In Vivo Xenograft Study

All experiments with animals were conducted in accordance with the Guidelines of the Yakult Central Institute and protocols approved by the Animal Experimental Committee of the Yakult Central Institute.

For evaluation of antitumor activity, MV-4-11 (5 × 10⁶ cells) or PC-3 cells (2 × 10⁶ cells) were injected subcutaneously in the inguinal region of 6-week-old male BALB/cSlc-nu/nu mice (Japan SLC, Inc., Shizuoka, Japan), with Matrigel (BD Biosciences, San Jose, CA, USA). When the average tumor volume (length × width² × 1/2) had reached 40 to 120 mm³, animals were randomized into groups of seven mice for vehicle control group of MV-4-11 xenograft model or five mice for the other groups (day 1). Test compounds were injected into the tail vein every 4 days from day 1, with an administration volume of 10 ml/kg. Tumor volumes were measured 2 times per week and body weights of the mice were monitored on 2 or 3 times per week as an indicator of tolerability. On day 22 or 16, antitumor activity was evaluated by weighing tumor tissues. Tumor growth inhibition, TGI (%), was calculated according to formula (1 − T/C) × 100, where T and C are the mean tumor weights for the experimental and vehicle control groups, respectively. The differences between the mean tumor weights for comparing groups were analyzed using Dunnett’s test, where P < 0.05 was considered to be significant.

For the pharmacodynamics study, mice with PC-3 tumors were treated with vehicle or YPC-21817 MsOH, with 5 mice per time point. Tumors were resected 1, 2, 6, or 24 h after injection and lyzed for immunoblot analysis.