In silico, in vitro and in vivo toxicity assessment of the antitumoral peptide GK-1

Peptide drugs have emerged as an attractive alternative for cancer treatment due to their potency, high specificity, general safety and low cost. GK-1 is a linear 18 amino acid peptide with proven immunomodulator, antitumor and antimetastatic capacity in animal models. Preclinical toxicity studies for its use as a vaccine adjuvant demonstrated its safety in various assay systems, but a comprehensive exploration of its toxicity profile is required to be used in cancer immunotherapy. Therefore, in the present work, the potential toxicity of GK-1 was predicted with ToxinPred 3.0 software, and its in vitro cytotoxicity, and single-dose and repeated-dose toxicity by subcutaneous route in mice were experimentally assessed. GK-1 peptide was predicted as a non-toxic and did not exhibit in vitro cytotoxicity for several non-tumor and tumor cell lines and primary cell cultures at concentrations up to 500 µM, reinforcing previous studies pointing that the antitumoral effect of GK-1 was not mediated by tumor cell cytotoxicity. The single-dose toxicity study did not evidence local or systemic toxicity up to the maximum tested dose of 1000 mg/kg. Moreover, no toxic effects were observed in the repeated-dose toxicity study based on four doses administered weekly at up to 300 mg/kg. Considering that GK-1 is effective in triple-negative breast cancer and melanoma models in mice at doses as low as 5 mg/kg, the present results support the safety of GK-1 as an antitumoral peptide candidate.