67 at 1 : 100 for 1 h at room temperature, DAKO