) under the luminescence setting. Viability at each motesanib or imatinib concentration was expressed as a percentage of the vehicle control (0.2% DMSO). Results In Vitro Inhibition of Wild-Type Kit by Motesanib Motesanib potently inhibited SCF-find more induced autophosphorylation of Kit in CHO cells stably transfected with the wild-type KIT gene (IC50 = 36 nM). In comparison,
imatinib inhibited wild-type Kit with an IC50 of 165 nM. Inhibition of Wild-Type Kit Activity in Mice by Motesanib Hair depigmentation was used as a surrogate marker to assess the ability of motesanib to inhibit Kit activity in vivo . Following depilation, female C57B6 mice were administered either 75 mg/kg motesanib (n = 8) or vehicle (n = 8) twice daily for 21 days. In mice receiving motesanib, hair regrowth was markedly depigmented compared with mice receiving SN-38 chemical structure Akt inhibitor drugs vehicle (Figure 1). This effect was reversible. Following the cessation of motesanib treatment on day 21, the mice were depilated again on day 28. There was no apparent depigmentation of regrown hair on day 35. Similar results were obtained in male mice (data not shown). Figure 1 Effect of treatment with motesanib or vehicle on hair depigmentation, a surrogate marker of Kit activity , in female C57B6 mice. Anesthetized animals were depilated and immediately treated with
either vehicle (water; left panels) or motesanib 75 mg/kg BID (right panels) for 21 days. On day 21, hair depigmentation was assessed. Depilation was repeated on day 28 and hair depigmentation was again assessed on day 35. Representative images from each treatment group for the day-21 and day-35 time points are shown. BID = twice daily. Characterization of Kit Mutants Figure 2 summarizes the results from the autophosphorylation experiments using CHO cells stably transfected with the wild-type KIT gene or various KIT mutant genes. Tyrosine phosphorylation of wild-type Kit was
dose-dependent, with the greatest intensity of autophosphorylation occurring after a 30 minute incubation of the cells with 300 ng/mL of SCF. In contrast, tyrosine phosphorylation of activated Etomidate Kit mutants occurred in the absence of SCF with no further phosphorylation induced by treatment with SCF. Figure 2 Effect of stem cell factor (SCF) treatment on tyrosine phosphorylation of wild-type Kit and mutant Kit isoforms stably expressed in Chinese hamster ovary cells. Chinese hamster ovary cells stably transfected with wild-type (WT) or mutant KIT isoforms were stimulated with single serial dilutions of stem cell factor, and Kit phosphorylation was assessed. For mutant Kit isoforms, data are expressed as the percentage of vehicle control. For wild-type Kit, data are expressed as the percentage of phosphorylation observed following stimulation with 300 ng/mL SCF. The results of a single experiment are shown.