The Rational Design of Therapeutic Peptides for Aminopeptidase N using a Substrate-Based Approach

Joshi, Shilvi; Chen, Lang; Winter, Michael B.; Lin, Yi-Lun; Yang, Yang; Shapovalova, Mariya; Smith, Paige M.; Liu, Chang; Li, Fang; LeBeau, Aaron M.

In vitro and in vivo therapeutic studies. The clonogenic survival assays were conducted as previously described 41 . Mouse care and treatment was approved by and performed in accordance with the guidelines of the University of Minnesota Institutional Animal Care and Use Committee. Cells maintained under standard conditions were detached by treatment with 0.25% trypsin-EDTA solution and washed in Hank's balanced salt solution (HBSS). They were then suspended in a 60% mixture of Matrigel Matrix (BD Biosciences) in HBSS at a concentration of 1.0 × 10 6 cells per 100 μL of solution. The cells were then injected into the subcutis overlying the rear flanks of 6-week-old male nude mice (Harlan). Once the tumors were established, the animals were randomized into three treatment groups per xenograft. The both cyc-LHSPW and cyc-NGR were dissolved in DMSO and diluted in sterile PBS to a final DMSO concentration of 5% v/v. The mice were then dosed via tail vein injection with 40 mg/kg of the cyclic peptides or saline at days three times a week for four weeks total. Tumor measurements were made twice weekly with calipers and the tumor volume (in mm 3 ) was calculated by the formula 0.5236 × length (L) × width (W) × height (H). The endpoint of the study was either five weeks after the first treatment dose or when the tumors reached a volume of 1,000 mm 3 as dictated by our animal protocol. PC3 tumors were removed from euthanized animals, formalin fixed and stained for Ki67 using the manufacturer's protocol.