**5. Water solubility and hydrolysis of the MKH esters**

The hydrochloride salts of the esters showed much improved aqueous solubility. The solubilities of MKH-1-DMG and MKH-4-DMG in water were 24 and 5.7 mM respectively and that of MKH-bis-DMG was >50 mM. In contrast, the solubility of MK-4 in water was <2.3 mM. Improving the low water solubility of MK-4 enables a bolus dose necessary for cancer chemotherapy without any surfactant. *In vitro* studies have confirmed that MKH derivatives can be hydrolyzed with esterases located in the rat and human liver and that the resultant MKH acts as cofactor for GGCX without the reductive activation process. The first-order rate constants observed for the hydrolysis of the MKH derivatives in the liver homogenate supernatant are listed in **Table 1**, along with the degradation rate constants of the derivatives in phosphate buffer. The rate of hydrolysis of MKH-1-DMG was about 24-fold and 5.7-fold faster than that of MKH-4-DMG in rat and human liver homogenate supernatant, respectively.

**Figure 2.** Hydrolytic pathway of MKH-bis-DMG. Adapted from Ref. [34].

**6. Vitamin K-dependent carboxylation** *in vitro*

without the reductive activation process of MK-4.

by rat and human liver esterases.

k1 and k2 accordance with **Figure 2**.

Disappearance of MKH-bis-DMG. Adapted from Refs. [33, 34].

a

**Medium kobsa**

As shown in **Table 2**, the rate of hydrolysis of MKH-bis-DMG at the 1-position (k1) was about 2.6-fold and 5.1-fold faster than that at the 4-position (k2) in rat and human liver homogenate supernatant, respectively. The rates of hydrolysis of MKH-1-DMG, MKH-4-DMG, and MKHbis-DMG in the liver homogenate supernatant were significantly reduced in the presence of physostigmine, a liver carboxylesterase inhibitor. Consequently, MKH esters were hydrolyzed

Rat liver homogenate 17.2 ± 1.0 11.9 ± 0.39 4.39 ± 0.18 Human liver homogenate 1.00 0.794 ± 0.019 0.156 ± 0.008

**Table 2.** Rate constants for the hydrolysis of MKH–DMG in human and rat liver homogenate supernatant.

To provide evidence that confirmed the bioreductive activation-independent delivery system of MKH was working properly, carboxylation activity was measured with the incorporation of 14CO2 in the synthetic tripeptide BOC-Glu-Glu-Leu-OMe. The accelerated carboxylation of MK-4 was only observed in the presence of dithiothreitol (DTT), an artificial reducing agent for MK-4, and not in its absence. Conversely, MKH esters stimulated carboxylase activity in the absence of DTT [33], clearly indicating that the MKH esters can stimulate carboxylation

 **(×10−2min−1) k1 (×10−2min−1) k2 (×10−2min−1)**

Enhanced Intracellular Delivery and Improved Antitumor Efficacy of Menaquinone-4

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

317


**Table 1.** Apparent first-order rate constants for the hydrolysis of the MKH derivatives, and regeneration half-lives of MK-4 in human and rat liver homogenate supernatant and phosphate buffer (pH 7.4).

The formation of MK-4 from MKH-bis-DMG should proceed through the intermediates MKH-1-DMG, MKH-4-DMG, and MKH. The pseudo-first-order rate constants for the interconversion of the species are assumed, as shown in **Figure 2**. Detection of MKH was unsuccessful because of its high susceptibility to oxidation.

Enhanced Intracellular Delivery and Improved Antitumor Efficacy of Menaquinone-4 http://dx.doi.org/10.5772/63343 317

**Figure 2.** Hydrolytic pathway of MKH-bis-DMG. Adapted from Ref. [34].

**5. Water solubility and hydrolysis of the MKH esters**

**Compound Without physostigmine**

Rat liver homogenate

316 Vitamin K2 - Vital for Health and Wellbeing

Human liver homogenate

a

Isotonic phosphate buffer of pH7.4

Disappearance of MKH-bis-DMG. Adapted from Refs. [33, 34].

MKH-1-DMG 0.0203 MKH-4-DMG 0.0295 MKH-bis-DMG 0.0500a

**(×10−2min−1)**

MKH-1-DMG 27.2 0.261 2.55 MKH-4-DMG 1.14 0.315 60.6 MKH-bis-DMG 17.2a 0.328a 39.7a

MKH-1-DMG 2.70 0.0714 25.7 MKH-4-DMG 0.476 0.0431 146 MKH-bis-DMG 1.00a 0.117a 227a

**Table 1.** Apparent first-order rate constants for the hydrolysis of the MKH derivatives, and regeneration half-lives of

The formation of MK-4 from MKH-bis-DMG should proceed through the intermediates MKH-1-DMG, MKH-4-DMG, and MKH. The pseudo-first-order rate constants for the interconversion of the species are assumed, as shown in **Figure 2**. Detection of MKH was

MK-4 in human and rat liver homogenate supernatant and phosphate buffer (pH 7.4).

unsuccessful because of its high susceptibility to oxidation.

The hydrochloride salts of the esters showed much improved aqueous solubility. The solubilities of MKH-1-DMG and MKH-4-DMG in water were 24 and 5.7 mM respectively and that of MKH-bis-DMG was >50 mM. In contrast, the solubility of MK-4 in water was <2.3 mM. Improving the low water solubility of MK-4 enables a bolus dose necessary for cancer chemotherapy without any surfactant. *In vitro* studies have confirmed that MKH derivatives can be hydrolyzed with esterases located in the rat and human liver and that the resultant MKH acts as cofactor for GGCX without the reductive activation process. The first-order rate constants observed for the hydrolysis of the MKH derivatives in the liver homogenate supernatant are listed in **Table 1**, along with the degradation rate constants of the derivatives in phosphate buffer. The rate of hydrolysis of MKH-1-DMG was about 24-fold and 5.7-fold faster than that of MKH-4-DMG in rat and human liver homogenate supernatant, respectively.

**With physostigmine**

**Regeneration half-life**

**(min)**

**(×10−2min−1)**

As shown in **Table 2**, the rate of hydrolysis of MKH-bis-DMG at the 1-position (k1) was about 2.6-fold and 5.1-fold faster than that at the 4-position (k2) in rat and human liver homogenate supernatant, respectively. The rates of hydrolysis of MKH-1-DMG, MKH-4-DMG, and MKHbis-DMG in the liver homogenate supernatant were significantly reduced in the presence of physostigmine, a liver carboxylesterase inhibitor. Consequently, MKH esters were hydrolyzed by rat and human liver esterases.


**Table 2.** Rate constants for the hydrolysis of MKH–DMG in human and rat liver homogenate supernatant.

#### **6. Vitamin K-dependent carboxylation** *in vitro*

To provide evidence that confirmed the bioreductive activation-independent delivery system of MKH was working properly, carboxylation activity was measured with the incorporation of 14CO2 in the synthetic tripeptide BOC-Glu-Glu-Leu-OMe. The accelerated carboxylation of MK-4 was only observed in the presence of dithiothreitol (DTT), an artificial reducing agent for MK-4, and not in its absence. Conversely, MKH esters stimulated carboxylase activity in the absence of DTT [33], clearly indicating that the MKH esters can stimulate carboxylation without the reductive activation process of MK-4.
