**1. Introduction**

The anticoagulant warfarin, which has a coumarin structure, causes hemorrhage by inhibiting the vitamin K (VK)–dependent synthesis of blood coagulation factors [1]. Blood coagulation factors undergo γ-carboxylation and have the ability to bind to calcium ions on the platelet surface, under the presence of VK and molecular oxygen. Vitamin K1 2,3-epoxide (VKO), which is produced while this γ-carboxylation, is converted to vitamin K1 (VK1) by vitamin K1 2,3-

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

epoxide reductase (VKOR) and is then recycled. Warfarin arrests the regeneration of VK by inhibiting VKOR and induces hemorrhage due to a VK deficiency.

DHA-S, which occurred in multiple organs and showed differences in sensitivity among individuals, was similar to that caused by warfarin. The prothrombin time (PT) and activated partial thromboplastin time (APTT) were significantly prolonged in each surviving rat in the 100 and 200 mg/kg/day groups (**Figure 1**). These results indicated that the repeated dosing of

Toxicological and Pharmacological Effects of VKOR Inhibitors

http://dx.doi.org/10.5772/63512

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**Figure 1.** Effects on PT and APTT in male rats received the repeated oral administration of DHA-S at doses of 50, 100, and 200 mg/kg/day. ( ) Vehicle; ( ) 50 mg/kg/day; ( ) 100 mg/kg/day; ( ) 200 mg/kg/day. Each value is presented

On the other hand, DHA-S-treated rats showed no significant decrease in PLT. In the histopathological examination of the liver, marked changes, such as fibrin thrombi in the vascular system and single cell necrosis, were not observed. Therefore, hemorrhage was assumed to be caused by a deficiency in VK. In order to demonstrate this hypothesis, the effects of VK on hemorrhage were investigated using rats that received a single subcutaneous injection of VK2 following the repeated oral administration of DHA-S at 200 mg/kg/day for 5 days. In rats, dietary VK1 was converted to menadione in the gut and menadione was absorbed and converted to VK2 [16]. Consequently, VK2 (menaquinone-4) reached a maximum concentration in the liver in rats faster than after the intravenous or intraperitoneal administration of VK1 [17]. VK2 was also reported to exert stronger preventive effects against hemorrhage than other VK products, such as VK1 and VK3. As a result, DHA-S prolonged blood coagulation parameters (PT, APTT, the thrombo test (TTO), and hepaplastin test (HPT), in rats that received 200 mg/kg/day for 5 days (**Figure 2**). However, the prolongation of these parameters was suppressed in animals injected subcutaneously with VK2 after DHA-S dosing. Since TTO and HPT are sensitive indexes of a deficiency in VK-dependent blood coagulation factors [18, 19], it was

as the mean ± SD. \* *P* < 0.05, \*\* *P* < 0.01: Significantly different from controls at 5% and 1%.

concluded that DHA-S induced hemorrhage via a deficiency in VK.

DHA-S-induced hemorrhage in rats [8].

Sodium dehydroacetate (DHA-S) has a wide spectrum of antimicrobial activity that accounts for its use in cosmetic products, as a preservative, and as an antimicrobial agent [2]. In Japan, it has also been used as a food preservative for cheese, butter, and margarine at a concentration of 0.5 g/kg or less as dehydroacetic acid [3, 4]. This concentration is the highest dose allowed under the Food Sanitation Law; however, the risk assessment of DHA-S has not been sufficiently performed for a food additive, and its acceptable daily intake has not yet been established. On the other hand, the toxicity of DHA-S has been widely studied. In an acute toxicity study of DHA-S using dogs, clinical signs such as salivation, vomiting, convulsions, and ataxia were reported [5]. In a subchronic toxicity study of DHA-S using dogs, body weight loss, gastric hemorrhage, and an increase in blood urea nitrogen were observed [6]. These findings were considered to be primarily due to a lack of appetite and subsequent weight loss [6]. However, the main structure of DHA-S, 2H-pyran-2-on, is included in a coumarin structure. It is reported that some derivatives of 4-hydroxy-2-pyrone, which includes similar structure of DHA-S, exhibit anti-blood coagulant activities in rats [7]. These findings suggest that DHA-S also shows the anticoagulant activity. Therefore, we investigated the effects of repeated administration of DHA-S in rats, and the amelioration by VK against DHA-S-induced hemorrhage [8].

In Thy-1 glomerulonephritis (GN) rats, the expression of growth arrest-specific gene 6 (Gas6) and Axl was found to be markedly increased in glomeruli and paralleled the proliferation of mesangial cells [9, 10]. VKOR is also related with mesangial cell proliferation through the vitamin K–dependent activation of Gas6 [10]. Gas6 is as an autocrine growth factor for mesangial cells through binding to its cell surface receptor Axl. These findings suggest that the Gas6/Axl pathway plays an important role in mesangial cell proliferation in GN [11]. VKOR inhibitors including warfarin is effective at blocking mesangial cell proliferation and improving renal function [11]; however, it inhibits the production of blood coagulation factors in the liver and has been able to cause bleeding [12]. Therefore, low-dose treatments of warfarin are used in renal failure patients, and the control of its blood concentration or the conformation of blood coagulation activities have to been required [13]. We are discovering new compounds that inhibit mesangial cell proliferation, but not hepatic production of blood coagulation factors using Thy-1 GN rats **Table 2**. Thy-1 GN rats have been used as an acute model of GN accompanied with mesangial cell proliferation, matrix expansion, and moderate decline of renal function [14]. We indicated the potential of a new VKOR inhibitor, 3-acetyl-5-methyltetronic acid (AMT), as an inhibitor of mesangial cell proliferation and suppressor of renal disease [15].
