Cell experiment:

MTT assays are applied to test cell viability. In brief, 3×103 cells per well are seeded in a 96-well plate and incubated with various concentration of TNP-470 for 24, 48, and 72 h at 37℃, 5% CO2. For comparison, cells cultured in the absence of TNP-470 are used as a control. After an incubation period, 10 μL MTT (0.5 mg/mL final concentration) is added to each well. After 4 h of additional incubation, 100 μL of 0.01 N HCl in isopropanol is added to dissolve the crystals. Absorption at 570 nm is determined by ELISA plate reader[1].

Animal experiment:

Individually housed, 4 wk old male C57BL/6 mice are used in this study. After a 1 wk acclimation period, mice are randomly allocated to receive either standard chow diet or high-fat diet for 6.5 wk. Throughout the high-fat feeding period the mice are treated with TNP-470 at a dose of 20 mg/kg body weight, injected subcutaneously every other day (TNP; n=7) or a vehicle injection of an equivalent volume (HFF controls; n=7). Vehicle injections contain 3% ethanol in phosphate-buffered saline. Chow-fed control mice (chow; n=8) are sham injected. Mice are fed ad libitum with food replaced every 2 or 3 days. Body weights are collected three times per week. After 6.5 wk of feeding, animals are fasted for 16-h and sacrificed. Final body, liver, and epididymal adipose tissue weights are measured. Liver and adipose tissue samples are frozen in liquid nitrogen and stored at -80℃ for subsequent analysis[2].

IC50: 2 μM

TNP-470 is a methionine aminopeptidase-2 (MetAP2) inhibitor.

MetAP2 is present in all organisms and is important due to its key role in tissue repair and protein degradation. Moreover, MetAP2 is of particular interest since it plays a critical role in angiogenesis, which is necessary for the progression of diseases, such as solid tumor cancers and rheumatoid arthritis.

In vitro: Antitumor activity of TNP-470 was evaluated in eight human cultured cell lines derived from choriocarcinoma; ovarian cancer; TYK and Nakajima; and uterine endometrial cancer. Results showed that after 7-day culture with TNP-470, in medium at the concentration of 10 to 10-2 μg/ml, the inhibition of growth was observed in all of the eight cell lines. The half inhibitory concentration of choriocarcinoma cell lines was at an extremely low level compared to that of e uterine endometrial cancer and pithelial ovarian cancer [1].

In vivo: The anti-tumor effect of TNP-470 was studied using nude mice with tumors of GCH-1(m), NUC-1, or Nakajima cells. When the size of the transplanted tumor reached 100–200 mm3, 3, 10, or 30 mg/kg of TNP-470 was injected every other day. It was found that the inhibitory effect of TNP-470 was obtained by the administration of 10 and 30 mg/kg in GCH-1(m) and NUC-1 cells, respectively, while in Nakajima cells there was no significant effect. In addition, in nude mice treated with 30 mg/kg of TNP-470, lung metastasis of GCH-1(m) cells was greatly inhibited both in the number and the size of tumor nodules, suggesting that the capillary growth in the originally developed tumor was also reduced [1].

Clinical trial: A phase I study found that the effects of TNP-470 could be evaluated in 32 of the 33 patients. The dose-limiting toxic effect was a characteristic neuropsychiatric symptom complex. No definite anti-tumor activity of TNP-470 was observed; however, transient stimulation of the serum prostate-specific antigen concentration occurred in some of the patients treated [2].

References:
[1] T. Yanase, M. Tamura, K. Rujita, et al. Inhibitory effect of angiogenesis inhibitor TNP-470 on tumor growth and metastasis of human cell lines in vitro and in vivo. Cancer Research 53(11), 2566-2570 (1993).
[2] C J Logothetis et al. Phase I trial of the angiogenesis inhibitor TNP-470 for progressive androgen-independent prostate cancer. Clinical Cancer Research 7(5):1198-203.