Depilation induced anagen as a model to study hedgehog pathway antagonist IPI-926: Implications for biomarker development

2827 The Hedgehog cell signaling pathway is normally active during embryonic development and plays a critical role in controlling the growth and differentiation of pluripotent progenitor cells in many tissues, including the skin. The Sonic Hedgehog ligand (SHH) is a key regulator of hair follicle growth and cycling and serves as a switch between the resting (telogen) and the growth (anagen) stage of the hair cycle. The hair cycle in the C57BL/6 mouse has been extensively characterized and post depilation provides a highly standardized model in which to explore Hedgehog pathway biology. Using 7 week old C57BL/6 mice which are in the telogen phase of the hair cycle, anagen and subsequently hair re-growth were initiated via chemical depilation with Nair. As a result of anagen initiation, the hair follicle cycle is synchronized allowing for reproducible measurement of Hedgehog target gene expression over time in the skin. Shaved skin, containing hair follicles which remain in telogen, serves as a control. From days 6 through 14 post depilation, corresponding to mid to late anagen, Hedgehog target gene expression was measured by RT-PCR. The Hedgehog signaling pathway is active during anagen as SHH, Gli-1, Gli-2, HHIP and PTCH-1 were all up-regulated in the depilated but not the shaved skin samples. The highest level of Hedgehog target gene expression was noted on day 10 post depilation. Smoothened (SMO) levels remained constant throughout the study and did not differ between telogen and anagen. Having established the up-regulation of Hedgehog target genes during depilation induced anagen, our novel SMO antagonist IPI-926 was evaluated in this model. IPI-926 is an orally bioavailable cyclopamine derivative with favorable PK properties and is a potent inhibitor of the Hedgehog pathway. To test compound activity, a single oral dose of either vehicle or IPI-926 was administered on day 10 post depilation. At various time points post dose, both shaved and depilated skin samples were collected to evaluate gene expression. In a dose-proportional manner, IPI-926 completely inhibited GLI-1 up-regulation in the depilated skin as early as 8 hours post dose and maintains complete inhibition out to 48 hours. IPI-926 also inhibits GLI-1 expression induced as a result of natural anagen, which occurs at approximately 12 weeks of age. Of note, after hair follicle synchronization, onset of melanogenesis occurs on Day 9 post depilation and hair re-growth by day 14. Daily administration of IPI-926 or BID administration of the SHH blocking antibody, 5E1, both inhibited hair re-growth post depilation. Collectively this data suggests that Hedgehog target gene expression and regulation in the hair follicle offers an attractive and obtainable biomarker for Hedgehog pathway antagonists under evaluation in the clinic as anti-cancer agents.