Nemorubicin

Cell experiment:

9L and CHO cells are plated in triplicate wells of a 96-well plate at 3000 cells per well 24 hr prior to drug treatment. Cells are treated with various concentrations of Nemorubicin or IFA for 4d. Cells are then stained with crystal violet (A595) and relative cell survival is calculated. IC50 values are determined from a semi-logarithmic graph of the data points using Prism 4[4].

Animal experiment:

9L and 9L/3A4 cells are grown as solid tumors in male ICR/Fox Chase SCID mice. Cells cultured in DMEM medium to 75% confluence are trypsinized and washed in PBS and then adjusted to 2 × 107 cells/mL of FBS-free DMEM. Four-week-old SCID mice (18-20 g) are implanted with either 9L or 9L/3A4 tumor cells by injection of 4 × 106 cells/0.2 mL of cell suspension, s.c. on each hind flank. Tumor sizes (length and width) are measured twice a week using Vernier calipers beginning 7d after tumor implantation. When the average tumor size reach 300 to 400 mm3, Nemorubicin dissolved in PBS is administered by tail vein injection (i.v.) or by direct intratumoral (i.t.) injection (three injections spaced 7 d apart, each at 60 µg Nemorubicin per kg body weight). Intratumoral injections are performed using a syringe pump set a 1 µL/s with a 30-gauge needle. Each i.t. treatment dose is divided into three injections per tumor, with the injected volume set at 50 µL per tumor per 25 g mouse. Thus, for a 30 g mouse, a total of 120 µL of 15 µg/mL of Nemorubicin solution is administered: 20 µL per site × 3 sites per tumor × 2 tumors/mouse. Drug-free controls are injected i.t. with the same vol of PBS. In some experiments, Nemorubicin is administered by i.p. injection at 40 or 60 µg/kg body weight. Tumor sizes and body weights are measured twice/wk for the duration of the study. Tumor volumes are calculated using the formula: V = π/6 (L × W)3/2. Percent tumor regression is calculated as 100 × (V1-V2)/V1, where V1 is the tumor vol on the day of drug treatment and V2 is the vol on the day when the largest the decrease in tumor size is seen following drug treatment. Tumor doubling time is calculated as the time required for tumors to double in vol after drug treatment[4].

References:

[1]. Quintieri L, et al. Formation and antitumor activity of PNU-159682, a major metabolite of nemorubicin in human liver microsomes. Clin Cancer Res. 2005 Feb 15;11(4):1608-17.
[2]. Quintieri L, et al. In vitro hepatic conversion of the anticancer agent nemorubicin to its active metabolite PNU-159682 in mice, rats and dogs: a comparison with human liver microsomes. Biochem Pharmacol. 2008 Sep 15;76(6):784-95.
[3]. Sabatino MA, et al. Down-regulation of the nucleotide excision repair gene XPG as a new mechanism of drug resistance in human and murine cancer cells. Mol Cancer. 2010 Sep 24;9:259.
[4]. Lu H, et al. Potentiation of methoxymorpholinyl doxorubicin antitumor activity by P450 3A4 gene transfer. Cancer Gene Ther. 2009 May;16(5):393-404.