We thank the reviewer for this question. The concept of MACC1 inhibition was originally developed by our group for the treatment of MACC1 positive colorectal cancers and was recently published (Kobelt et al. Oncogene 2021). In this study we transferred our experience with MACC1 inhibition by selumetinib on AGE/S. Therefore, we chose the same doses for in vitro and in vivo experiments, and which was also used by other authors (Huynh et al. Mol Cancer Therapy. 2007). Further, we knew that the selected selumetinib concentrations of 10 µM has no cytotoxic effect in vitro and showed no reduction of migration in the MACC1 negative clones (FLO1-EV and OEshMACC1). Regarding the different dosages in the context of xenografted mice treatment in vivo and the clinically used dosages, the FDA recommends multiplying the dosage by a factor of 0.08 when transferring from mouse to human (Niar et al. Journal of basic and clinical pharmacy 2016). According to this calculation, the dosage of 50 mg selumetinib /kg mouse used would equal a dosage of 4 mg/kg in humans. The clinical dose of 75mg twice daily corresponds to a dose of 2x1mg per kg assuming a patient weight of 75kg. Using the aforementioned calculation, this dose equals then 12.5 mg/kg for treatment of mice. In our study, we refer again to the work of Huynh et al. Here, in which a dosage of 12 mg/kg selumetinib showed only a low level of effectiveness. At 100 mg/kg, a very good effectiveness was seen, however combined with an increased toxicity. Thus, we used in our study the dosage of 50 mg/kg as an effective dosage with low toxicity for the mice. Overall, we are considering here a pre-clinical model. The possibility of MEK1 inhibition to inhibit MACC1-induced effects in AGE/S was examined. Further, extended in vivo studies might then reveal the ideal dosage in vivo, being thereafter translated to use in humans. The use of trametinib is an interesting suggestion and might well be adapted to its use in forthcoming studies References: Kobelt, D., Perez-Hernandez, D., Fleuter, C., Dahlmann, M., Zincke, F., Smith, J., ... & Stein, U. (2021). The newly identified MEK1 tyrosine phosphorylation target MACC1 is druggable by approved MEK1 inhibitors to restrict colorectal cancer metastasis. Oncogene, 40(34), 5286-5301. Huynh H, Soo KC, Chow PK, Tran E. Targeted inhibition of the extracellular signal-regulated kinase kinase pathway with AZD6244 (ARRY-142886) in the treatment of hepatocellular carcinoma. Mol Cancer Ther. 2007 Jan;6(1):138-46.. Nair, Anroop B.; Jacob, Shery. (2016). A simple practice guide for dose conversion between animals and human. Journal of basic and clinical pharmacy, 7. Jg., Nr. 2, S. 27.
It would be nice to know the selumetinib sensitivity of the two used cell lines. The concentration used from selumetinib both for the cell line experiments (10 microM) and for the animal experiments (50mg/kg) are quite high. Please discuss how the applied concentrations are related to the clinicaly used dosage (75 mg administered orally twice https://clinicaltrials.gov/ct2/show/NCT02448290) and why not the more efficient MEK1 inhibitor trametinib was used for the experiments.