Eldecalcitol replaces endogenous calcitriol but does not fully compensate for its action in vivo

Calcitriol (1α,25-dihydroxyvitamin D3, 1α,25(OH)2D3) is an essential hormone that works in cooperation with parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF-23) to regulate calcium and phosphorus homeostasis. Previous in vivo studies in rats have shown that eldecalcitol, a vitamin D analog, is more active than calcitriol in stimulating calcium and phosphorus absorption in the intestine and in increasing serum FGF-23, but is not as active in suppressing blood PTH. However, those results are problematic because administration of exogenous eldecalcitol or calcitriol affects the synthesis and degradation of endogenous calcitriol, and competes for binding to vitamin D receptor (VDR) in target tissues. Therefore, we tried to evaluate the 'true biological activity in vivo' of each compound by comparing their biological activities with respect to their blood concentrations. In VDR gene knockout mice, calcitriol and eldecalcitol did not affect either serum or urinary calcium levels, and also did not induce the expression of target genes. These results indicate that the actions of eldecalcitol are mediated by VDR. In normal rats, concentrations of both calcitriol and eldecalcitol in the blood increased dose-dependently and had a linear correlation with administered dosage. The concentration of calcitriol in the blood was reduced by eldecalcitol treatment, falling to below the limit of detection at 0.1μg/kg eldecalcitol. Based on the concentration of each compound in the blood, eldecalcitol had approximately 1/4 to 1/7 the activity of calcitriol to increase serum calcium, FGF-23, and urinary calcium excretion, and to suppress blood PTH. Eldecalcitol dose-dependently increased urinary phosphorus excretion and reduced serum phosphorus. However, calcitriol did not change serum phosphorus. In accordance with serum chemistry and hormones, a concentration of eldecalcitol in the blood of 3-8 times that of calcitriol was required to stimulate target gene expressions in the kidneys (VDR, TRPV5, and calbindin-D28k) and bone (VDR, FGF-23, and RANKL). On the other hand, the blood concentrations of eldecalcitol needed to stimulate target genes in the intestine (TRPV6, calbindin-D9k, and VDR) were comparable to those of calcitriol. These results indicate that oral administration of eldecalcitol stimulates target gene expression in the intestine similarly to calcitriol, but to a much lesser extent than calcitriol in the kidneys and bones. The major finding of the present study is that eldecalcitol suppresses endogenous calcitriol and replaces it. However, it may not fully compensate for the action of calcitriol in vivo. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.


Introduction
Calcitriol (1␣,25-dihydroxyvitamin D 3 , 1␣,25(OH) 2 D 3 ) exerts a wide variety of biological actions in many target organs. Calcitriol regulates calcium and phosphorus homeostasis, mineral metabolism, and bone metabolism. Through the action of calcitriol, in cooperation with parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF-23), the absorption of intestinal calcium and phosphorus, resorption of bone phosphorus and calcium, and reabsorption of renal calcium and phosphorus are increased, resulting in a rise in the serum calcium and phosphorus available for bone mineralization [1][2][3].
The biological actions of calcitriol are mediated through vitamin D receptor (VDR). VDR is a member of the nuclear hormone receptor gene family and is a ligand-dependent transcription factor [8][9][10]. The physiological importance of VDR in maintaining the integrity of mineral metabolism is indicated by the observation that patients with vitamin D deficiency and VDR gene knockout (VDRKO) mice both develop hypocalcemia and rickets or osteomalacia [11][12][13].
The intestinal and renal transepithelial transport of calcium in response to calcitriol is mediated by apical calcium ion channels of the transient receptor potential vanilloid subfamily 5 and 6 (TRPV5 and TRPV6), followed by cytosolic transport by calcium binding proteins (calbindin-D9k and calbindin-D28k) and extrusion across the basolateral membrane into the extracellular fluid by plasma membrane calcium ATPase (PMCA1b) and/or sodiumcalcium exchanger (NCX1) [14].
Eldecalcitol (1␣,25-dihydroxy-2␤-(3-hydroxypropyloxy) vitamin D 3 ), a new active vitamin D 3 analog, has recently been approved for the treatment of osteoporosis in Japan. A Phase III clinical trial in patients with osteoporosis showed that eldecalcitol increased bone mineral density (BMD) and reduced the incidence of vertebral fracture with an efficacy greater than that of alfacalcidol [15]. It has also been shown that eldecalcitol promotes urinary calcium excretion similarly to alfacalcidol, but has a lower potency to suppress blood PTH [16]. Eldecalcitol increases BMD and reduces bone turnover markers in normal, ovariectomized (OVX), and steroid-treated rats, and also in patients with osteoporosis [17][18][19][20][21]. Eldecalcitol is more active than calcitriol in stimulating calcium and phosphorus absorption in the intestine, as well as in increasing serum FGF-23 in normal rats [22]. However, administration of exogenous eldecalcitol or calcitriol affects the synthesis and/or degradation of endogenous calcitriol, and exogenous eldecalcitol or calcitriol competes with endogenous calcitriol for binding to VDR in target tissues. In the current study, we tried to evaluate the 'true biological activity in vivo' of each compound by comparing their biological activities with respect to their blood concentrations.

Experiment 1
VDRKO mice were kindly provided by Dr. S. Kato [11]. VDRKO mice were fed ad libitum with a rescue diet containing 2% calcium, 1.25% phosphorus, and 20% lactose (CLEA Japan, Tokyo, Japan) [23]; wild-type (WT) mice were fed normal rodent chow (CE-2; CLEA Japan). All animals were given free access to tap water and were maintained under specific pathogen free conditions with a 12-h light and dark cycle at 20-26 • C and humidity of 35-75%. The 9week-old VDRKO and WT mice were divided into 3 groups based on body weight. Mice were administered either a vehicle control (medium chain triglyceride; The Nisshin Oillio Group, Tokyo, Japan) (MCT), eldecalcitol (0.2 g/kg), or calcitriol (2 g/kg) by once-a-day oral gavage for 14 days (n = 5). Blood, kidney, and intestine samples were collected 6 h after the last dosing.

Experiment 2
Six-week-old male Sprague-Dawley rats were purchased from CLEA Japan. Animals were fed with normal rodent chow and tap water and acclimated to the above conditions for 1 week. Rats were divided into 11 groups based on body weight. Various doses of eldecalcitol (0.025, 0.05, 0.1, 0.25, and 0.5 g/kg), calcitriol (0.25, 0.5, 1, 2.5, and 5 g/kg), or MCT vehicle were administered by oncea-day oral gavage for 14 days (n = 6). On the 13th day, rats were transferred to and kept in metabolic cages for 24 h to collect urine samples. Blood, bone, kidney, and intestine samples were collected at 6 h after the last dosing on the 14th day.
Both animal studies were carried out in accordance with Chugai Pharmaceutical's ethical guidelines of animal care, and the experimental protocols were approved by the animal care committee of the institution.

Biochemical analysis
Levels of calcium, phosphorus, and creatinine in serum and urine were determined by using an automatic analyzer (TBA-120FR; Toshiba Medical Systems, Tochigi, Japan). PTH in plasma was measured by rat intact PTH ELISA kit (Immutopics International, San Clemente, CA, USA). FGF-23 in serum was measured by FGF-23   ELISA kit (Kainos Laboratories, Tokyo, Japan). Calcitriol in serum was measured by 1,25(OH) 2 D RIA kit (TFB, Tokyo, Japan). Measurement of eldecalcitol in plasma was performed at the BoZo Research Center (Tokyo, Japan).

Quantitative RT-PCR
The right femur, intestine, and kidneys of mice, and the right femur, intestine, and kidneys of rats were excised and immediately frozen in liquid nitrogen. A small portion of each of the frozen tissues was soaked in TRIzol (Invitrogen, Carlsbad, CA, USA) and crushed in a homogenizer (Physcotron NS-310E; Microtec, Chiba, Japan). Total RNA was extracted with an RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA was synthesized from 200 ng of total RNA by reverse transcription PCR using TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, USA). The reaction was performed at 37 • C for 1 h. Expression of mRNA in the tissues was detected using TaqMan Gene Expression Assays (Applied Biosystems). Target cDNA was amplified by 40 cycles (1 cycle: 95 • C for 15 s, 60 • C for 1 min) of PCR in an ABI PRISM 7000 Sequence Detector System (Applied Biosystems). The TaqMan probes used in this study were TRPV5, TRPV6, calbindin-D28k, and calbindin-D9k

Effects of eldecalcitol on calcium metabolism in VDRKO mice
A dose of 2 g/kg calcitriol or 0.2 g/kg eldecalcitol administered daily by oral gavage for 14 days significantly increased serum calcium and urinary calcium excretion compared with vehicle administration in WT mice. However, neither eldecalcitol nor calcitriol affected serum or urinary calcium in the VDRKO mice ( Fig. 1A and B). Calcitriol and eldecalcitol significantly increased the expression of renal TRPV5 and calbindin-D28k mRNA and the expression of intestinal TRPV6 and calbindin-D9k mRNA in the WT mice. On the other hand, the expression of these genes in the VDRKO mice was not altered by the treatment (Fig. 1C-F). These results indicate that the calcemic actions of calcitriol and eldecalcitol are mediated by VDR.

Effects of eldecalcitol on calcium and phosphorus metabolism in normal rats
Eldecalcitol (0.025, 0.05, 0.1, 0.25, and 0.5 g/kg) or calcitriol (0.25, 0.5, 1, 2.5, and 5 g/kg) administered daily by oral gavage for 14 days dose-dependently increased the blood concentration of each compound. The blood concentration of each compound correlated well with the administered dosage (eldecalcitol: y (pmol/L) = 29,834x (g/kg) + 646.3, R 2 = 0.996; calcitriol: y (pmol/L) = 681.81x (g/kg) + 402.1, R 2 = 0.971) ( Fig. 2A and B). This result indicates that in order to reach the same concentration in the blood, the amount of eldecalcitol required is approximately 1/40 that of calcitriol. In the eldecalcitol-treated rats, serum concentration of calcitriol dose-dependently decreased and fell to below the limit of detection at 0.1 g/kg (Fig. 2C). Treatment with eldecalcitol and calcitriol significantly reduced renal CYP27B1 gene expression and dose-dependently increased renal CYP24A1 gene expression ( Fig. 2D and E). These results suggest that the administration of eldecalcitol and calcitriol reduces endogenous production of calcitriol and stimulates degradation of calcitriol in the kidneys.
Blood concentrations of eldecalcitol and calcitriol correlated with urinary phosphorus excretion. Serum phosphorus slightly decreased along with the increase in eldecalcitol concentration in blood, whereas calcitriol concentration did not alter serum phosphorus ( Fig. 3A and B). Serum calcium was significantly elevated at higher blood concentrations of eldecalcitol (≥7520 pmol/L) and calcitriol (≥1170 pmol/L) (Fig. 3C). Urinary calcium excretion correlated with blood concentrations of calcitriol and eldecalcitol (Fig. 3D). Serum FGF-23 increased at 15,800 pmol/L of eldecalcitol and at ≥2480 pmol/L of calcitriol in blood (Fig. 3E). High concentrations of eldecalcitol in the blood (≥7520 pmol/L) suppressed plasma PTH concentration, whereas plasma PTH concentration was reduced from low blood calcitriol concentrations (≥590 pmol/L) (Fig. 3F).

Effects of eldecalcitol on target gene expression in normal rats
In the intestine, TRPV6 gene expression was induced from relatively low blood concentrations of eldecalcitol (≥1220 pmol/L) (Fig. 4A). Similarly, induction of TRPV6 gene expression was observed from low concentrations of calcitriol (590 pmol/L and ≥2480 pmol/L). Calbindin-D9k gene expression was unchanged by the administration of calcitriol or eldecalcitol (Fig. 4B). In the kidneys, TRPV5 mRNA expression was significantly elevated at the highest concentration of eldecalcitol (15,800 pmol/L) and at high concentrations of calcitriol (≥1170 pmol/L) (Fig. 4C). Calbindin-D28k mRNA was increased at the higher blood concentrations of eldecalcitol (≥7520 pmol/L) and calcitriol (≥1170 pmol/L) (Fig. 4D). In bone, blood concentrations of calcitriol correlated with RANKL and FGF-23 gene expression; however, only the highest concentration of eldecalcitol (15,800 pmol/L) induced RANKL and FGF-23 gene expression (Fig. 4E).
Blood concentration of calcitriol correlated with VDR gene expression in the kidneys and bone ( Fig. 5B and C), but calcitriol did not affect VDR gene expression in the intestine (Fig. 5A). Induction of VDR gene expression in the intestine and kidneys were associated with increasing concentration of eldecalcitol in the blood (Fig. 5A and B). In bone, significant induction of VDR gene expression was observed only at the highest concentration of eldecalcitol (Fig. 5C).
Taken together, these results show that, in comparison to calcitriol, relatively higher concentrations of eldecalcitol in the blood were required to stimulate expression of vitamin D target genes in the kidneys (VDR, TRPV5, and calbindin-D28k) and bone (VDR, RANKL, and FGF-23).

Comparison of the 'true in vivo biological activity' of eldecalcitol and calcitriol
In order to compare the true biological activity of calcitriol and eldecalcitol in vivo, the blood concentration required to elicit a 50% response in each activity was calculated from the raw data above. The ratio of biological activity was obtained by dividing the 50% response concentration of calcitriol by that of eldecalcitol. Based on these calculations, eldecalcitol was approximately 1/4 to 1/7 as active as calcitriol in increasing serum calcium and FGF-23, in stimulating urinary calcium excretion, and in suppressing plasma PTH (Table 1). Eldecalcitol was approximately 1/3 to 1/8 as active as calcitriol in stimulating expression of target genes in the kidneys (VDR, TRPV5, and calbindin-D28k) and bone (VDR, FGF-23, and RANKL). The biological activities of eldecalcitol in increasing intestinal TRPV6 and VDR gene expression were comparable to those of calcitriol (Figs. 4A and 5A).

Discussion
The half-life of eldecalcitol in the blood is much longer than it is for calcitriol [23]. Although eldecalcitol strongly induces CYP24A1 in the intestine and kidneys [24], eldecalcitol itself is hardly degraded by CYP24A1 [25]. At the same time, eldecalcitol strongly suppresses CYP27B1 in the kidneys. Blood concentration of eldecalcitol during treatment in clinical trials was reported to be relatively high (200-250 pg/mL) in comparison to the normal range of calcitriol (30-60 pg/mL) in humans [15]. An approximately 50% reduction in blood calcitriol was observed during eldecalcitol treatment in the clinical trial. In the present study, we demonstrated by using VDRKO mice that the calcemic actions of calcitriol and eldecalcitol were mediated solely by VDR. Administration of small amounts of eldecalcitol in rats markedly reduced serum concentration of calcitriol, which fell to below the limit of detection at 0.1 g/kg eldecalcitol. Plasma concentration of eldecalcitol increased dose-dependently and reached 3820 pmol/L by 0.1 g/kg eldecalcitol administration. These observations indicate that, after administration of eldecalcitol, the eldecalcitol rapidly replaces calcitriol in blood and exerts biological activities in target organs. It was observed in an earlier study that the binding activity of eldecalcitol to VDR is approximately 1/8 of that of calcitriol in vitro [26] and that the distribution capacity of eldecalcitol to target organs is much lower than that of calcitriol in rats.
In this study, based on the concentration of each compound in the blood, the relative biological activities of eldecalcitol, such as its activity in increasing serum calcium, FGF-23, and urinary calcium excretion, and in suppressing plasma PTH in vivo were only 15-26% of that of calcitriol (Table 1). Eldecalcitol stimulated the expression of target genes in the kidneys (VDR, TRPV5, and calbindin-D28k) and bone (VDR, FGF-23, and RANKL) much less than did calcitriol. Stimulation of target genes in the intestine by eldecalcitol treatment was comparable to that of calcitriol. These results indicate that eldecalcitol is primarily a weak agonist of VDR as compared with calcitriol in vivo.
Thus, we conclude that administration of eldecalcitol rapidly suppresses endogenous calcitriol and replaces it. However, eldecalcitol may not fully compensate for the action of calcitriol in the kidneys and bone.