**4. Improvement of the antiproliferative effect of vitamin K2**

#### **4.1. A novel chemosynthetic vitamin K derivative**

Carr et al. demonstrated that a new, chemically synthesized vitamin K analog, compound 5 (Cpd5), inhibited Cdc25A phosphatase activity and particularly reduced HCC cell growth through arrest of the G1/S phase of the cell cycle. Inhibition of Cdc25A by Cpd5 results in prolonged tyrosine phosphorylation and activation of ERK1/2, which could be triggered by upstream epidermal growth factor receptor signaling pathway molecules [23]. Suhara et al. synthesized vitamin K2 analogues with hydroxyl or phenyl groups at the ω-terminal of the side chain, and with dual side chains at the C-2 and C-3 positions. They found that modifying the side chain of vitamin K affects the SXR-mediated transcriptional activity [24, 25]. The novel biological activities of MK-4 include tumor suppressive effects related to gene transcription through the SXR. The new derivatives of MK-4 have shown some efficacy against HCC, but a lengthy development process is still necessary to yield safe and effective clinical products.

in HCC. MKH is mainly generated by a vitamin K 2, 3-epoxide reductase complex subunit 1 like-1 (VKORC1L1) in mammalian cells [38]. Key reasons for further development are that MK-4 delivery to HCC cells is poor and reductive activation of MK-4 is low in HCC cells. We hypothesized that effective delivery of MKH into HCC cells is essential in regulating HCC growth and metastasis. However, MKH cannot be used as a therapeutic agent, because it is easy to be oxidized and converted to MK-4. In addition, the production of MKH depends on VKORC1L1 activity in HCC cells. As such, MK-4 does not display sufficient antitumor activity

Enhanced Intracellular Delivery and Improved Antitumor Efficacy of Menaquinone-4

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

315

In light of these findings, we synthesized three types of *N,N*-dimethylglycine esters of MKH (MKH-1-DMG, MKH-4-DMG, and MKH-bis-DMG) and assessed their potential as watersoluble prodrugs for a bioreductive activation-independent delivery system of MKH [33–35]

**Figure 1.** Structure of the MKH derivatives and schematic illustration of the concept of the MKH delivery system. (A) Chemical structures of menaquinone-4 (MK-4), menahydroquinone-4 (MKH) and MKH *N,N*-dimethylglycinate (MKH–DMG). (B) Schematic illustration of the vitamin K cycle and the concept of the MKH delivery system. Adapted

even at high doses.

(**Figure 1**).

from Ref. [32].

#### **4.2. Synergistic drug combinations**

Yoshiji et al. [26, 27] reported that combined treatment with MK-4 and angiotensin-converting enzyme inhibitor significantly suppressed experimental hepatocarcinogenesis. Further to this, they reported that the combined treatment with MK-4 and angiotensin-converting enzyme inhibitor may suppress the cumulative recurrence of HCC after the curative therapy, at least partly through suppression of the vascular endothelial growth-mediated neovascularization. Kanamori et al. [28] demonstrated that a combination of MK-4 and acyclic retinoid synergistically inhibited the growth of Huh7 HCC cells by increasing apoptosis. When combined with acyclic retinoid, MK-4 synergistically inhibits Ras activation and inhibits phosphorylation of retinoid X receptor α. Zhang H et al. [29] showed that MK-4 enhanced the inhibition of 5 fluorouracil-induced cell growth in HepG2, Huh7, HLE, and Hep3B HCC cells, and via G1 cell-cycle arrest through induced expression of p21 and p27 and inhibited expression of cyclin D1. Zhang et al. reported that a combination of MK-4 and sorafenib work synergistically to inhibit growth of HepG2, Hep3B, and HuH7 HCC cells. They also demonstrated that the levels of cyclin D1 expression are clearly reduced in HepG2 cells treated with a combination treatment of MK-4 and sorafenib [30]. A clinical trial to test the efficacy of the combination of MK-4 and sorafenib in HCC was attempted [31].

#### **4.3. Delivery of menahydroquinone-4, the active form of MK-4**

We have previously synthesized the ester derivatives of menahydroquinone-4 (MKH), the fully reduced form of MK-4, and revealed their effective antiproliferative activity against HCC cell lines [32–35]. The menaquinone (MK-4 to MK-10) concentrations were significantly lower in HCC tissues, from patients with or without increased plasma concentrations of DCP, than in the surrounding normal liver tissue. There was no significant difference between PK and PK epoxide concentration in HCC tissues without increased plasma concentrations of DCP and normal liver tissue [36]. Furthermore, the rate of uptake into MH7777 cells (vitamin K2 sensitive HCC) was lower than for normal hepatocytes. In addition, the rate of uptake into H4IIE cells (vitamin K2-resistant HCC) was negligible compared with that for MH7777 cells and normal hepatocytes. Further, hepatocytes from diethylnitrosamine-induced liver nodules exhibited a significantly lower rate of vitamin K2 uptake than that for normal hepatocytes [37].

MKH acts as a cofactor for *γ*-glutamyl carboxylase (GGCX), which catalyzes the carboxylation of specific Glu residues (*γ*-carboxylation) of substrate proteins such as prothrombin. Thus, decreased MKH availability in HCC cells is a possible causative mechanism of DCP production in HCC. MKH is mainly generated by a vitamin K 2, 3-epoxide reductase complex subunit 1 like-1 (VKORC1L1) in mammalian cells [38]. Key reasons for further development are that MK-4 delivery to HCC cells is poor and reductive activation of MK-4 is low in HCC cells. We hypothesized that effective delivery of MKH into HCC cells is essential in regulating HCC growth and metastasis. However, MKH cannot be used as a therapeutic agent, because it is easy to be oxidized and converted to MK-4. In addition, the production of MKH depends on VKORC1L1 activity in HCC cells. As such, MK-4 does not display sufficient antitumor activity even at high doses.

prolonged tyrosine phosphorylation and activation of ERK1/2, which could be triggered by upstream epidermal growth factor receptor signaling pathway molecules [23]. Suhara et al. synthesized vitamin K2 analogues with hydroxyl or phenyl groups at the ω-terminal of the side chain, and with dual side chains at the C-2 and C-3 positions. They found that modifying the side chain of vitamin K affects the SXR-mediated transcriptional activity [24, 25]. The novel biological activities of MK-4 include tumor suppressive effects related to gene transcription through the SXR. The new derivatives of MK-4 have shown some efficacy against HCC, but a lengthy development process is still necessary to yield safe and effective clinical products.

Yoshiji et al. [26, 27] reported that combined treatment with MK-4 and angiotensin-converting enzyme inhibitor significantly suppressed experimental hepatocarcinogenesis. Further to this, they reported that the combined treatment with MK-4 and angiotensin-converting enzyme inhibitor may suppress the cumulative recurrence of HCC after the curative therapy, at least partly through suppression of the vascular endothelial growth-mediated neovascularization. Kanamori et al. [28] demonstrated that a combination of MK-4 and acyclic retinoid synergistically inhibited the growth of Huh7 HCC cells by increasing apoptosis. When combined with acyclic retinoid, MK-4 synergistically inhibits Ras activation and inhibits phosphorylation of retinoid X receptor α. Zhang H et al. [29] showed that MK-4 enhanced the inhibition of 5 fluorouracil-induced cell growth in HepG2, Huh7, HLE, and Hep3B HCC cells, and via G1 cell-cycle arrest through induced expression of p21 and p27 and inhibited expression of cyclin D1. Zhang et al. reported that a combination of MK-4 and sorafenib work synergistically to inhibit growth of HepG2, Hep3B, and HuH7 HCC cells. They also demonstrated that the levels of cyclin D1 expression are clearly reduced in HepG2 cells treated with a combination treatment of MK-4 and sorafenib [30]. A clinical trial to test the efficacy of the combination of

We have previously synthesized the ester derivatives of menahydroquinone-4 (MKH), the fully reduced form of MK-4, and revealed their effective antiproliferative activity against HCC cell lines [32–35]. The menaquinone (MK-4 to MK-10) concentrations were significantly lower in HCC tissues, from patients with or without increased plasma concentrations of DCP, than in the surrounding normal liver tissue. There was no significant difference between PK and PK epoxide concentration in HCC tissues without increased plasma concentrations of DCP and normal liver tissue [36]. Furthermore, the rate of uptake into MH7777 cells (vitamin K2 sensitive HCC) was lower than for normal hepatocytes. In addition, the rate of uptake into H4IIE cells (vitamin K2-resistant HCC) was negligible compared with that for MH7777 cells and normal hepatocytes. Further, hepatocytes from diethylnitrosamine-induced liver nodules exhibited a significantly lower rate of vitamin K2 uptake than that for normal hepatocytes [37].

MKH acts as a cofactor for *γ*-glutamyl carboxylase (GGCX), which catalyzes the carboxylation of specific Glu residues (*γ*-carboxylation) of substrate proteins such as prothrombin. Thus, decreased MKH availability in HCC cells is a possible causative mechanism of DCP production

**4.2. Synergistic drug combinations**

314 Vitamin K2 - Vital for Health and Wellbeing

MK-4 and sorafenib in HCC was attempted [31].

**4.3. Delivery of menahydroquinone-4, the active form of MK-4**

In light of these findings, we synthesized three types of *N,N*-dimethylglycine esters of MKH (MKH-1-DMG, MKH-4-DMG, and MKH-bis-DMG) and assessed their potential as watersoluble prodrugs for a bioreductive activation-independent delivery system of MKH [33–35] (**Figure 1**).

**Figure 1.** Structure of the MKH derivatives and schematic illustration of the concept of the MKH delivery system. (A) Chemical structures of menaquinone-4 (MK-4), menahydroquinone-4 (MKH) and MKH *N,N*-dimethylglycinate (MKH–DMG). (B) Schematic illustration of the vitamin K cycle and the concept of the MKH delivery system. Adapted from Ref. [32].
