**10. Antiproliferative effects of MKH-bis-DMG in a spleen–liver metastasis mouse model**

To assess the pharmacological effects of the MKH delivery system *in vivo*, we assessed the effects of oral administration of MKH-bis-DMG on hepatic metastasis and proliferation of PLC/ PRF/5 cells in a spleen–liver metastasis model [39]. MKH-bis-DMG treatment significantly suppressed the increase of liver weight caused by tumor growth (**Figure 4A**). The percentage surface area of the cancer compared with the total surface area of the liver was significantly lower in the spleen-liver metastasis model treated with the MKH-bis-DMG than with the vehicle (**Figure 4B**).

Plasma DCP production was completely suppressed after MKH–DMG administration, while liver metastasis of HCC was not completely prevented (**Figure 4C**). These results suggest that the level of influence of DCP suppression on the antiproliferative effects of MKH-bis-DMG was severely limited. No obvious side effects, such as body weight loss, were observed in the MKH-di-DMG treated animals.

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**Figure 4.** HCC growth inhibitory effects of MKH–DMG in a spleen-liver metastasis mouse model. (A) Total liver weight. (B) Percentage of cancer surface area/total liver surface area. (C) DCP levels in plasma. Central horizontal line, mean; error bar, SD. Vehicle group, n = 15; MKH–DMG group, n = 15. Doses were 0.2 μmol/head/day for 50 days. Adapted from Ref. [32].

#### **11. Conclusion and perspectives**

In previous studies, we have indicated that MKH-bis-DMG can be delivered to the liver through intravenous and oral administration and that thereafter it is transported and converted to MKH with enzymatic hydrolysis in HCC cells. Regarding the effective delivery of MKH into the HCC cells, MKH-bis-DMG enhanced the inhibition of HCC cell growth compared with that from MK-4 and suppressed metastasis of HCC. Given these results, we suggest that MKH-bis-DMG is a potential candidate for chemoprevention that can be safely administered over long periods, and can reduce or eliminate the recurrence and metastasis of HCC. This MKH prodrug approach was our original method to improve the antitumor effect of MK-4, and this approach may be applied to improve HCC treatment in the future. However, further studies are required to develop MKH–DMG for use in human HCC treatment.

#### **Author details**

Kazuhisa Matsunaga\* , Munechika Enjoji, Yoshiharu Karube and Jiro Takata

\*Address all correspondence to: k-matsu@fukuoka-u.ac.jp

Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan
