It has been reported that vitamin K2 (menaquinone-4) promoted 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-induced mineralization and enhanced gamma-carboxyglutamic acid (Gla)-containing osteocalcin accumulation in cultured human osteoblasts. In the present study, we investigated whether menaquinone-4 (MK-4) was metabolized in human osteoblasts to act as a cofactor of gamma-glutamyl carboxylase. Both conversions of MK-4 to MK-4 2,3-epoxide (epoxide) and epoxide to MK-4 were observed in cell extracts of cultured human osteoblasts. The effect of 1,25(OH)2D3 and warfarin on the vitamin K cycle to cultured osteoblasts were examined. With the addition of 1 nM 1,25(OH)2D3 or 25 microM warfarin in cultured osteoblasts, the yield of epoxide from MK-4 increased. However, the conversion of epoxide to MK-4 was strongly inhibited by the addition of warfarin (2.5-25 microM), whereas it was almost not inhibited by 1,25(OH)2D3 (0.1-10 nM). To clarify the mechanism for this phenomenon, a cell-free assay system was studied. Osteoblast microsomes were incubated with 10 microM epoxide in the presence or absence of warfarin and 1,25(OH)2D3. Epoxide reductase, one of the enzymes in the vitamin K cycle was strongly inhibited by warfarin (2.5-25 microM), whereas it was not affected by 1,25(OH)2D3 (0.1-1 nM). Moreover, there was no effect of pretreatment of osteoblasts with 1 nM 1,25(OH)2D3 on the activity of epoxide reductase. However, the activity of epoxidase, that is the gamma-glutamyl carboxylase was induced by the pretreatment of osteoblasts with 1 nM 1,25(OH)2D3. In the present study, it was demonstrated that the vitamin K metabolic cycle functions in human osteoblasts as well as in the liver, the post-translational mechanism, by which 1,25(OH)2D3 caused mineralization in cooperation with vitamin K2 was clarified.

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1. We have examined changes in caffeine and trimethadione (TMO) metabolism in vivo, agents which are used as probe drugs. In this study the total body clearance (Cl) of caffeine and TMO was low 1 week after birth (week 1), increased rapidly from week 3, peaked and then decreased gradually until reaching the level for the mature, adult dog. The elimination half-life (t1/2) of caffeine and TMO was prolonged during week 1; however, it then gradually became shorter. Gradually it became longer and reached the level for the adult dog. The apparent volume of distribution (Vd) of caffeine did not change throughout the study. However, the Vd of TMO was only high during week 1. 2. The in vitro changes in a variety of typical substrates for seven different cytochrome P450 (CYP) isozymes were examined. In this study three different patterns of metabolism can be identified: (1) activity is low immediately after birth, increases, peaks and then decreases to the adult dog level (p-nitroanisole; CYP1A1, caffeine; CYP1A2, benzphetamine; CYP3A/2B(?), aniline; 2E1 and TMO; CYP2C9/2E1/3A4); (2) activity generally increases rapidly soon after birth, continues to increase, peaks and then gradually decreases to the adult level (phenytoin; CYP2C9); and (3) activity is high (about the same level as the adult) immediately after birth, decreases and then gradually increases to the adult level (erythromycin; CYP3A4/5). 3. The results of these in vivo and in vitro studies suggest that changes in enzyme activity are due to differences in P450 isoenzymes during development.

Summary: [14C]menaquinone-4, a therapeutic agent for osteoporosis, was administered orally to male dogs at a dose of 4 mg/kg, and the absorption, distribution, metabolism and excretion were investigated.1. The plasma concentration of radioactivity reached the maximum level (Cm) of 4540.4 ng eq./ml at 1 to 2 hours after administration and thereafter decreased slowly. At Tmax, the unchanged menaquinone-4 in the plasma corresponded to 79.6% of the total radioactivity.2. Within 168 hours, 2.3 and 78.1% of the radioactivity was excreted into the urine and feces, respective ly. Unchanged menaquinone-4 corresponded to 28.5 0/ of the radioactivity in the feces excreted within 24 hours.3. The liver (33164.0 ng eq./g) and spleen (12542.2 ng eq./g) showed much higher concentrations of radioactivity than the other tissues and plasma (2994.5 ng eq./ml) at 1.5 hours after administration. The concentration in bile (109392.1 ng eq./ml) was also high, suggesting that the menaquinone-4 was immedi ately excreted into the bile. The elimination of radioactivity from tissues was relatively slow compared to that from plasma. In bone tissues, the target organ of the drug, the marrow (1424.9 ng eq./g) and cancel lous tissue (2465.7 ng eq./g) of thighbone and the marrow (3533.4 ng eq./g) of ribs showed levels as high as the plasma concentrations at 1.5 hours and were 2 to 3 times higher than that of the plasma at 24 hours af ter dosing.4. The unchanged menaquinone-4 was the major form present in the plasma, liver, kidney, spleen, adrenal, adipose and cancellous tissue of thighbone at 1.5 hours after administration. The known metabo lites, co-COOH, K acid I and K acid II, were found in these tissues at 1.5 hours. In the adipose and cancel lous tissue of thighbone, the unchanged menaquinone 4 was the major form up to 168 hours, while the rela tive amount of metabolites increased by 24 hours in the other tissues. Polar metabolites were found in the urine excreted within 24 hours and bile at 1.5 and 24 hours after administration. The unchanged menaqui none-4 was mainly found in the feces excreted within 24 hours.5. The levels of radioactivity in the marrow and cancellous tissue of thighbone and the marrow of ribs at 1.5 and 24 hours after dosing (1.5 hours: 3.3-8.0 x 10-6 M, 24 hours: 1.9-3.1 x 10-6 M) were comparable to the pharmacologically effective concentrations of menaquinone-4 (10-6-10-5 M) in in vitro studies on bone formation. This finding suggests that menaquinone-4 distributes sufficiently into bone tissues after a single oral administration in dogs, and can be expected to maintain pharmacologically effective concentrations by repeated administration as a therapeutic agent for osteoporosis.