Intestinal alkaline phosphatase (ALP); intestinal-type ALP [1]
Osteoprotegerin (OPG); Receptor activator of NF-κB ligand (RANKL) [2]
Menatetrenone targets multiple pathways. It serves as an anti-hemorrhagic agent by promoting blood coagulation through its role in the synthesis of clotting factors. It inhibits bone resorption through inhibition of prostaglandin E₂ synthesis and other mechanisms. It may act by modulating the signaling of certain tyrosine kinases, affecting transcription factors including c-myc and c-fos. It inhibits tumor cell growth by inducing apoptosis and cell cycle arrest.

In Caco-2 cells, menaquinone-4 (MK-4, 0, 1, 5, 10 μM) raises the activity of ALP. The intensity of hSI expression is greatly increased by menaquinone-4 (1 μM)[1].
Treatment with Menaquinone-4 (MK-4) significantly increased alkaline phosphatase (ALP) activities in human colon carcinoma Caco-2 cells, which are known to differentiate into small intestinal epithelial cells in vitro. [1] Inhibitor and thermal inactivation experiments demonstrated that the increased ALP induced by MK-4 had properties of intestinal-type ALP. [1] Semiquantitative reverse transcription-polymerase chain reaction analysis revealed that MK-4 highly enhanced the expression of human intestinal ALP mRNA and sucrase-isomaltase mRNA (intestinal differentiation markers) in Caco-2 cells. [1]
In vitro, menatetrenone raises the activity of alkaline phosphatase (ALP) in Caco-2 cells. Treatment with menaquinone-4 significantly increases alkaline phosphatase activities in human colon carcinoma Caco-2 cells, which differentiate into small intestinal epithelial cells. These effects suggest a role in promoting osteoblast differentiation and bone formation.

In addition to increasing mice's bone density, menaquinone-4 (K2, 0.2 g/kg diet) in the HF-K2 group causes C57BL/6J mice to develop epididymal fat[2].

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- In high-fat diet-induced obese mice, supplementation with Menaquinone-4 (Vitamin K2) (200 mg/1000 g diet for 12 weeks) significantly increased serum osteocalcin (OC) level compared to the high-fat diet-only group (51.02 ± 7.34 ng/ml vs. 41.84 ± 1.54 ng/ml, p<0.05). [2]
- Serum osteoprotegerin (OPG) level was higher in the high-fat diet + K2 group (2.90 ± 0.11 ng/ml) compared to the high-fat diet group (2.31 ± 0.31 ng/ml). [2]
- Serum RANKL level was significantly lower in the high-fat diet + K2 group (0.21 ± 0.03 ng/ml) compared to the high-fat diet group (0.40 ± 0.06 ng/ml, p<0.05). [2]
- The RANKL/OPG ratio was significantly reduced in the high-fat diet + K2 group (0.07 ± 0.01) compared to the high-fat diet group (0.21 ± 0.06, p<0.01). [2]
- Vitamin K2 supplementation reversed high-fat diet-induced bone deterioration by modulating osteoblast and osteoclast activities, and prevented bone loss. [2]
- Bone mineral density (BMD) in the high-fat diet + K2 group increased to 0.24 ± 0.00 compared to the high-fat diet group (0.22 ± 0.00, p<0.05). [2]
- Trabecular number (Tb.N) was higher in the high-fat diet + K2 group (1.10 ± 0.07 1/mm) compared to the high-fat diet group (1.03 ± 0.07 1/mm). [2]
- Total fat amount (epididymal, perirenal, retroperitoneal) was significantly lower in the high-fat diet + K2 group (1.95 ± 0.33 g) compared to the high-fat diet group (2.89 ± 0.15 g, p<0.05). [2]
- Epididymal fat was significantly lower in the high-fat diet + K2 group (1.36 ± 0.23 g) compared to the high-fat diet group (2.04 ± 0.13 g, p<0.05). [2]
- Perirenal fat was lower in the high-fat diet + K2 group (0.16 ± 0.07 g) compared to the high-fat diet group (0.26 ± 0.02 g, p<0.05). [2]
In vivo, menatetrenone is used as a hemostatic agent and as adjunctive therapy for the pain of osteoporosis. It inhibits bone resorption and promotes bone health. In Japan, it is marketed by Eisai Co. under the trade name Glakay for the treatment of osteoporosis. It has been investigated for its therapeutic potential beyond coagulation, including applications in oncology and neurology.

Inhibitor and thermal inactivation experiments were performed to characterize the increased ALP activity. The results showed that the ALP induced by Menaquinone-4 (MK-4) had properties consistent with intestinal-type ALP. [1]
In vitro enzyme/receptor binding studies for menatetrenone are not typical, as it is a vitamin K2 analog. Its activity is assessed by measuring its effects on bone-related enzymes such as alkaline phosphatase (ALP) in osteoblast-like cells. Its effects on osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) levels may also be measured.

- Human colon carcinoma Caco-2 cells, known to differentiate into small intestinal epithelial cells in vitro, were treated with Menaquinone-4 (MK-4). After treatment, ALP activities were measured and showed significant increases compared to untreated controls. [1]
- Semiquantitative reverse transcription-polymerase chain reaction analysis was performed to examine the expression of human intestinal ALP and sucrase-isomaltase (intestinal differentiation markers) in Caco-2 cells following MK-4 treatment. Results revealed that MK-4 highly enhanced the expression of both markers. [1]
In vitro cellular assays for menatetrenone involve culturing osteoblast-like cells such as Caco-2 or MC3T3-E1 in the presence of serial dilutions of the compound. Alkaline phosphatase activity is measured as a marker of osteoblast differentiation. Cell viability and proliferation are assessed using standard assays. Gene expression of bone-related markers is measured by qRT-PCR.

- Four-week-old male C57BL/6J mice were fed a 45% kcal high-fat diet supplemented with Menaquinone-4 (Vitamin K2) at 200 mg per 1000 g of diet for 12 weeks. The diet was provided in pellet form. Food intake was measured every other day, and body weight was measured once a week. [2]
- After 12 weeks of feeding, animals were fasted for 12 hours, sacrificed with ether, and blood was collected from the aorta and orbital veins. Blood samples were centrifuged at 3,000 rpm for 15 minutes and stored at -70°C until analysis. Femurs were removed and stored in formalin for bone microstructure analysis. [2]
- Bone microstructure was analyzed using high-resolution 3D micro-focus computed tomography. The region of interest was set at 2.35 mm from the cartilage above the growth plate. Parameters measured included bone mineral density, bone volume, bone-specific surface, percent bone volume, trabecular thickness, trabecular number, trabecular spacing, structure model index, and connectivity density. [2]
In vivo animal studies for menatetrenone include models of osteoporosis, such as ovariectomized rats. Animals are treated with menatetrenone via oral administration. Endpoints include bone mineral density, bone turnover markers, and histomorphometric analysis of bone. Its hemostatic effects may be assessed in models of bleeding disorders.

Pharmacokinetic properties of menatetrenone include a molecular weight of 444.6 and a molecular formula of C₃₁H₄₀O₂. It is a lipophilic molecule and is absorbed with dietary fats. It is the predominant form of vitamin K2 in animal tissues. It is stored in the liver and other tissues. Its half-life is longer than that of phylloquinone (vitamin K1).

Menatetrenone is generally well-tolerated at therapeutic doses. As a vitamin K analog, it can interfere with anticoagulant therapy, particularly with warfarin. It may cause gastrointestinal upset in some individuals. It is contraindicated in patients taking anticoagulants without medical supervision. High doses may lead to an increased risk of thrombosis in susceptible individuals.

[1]. Menaquinone-4 (vitamin K2) up-regulates expression of human intestinal alkaline phosphatase in Caco-2 cells. Nutr Res. 2016 Nov;36(11):1269-1276.
[2]. Vitamin K1 (phylloquinone) and K2 (menaquinone-4) supplementation improves bone formation in a high-fat diet-induced obese mice. J Clin Biochem Nutr. 2013 Sep;53(2):108-13

Menadione-4 is a menadione compound with four all-trans isoprene units in its side chain. It plays a role in maintaining bone density, acting as a metabolite, an antioxidant, an anti-inflammatory agent, and a neuroprotective agent. Menadione-4 has been used in clinical trials for the treatment of diabetes, osteoporosis, prediabetes, and hepatocellular carcinoma. Menadione-4 is a menadione compound and a form of vitamin K2, possessing potential antitumor activity. Menadione-4 may exert its effects by modulating the signaling of certain tyrosine kinases, thereby affecting multiple transcription factors, including c-myc and c-fos. This drug inhibits tumor cell growth by inducing apoptosis and cell cycle arrest.

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- Menaquinone-4 (Vitamin K2) plays an important role in improving bone metabolism. It promotes osteocalcin carboxylation of γ-glutamic acid produced by osteoblasts, aiding in bone formation. [2]
- Vitamin K2 supplementation in patients with osteoporosis necessitated by glucocorticoid administration inhibited OPG decrease and had bone loss prevention effects. [2]
- Vitamin K2 supplementation in patients with rheumatoid arthritis accompanied by osteoporosis decreased RANKL levels and inhibited osteoclast activation. [2]
- In this study, vitamin K2 had a greater effect on preventing bone density decrease than vitamin K1 in high-fat diet-induced obese mice. [2]

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- Alkaline phosphatase (ALP) hydrolyzes several monophosphate esters into inorganic acid and alcohol. In humans, four kinds of ALP isozymes have been identified: tissue-nonspecific ALP, intestinal ALP, placental ALP, and germ cell ALP. Intestinal ALP is expressed at a high concentration in the brush border membrane of intestinal epithelial cells and is known to be affected by several kinds of nutrients, such as lipids, but its physiological function has remained elusive. [1]
- Vitamin K is an essential cofactor for the posttranslational carboxylation of glutamate residues into γ-carboxy glutamate. [1]
- Menaquinone-4 (MK-4) with 4 isoprene units (vitamin K2) has been previously shown to induce bone-type ALP activity and osteoblastogenesis in human bone marrow cells. [1]
- This is the first report concerning ALP messenger RNA expression induced by vitamin K2 in Caco-2 cells. Further studies on the physiological functions of human intestinal ALP will provide useful data on the novel effects of vitamin K. [1]
Menatetrenone (Vitamin MK4, E3100) is a menaquinone compound and a form of vitamin K2. It is also known as Menaquinone-4 (MK-4). It is one of the nine naturally occurring forms of vitamin K2. It is used as a hemostatic agent and as adjunctive therapy for osteoporosis. It is marketed in Japan for the treatment of osteoporosis. It is not approved in all jurisdictions.

C31H40O2

444.6481

444.302

C, 83.74; H, 9.07; O, 7.20

863-61-6

5282367

Light yellow to yellow <35°C solid powder,>35°C liquid

1.0±0.1 g/cm3

570.6±50.0 °C at 760 mmHg

350ºC

208.3±27.1 °C

0.0±1.6 mmHg at 25°C

1.539

10.94

0

2

11

33

855

0

O=C1C(C/C=C(\C)/CC/C=C(\C)/CC/C=C(\C)/CC/C=C(\C)/C)=C(C)C(=O)C2C=CC=CC1=2

DKHGMERMDICWDU-GHDNBGIDSA-N

InChI=1S/C31H40O2/c1-22(2)12-9-13-23(3)14-10-15-24(4)16-11-17-25(5)20-21-27-26(6)30(32)28-18-7-8-19-29(28)31(27)33/h7-8,12,14,16,18-20H,9-11,13,15,17,21H2,1-6H3/b23-14+,24-16+,25-20+

2-methyl-3-((2E,6E,10E)-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-yl)naphthalene-1,4-dione

E-3100 E 3100 E3100 Ea0167 Ea 0167 Ea-0167 MK4MK-4 MK 4Vitamin K-2(20) Vitamin MK-4 Vitamin K 2(20) Vitamin MK 4 Vitamin K2(20) Vitamin MK4

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~10 mg/mL (~22.49 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.68 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2 mg/mL (4.50 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2 mg/mL (4.50 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)