There is some concern that the effects of Sandalore® require exposure to relatively high concentrations for 6 days. Under those circumstances can the authors confidently rule out non- specific effects, possibly even membrane permeability to this volatile compound that may be at work?
Thanks for raising this important point. In our previous study (Busse et al., 2014), we had carefully addressed the specificity and receptor dependence of Sandalore® effects at the relatively high concentration tested in human HaCaT cells and primary human epidermal keratinocytes. For example, we had demonstrated that after 5 days of Sandalore® stimulation in the presence of specific antagonist of OR2AT4 or of relevant signalling pathway blockers of adenylyl cyclase (MDL-12.330A or SQ-22536) and CNG channel (L-cis Diltiazem), the Sandalore® effect in HaCaT cells or primary human epidermal keratinocytes was completely blocked, without any effects on cell morphology or any other cell biology parameter evaluated. In addition, the co-application of the specific OR2AT4 antagonist Phenirat® with Sandalore® (1:1, 500 mM) completely blocked Sandalore® -induced calcium signals in HaCaT cells indicating that the effects of Sandalore® are based on the activation of OR2AT4 inkeratinocytes. While these cell culture data demonstrated OR2AT4-dependency of the Sandalore® effects on human keratinocytes in vitro (for details, see Busse et al., 2014), we agree that even the Phenirat® + Sandalore® co-stimulation experiment (which showed that Sandalore® effects on the HF can be partially antagonized by Phenirat® only in the presence of Sandalore® ) does not “confidently rule out” that OR2AT4-INDEPENDENT effects, possibly including even changes in membrane permeability to this volatile compound, might also have been at work, given the relatively high concentration of Sandalore® and long duration ofHF organ culture. That is exactly why we went one key step further and silenced OR2AT4 in organ-cultured HFs (incidentally, to the best of our knowledge, this is the first time that any olfactory receptor has been successfully knocked-down in any human [mini-] organ). This clearly showed that – notably in the presence of high-dose Sandalore® ! – OR2AT4 knock-down exerted the opposite effects of agonist treatment alone and profoundly inhibited both, hair growth (i.e. OR2AT4 siRNA shortened anagen and prematurely induced catagen and up-regulated hair matrix keratinocyte apoptosis) and intrafollicular IGF-1 production. However, to alert readers to the important caveat raised by the expert reviewer, we have complemented the revised Discussion with the following supplementary text(supplementary text 5): “Previously, we had addressed the specificity and receptor dependence of Sandalore® effects at the relatively high concentration tested here in human HaCaT cells and primary human epidermal keratinocytes (Busse et al 2014). This had demonstrated that after 5 days of Sandalore® stimulation in the presence of specific antagonist of OR2AT4 or of relevant signalling pathway blockers (adenylyl cyclase inhibition by MDL-12.330A or SQ-22536; CNG channel inhibition by L-cis Diltiazem), the Sandalore® effect in HaCaT cells or primary human epidermal keratinocytes was completely blocked, without any effects on cell morphology or any other cell biology parameter evaluated. In addition, co-application of the specific OR2AT4 antagonist Phenirat® with Sandalore® (1:1, 500 mM) completely blocked Sandalore® -induced calcium signals in HaCaT cells (Busse et al. 2014), indicating that the effects of Sandalore® are based on the activation of OR2AT4 in keratinocytes. Most importantly, silencing OR2AT4 in organ-cultured HFs clearly showed that – notably in the presence of high-dose Sandalore® – OR2AT4 knock-down exerted the opposite effects of agonist treatment alone and profoundly inhibited both, hair growth (i.e. OR2AT4 siRNA shortened anagen and prematurely induced catagen and upregulated hair matrix keratinocyte apoptosis) and intrafollicularIGF-1 production. “