**8. Mechanism of growth inhibition of HCC cells by MKH esters**

One of the mechanisms of the antiproliferative effect of MK-4 was thought to involve G1/S cellcycle arrest via reduced protein expression of cyclin D1 and Cdk4, and through suppression of NF-κB activation [10, 12]. We investigated whether the antiproliferative activity of MKHbis-DMG was via cell-cycle arrest in HCC cells using flow cytometry and Western blotting [32]. MKH-bis-DMG-treated PLC/PRF/5 cells showed an increase in G1 phase cells and a decrease in S phase cells in flow cytometric analysis. Treatment of both DCP-positive and DCP-negative HCC cells with MKH-bis-DMG downregulated cyclin D1, cyclin D3, and Cdk4 expression after 24 h, and almost completely removed expression after 48 h. In comparison, the modest downregulation of cyclin D1, cyclin D3, and Cdk4 expression was observed after 48 h of MK-4 treatment in all tested HCC cell lines. NF-κB was downregulated after MKH-bis-DMG treatment in all tested HCC cell lines, but no effect was observed after MK-4 treatment in PLC/ PRF/5 and SK-Hep-1 cell lines at this dose.

These findings strongly support our hypothesis that the rapid and strong growth-inhibitory effects on cells resulted from the rapid and effective delivery of MKH into HCC cells by an MKH prodrug. The mechanism of the MKH-bis-DMG antiproliferative effect is the same as that of MK-4 and involves cell-cycle arrest. Therefore, MKH-bis-DMG is expected to be a safe antitumor agent and chemopreventive agent.

#### **9. Pharmacokinetics of MKH esters**

Plasma MKO levels can reflect the levels of MKH not only *in vitro* but also *in vivo*. This also assists the function of GGCX at the active site. The relative bioavailability for MKH (FMKH), after the parenteral administration of the MKH esters, relative to MK-4 solubilized with HCO-60, was calculated using AUCMKO as in Eq. (1), and is shown in **Table 4**. In Eq. (1), AUCMKO, MKH—DMG and AUCMKO, MK-4 are the AUCMKO values after the administration of MKH– DMG and MK-4, respectively. DMK-4 and DMKH—DMG are the doses of MK-4 and MKH–DMG, respectively. MKH-1-DMG and MKH-bis-DMG, but not MKH-4-DMG, showed an improvement in bioavailability compared with the MK-4 injection.


$$\mathbf{F}\_{\text{MKH}} = \frac{\mathbf{AUC}\_{\text{MKO},\text{MKH}-\text{DMG}} \cdot \mathbf{D}\_{\text{MK}-4}}{\mathbf{AUC}\_{\text{MKO},\text{MK}-4} \cdot \mathbf{D}\_{\text{MKH}-\text{DMG}}} \tag{1}$$

The distribution of MKO in the liver after the injection of MKH esters is the most important indicator for assessing the potential of MKH esters as the MKH delivery system for HCC. The values of AUCMKO and MRTMKO of MKH-1-DMG were larger than that for MK-4, which indicates that the MKH esters distribute successfully and provide prolonged deliver MKH to the liver. For the evaluation of MKH-1-DMG as a liver-specific delivery system for MKH, the

selective advantage value was defined as in Eq. (2). In Eq. (2), AUCMKO, MKH <sup>−</sup> DMG

Liver are the AUCMKO values in the liver after the administration of MKH–DMG and

Plasma

Enhanced Intracellular Delivery and Improved Antitumor Efficacy of Menaquinone-4

ï ï î þ <sup>=</sup> ì ü ï ï í ý

AUC

MKH DMG,MKH DMG Plasma MK 4,MK 4

ï ï î þ


Plasma and AUCMK−4, MK <sup>−</sup> <sup>4</sup>

plasma levels of MKH–DMG and MK-4 after the administration of MKH–DMG and MK-4, respectively. Remarkable site-specific delivery of MKH was observed after the intravenous

It is proposed that the intravenous injection of MKH esters is appropriate when a rapid and large quantity is necessary for cancer treatment, whereas oral administration of MKH esters is otherwise appropriate for cancer prevention. We performed a pharmacokinetic study of MKH-bis-DMG after oral administration, and found that MKH-bis-DMG was absorbed in the

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

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

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

injection of MKH-1-DMG. The selective advantage of MKH-1-DMG was 5.7 [35].

MKO,MKH DMG Liver MKO,MK 4

ì ü ï ï í ý


Liver

ester form, distributed to the liver and converted to MKH *in vivo* [32].

AUC AUC Selectiveadvantage AUC

AUCMKO, MK <sup>−</sup> <sup>4</sup>

**mouse model**

vehicle (**Figure 4B**).

MKH-di-DMG treated animals.

MK-4, respectively. AUCMKH−DMG, MKH <sup>−</sup> DMG

Liver and

http://dx.doi.org/10.5772/63343

(2)

321

Plasma are the AUC values of

a The values are the mean ± S.D. of 3 rats at a dose of 5 mg/kg equivalent for MK-4. b

Calculated from Eq. (1) using the mean values.

Adapted from Ref. [35].

**Table 4.** Pharmacokinetic parameters for MKO and MK-4 in plasma after the intravenous administration of the prodrugs, and for MK-4 in vitamin K cycle-inhibited rats.a

The distribution of MKO in the liver after the injection of MKH esters is the most important indicator for assessing the potential of MKH esters as the MKH delivery system for HCC. The values of AUCMKO and MRTMKO of MKH-1-DMG were larger than that for MK-4, which indicates that the MKH esters distribute successfully and provide prolonged deliver MKH to the liver. For the evaluation of MKH-1-DMG as a liver-specific delivery system for MKH, the selective advantage value was defined as in Eq. (2). In Eq. (2), AUCMKO, MKH <sup>−</sup> DMG Liver and AUCMKO, MK <sup>−</sup> <sup>4</sup> Liver are the AUCMKO values in the liver after the administration of MKH–DMG and MK-4, respectively. AUCMKH−DMG, MKH <sup>−</sup> DMG Plasma and AUCMK−4, MK <sup>−</sup> <sup>4</sup> Plasma are the AUC values of plasma levels of MKH–DMG and MK-4 after the administration of MKH–DMG and MK-4, respectively. Remarkable site-specific delivery of MKH was observed after the intravenous injection of MKH-1-DMG. The selective advantage of MKH-1-DMG was 5.7 [35].

These findings strongly support our hypothesis that the rapid and strong growth-inhibitory effects on cells resulted from the rapid and effective delivery of MKH into HCC cells by an MKH prodrug. The mechanism of the MKH-bis-DMG antiproliferative effect is the same as that of MK-4 and involves cell-cycle arrest. Therefore, MKH-bis-DMG is expected to be a safe

Plasma MKO levels can reflect the levels of MKH not only *in vitro* but also *in vivo*. This also assists the function of GGCX at the active site. The relative bioavailability for MKH (FMKH), after the parenteral administration of the MKH esters, relative to MK-4 solubilized with HCO-60, was calculated using AUCMKO as in Eq. (1), and is shown in **Table 4**. In Eq. (1), AUCMKO, MKH—DMG and AUCMKO, MK-4 are the AUCMKO values after the administration of MKH– DMG and MK-4, respectively. DMK-4 and DMKH—DMG are the doses of MK-4 and MKH–DMG, respectively. MKH-1-DMG and MKH-bis-DMG, but not MKH-4-DMG, showed an improve-

MKO,MKH DMG MK 4


**MK-4 MKH-1-DMG MKH-4-DMG MKH-bis-DMG**

<sup>×</sup> <sup>=</sup> <sup>×</sup> (1)

AUC D

AUC D

*Cmax* (nmol mL−1) 107 ± 2.07 2.41 ± 0.826 20.8 ± 5.24 11.1 ± 3.03 *tmax* (h) 0.125 0.125 0.125 0.125 *AUCMK-4* (nmol h mL−1) 31.7 ± 0.526 2.52 ± 0.387 6.01 ± 1.26 10.1 ± 1.15 *MRTMK-4*x (h) 0.338 ± 0.018 2.44 ± 0.032 0.924 ± 0.004 1.32 ± 0.063

*Cmax* (nmol mL−1) 1.92 ± 0.172 0.991 ± 0.131 0.518 ± 0.039 0.968 ± 0.180

*AUCMKO* (nmol h mL−1) 2.37 ± 0.115 4.45 ± 0.510 2.06 ± 0.322 3.20 ± 0.467 *MRTMKO* (h) 2.14 ± 0.091 3.78 ± 0.404 3.93 ± 0.463 2.80 ± 0.066

**Table 4.** Pharmacokinetic parameters for MKO and MK-4 in plasma after the intravenous administration of the prodrugs,

*tmax* (h) 1 2 0.5 1

*FMKH* 100 188 87 135

The values are the mean ± S.D. of 3 rats at a dose of 5 mg/kg equivalent for MK-4.

Calculated from Eq. (1) using the mean values.

and for MK-4 in vitamin K cycle-inhibited rats.a

MKO,MK 4 MKH DMG

antitumor agent and chemopreventive agent.

320 Vitamin K2 - Vital for Health and Wellbeing

**9. Pharmacokinetics of MKH esters**

ment in bioavailability compared with the MK-4 injection.

MKH

F

for MK-4

for MKO

a

b

Adapted from Ref. [35].

$$\text{Selocity} = \left\{ \begin{aligned} & \left\{ \frac{\text{AUC}\_{\text{MKO},\text{MKH}\text{-DMG}}^{\text{Liver}}}{\text{AUC}\_{\text{MKO},\text{MK}-4}} \right\} \\ & \end{aligned} \right\} \left\{ \begin{aligned} & \text{AUC}\_{\text{MKO},\text{MKH}-4}^{\text{PLiser}} \\ & \left\{ \frac{\text{AUC}\_{\text{MKH}-\text{DMG},\text{MKH}-\text{DMG}}^{\text{PLiser}}}{\text{AUC}\_{\text{MK}-4,\text{MK}-4}} \right\} \end{aligned} \tag{2}$$

It is proposed that the intravenous injection of MKH esters is appropriate when a rapid and large quantity is necessary for cancer treatment, whereas oral administration of MKH esters is otherwise appropriate for cancer prevention. We performed a pharmacokinetic study of MKH-bis-DMG after oral administration, and found that MKH-bis-DMG was absorbed in the ester form, distributed to the liver and converted to MKH *in vivo* [32].
