Cu(CQ)2 efficacy studies in the U251 and A2780-CP subcutaneous tumor models

U-251 cells were grown in culture for four to eight passages prior to inoculation. NRG mice (n = 6 per group) were inoculated subcutaneously using a 28-gauge needle into the right flank of the mouse with 5 × 10⁶ cells in a total volume of 50 μL. When the tumors reached 50–100 mm³, as measured using digital calipers, animals were given (i.v.) vehicle (SH buffer), CuSO₄-liposomes (Cu = 3.2 mg/kg), or Cu(CQ)₂ (CQ = 30 mg/kg, Cu = 3.2 mg/kg) on a Monday, Wednesday, and Friday for 2 weeks. The amount of Cu in Cu(CQ)₂ liposomes was equivalent to a dose of 3.2 mg/kg and this was the rationale used for dosing of the CuSO₄-liposome group.

A2780-CP cells were grown in culture for four to eight passages prior to inoculation. NRG mice (n = 8 per group) were inoculated subcutaneously using a 28-gauge needle with 1 × 10⁶ cells in a total volume of 50 μL. Treatment was initiated 4 days after cell inoculation and the treatment groups were the same as those indicated above for studies in animals with established U-251 tumors.

Tumor size and body weight were measured three times weekly throughout the study. Animals were terminated by CO₂ asphyxiation following isoflurane anesthesia when tumors reached a maximum size of 800 mm³ or when tumors ulcerated. The health status of the animals was monitored daily following an established standard operating procedure as described above.