Eldecalcitol Replaces Endogenous Calcitriol but Does Not Fully

Journal of Steroid Biochemistry & Molecular Biology 144 (2014) 189–196

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Original Article

Eldecalcitol replaces endogenous calcitriol but does not fully

ଝ

compensate for its action in vivo

∗,1 1

Hitoshi Saito , Suguru Harada

Medical Affairs Division, Chugai Pharmaceutical Co., Ltd., Tokyo 103-8324, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Calcitriol (1␣,25-dihydroxyvitamin D3, 1␣,25(OH)2D3) is an essential hormone that works in cooper-

Received 1 August 2013

ation with parathyroid hormone (PTH) and ﬁbroblast growth factor-23 (FGF-23) to regulate calcium

Received in revised form

and phosphorus homeostasis. Previous in vivo studies in rats have shown that eldecalcitol, a vitamin D

17 November 2013

analog, is more active than calcitriol in stimulating calcium and phosphorus absorption in the intestine

Accepted 19 November 2013

and in increasing serum FGF-23, but is not as active in suppressing blood PTH. However, those results

Available online 26 November 2013

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 tis-

Keywords:

Eldecalcitol sues. Therefore, we tried to evaluate the ‘true biological activity in vivo’ of each compound by comparing

VDR their biological activities with respect to their blood concentrations.

Calcium In VDR gene knockout mice, calcitriol and eldecalcitol did not affect either serum or urinary calcium

Phosphorus 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 con-

centration of calcitriol in the blood was reduced by eldecalcitol treatment, falling to below the limit of

detection at 0.1 ␮g/kg eldecalcitol. Based on the concentration of each compound in the blood, eldecalci-

tol 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 phos-

phorus 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’.

Contents

1. Introduction ...... 190

2. Materials and methods ...... 190

2.1. Chemicals ...... 190

2.2. Animals ...... 190

2.2.1. Experiment 1...... 190

2.2.2. Experiment 2...... 190

ଝ

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in

any medium, provided the original author and source are credited.

∗

Corresponding author at: Medical Affairs Division, Chugai Pharmaceutical Co., Ltd., 2-1-1 Nihonbashi-Muromachi, Chuo-ku, Tokyo 103-8324, Japan.

Tel.: +81 3 3516 5299; fax: +81 3 3281 0191.

E-mail addresses: [email protected], hitoshi [email protected] (H. Saito).

These authors contributed equally to this work.

190 H. Saito, S. Harada / Journal of Steroid Biochemistry & Molecular Biology 144 (2014) 189–196

2.3. Biochemical analysis...... 190

2.4. Quantitative RT-PCR ...... 191

2.5. Statistical analysis ...... 192

3. Results ...... 192

3.1. Effects of eldecalcitol on calcium metabolism in VDRKO mice ...... 192

3.2. Effects of eldecalcitol on calcium and phosphorus metabolism in normal rats ...... 193

3.3. Effects of eldecalcitol on target gene expression in normal rats ...... 194

3.4. Comparison of the ‘true in vivo biological activity’ of eldecalcitol and calcitriol ...... 195

4. Discussion ...... 195

References ...... 196

1. Introduction 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

Calcitriol (1␣,25-dihydroxyvitamin D3, 1␣,25(OH)2D3) exerts

biological activities with respect to their blood concentrations.

a wide variety of biological actions in many target organs. Cal-

citriol regulates calcium and phosphorus homeostasis, mineral

2. Materials and methods

metabolism, and bone metabolism. Through the action of calcitriol,

in cooperation with parathyroid hormone (PTH) and ﬁbroblast

2.1. Chemicals

growth factor-23 (FGF-23), the absorption of intestinal calcium and

phosphorus, resorption of bone phosphorus and calcium, and reab-

Calcitriol was purchased from Wako Pure Chemical Industries

sorption of renal calcium and phosphorus are increased, resulting

(Osaka, Japan). Eldecalcitol was synthesized by Chugai Pharmaceu-

in a rise in the serum calcium and phosphorus available for bone

tical Co., Ltd. (Tokyo, Japan).

mineralization [1–3].

Vitamin D3 is ﬁrst metabolized to 25-hydroxyvitamin D3

2.2. Animals

[25(OH)D3] in the liver, then to calcitriol (1␣,25(OH)2D3) in the

kidneys. In this pathway, renal 1␣-hydroxylation (by 25-OHD-1␣-

2.2.1. Experiment 1

hydroxylase, CYP27B1) is a rate-limiting step in the production of

VDRKO mice were kindly provided by Dr. S. Kato [11]. VDRKO

calcitriol. Degradation of 25(OH)D3 and calcitriol is mediated by

mice were fed ad libitum with a rescue diet containing 2% calcium,

renal 24-hydroxylase (CYP24A1). The concentration of calcitriol in

1.25% phosphorus, and 20% lactose (CLEA Japan, Tokyo, Japan) [23];

the blood is tightly regulated by a feedback loop controlling expres-

wild-type (WT) mice were fed normal rodent chow (CE-2; CLEA

sion of renal CYP27B1 and CYP24A1. Expression of these enzymes

Japan). All animals were given free access to tap water and were

is strictly regulated by PTH, FGF-23, and the vitamin D status of

maintained under speciﬁc pathogen free conditions with a 12-h animals [3–7].

◦

light and dark cycle at 20–26 C and humidity of 35–75%. The 9-

The biological actions of calcitriol are mediated through vitamin

week-old VDRKO and WT mice were divided into 3 groups based

D receptor (VDR). VDR is a member of the nuclear hormone receptor

on body weight. Mice were administered either a vehicle control

gene family and is a ligand-dependent transcription factor [8–10].

(medium chain triglyceride; The Nisshin Oillio Group, Tokyo, Japan)

The physiological importance of VDR in maintaining the integrity

(MCT), eldecalcitol (0.2 ␮g/kg), or calcitriol (2 ␮g/kg) by once-a-day

of mineral metabolism is indicated by the observation that patients

oral gavage for 14 days (n = 5). Blood, kidney, and intestine samples

with vitamin D deﬁciency and VDR gene knockout (VDRKO) mice

were collected 6 h after the last dosing.

both develop hypocalcemia and rickets or osteomalacia [11–13].

The intestinal and renal transepithelial transport of calcium

in response to calcitriol is mediated by apical calcium ion chan- 2.2.2. Experiment 2

nels of the transient receptor potential vanilloid subfamily 5 and Six-week-old male Sprague-Dawley rats were purchased from

6 (TRPV5 and TRPV6), followed by cytosolic transport by calcium CLEA Japan. Animals were fed with normal rodent chow and tap

binding proteins (calbindin-D9k and calbindin-D28k) and extru- water and acclimated to the above conditions for 1 week. Rats were

sion across the basolateral membrane into the extracellular ﬂuid divided into 11 groups based on body weight. Various doses of elde-

␮

by plasma membrane calcium ATPase (PMCA1b) and/or sodium- calcitol (0.025, 0.05, 0.1, 0.25, and 0.5 g/kg), calcitriol (0.25, 0.5,

␮

calcium exchanger (NCX1) [14]. 1, 2.5, and 5 g/kg), or MCT vehicle were administered by once-

Eldecalcitol (1␣,25-dihydroxy-2␤-(3-hydroxypropyloxy) vita- a-day oral gavage for 14 days (n = 6). On the 13th day, rats were

min D3), a new active vitamin D3 analog, has recently been transferred to and kept in metabolic cages for 24 h to collect urine

approved for the treatment of osteoporosis in Japan. A Phase III samples. Blood, bone, kidney, and intestine samples were collected

clinical trial in patients with osteoporosis showed that eldecalcitol at 6 h after the last dosing on the 14th day.

increased bone mineral density (BMD) and reduced the incidence Both animal studies were carried out in accordance with Chugai

of vertebral fracture with an efﬁcacy greater than that of alfacalci- Pharmaceutical’s ethical guidelines of animal care, and the experi-

dol [15]. It has also been shown that eldecalcitol promotes urinary mental protocols were approved by the animal care committee of

calcium excretion similarly to alfacalcidol, but has a lower potency the institution.

to suppress blood PTH [16]. Eldecalcitol increases BMD and reduces

bone turnover markers in normal, ovariectomized (OVX), and 2.3. Biochemical analysis

steroid-treated rats, and also in patients with osteoporosis [17–21].

Eldecalcitol is more active than calcitriol in stimulating calcium Levels of calcium, phosphorus, and creatinine in serum and urine

and phosphorus absorption in the intestine, as well as in increas- were determined by using an automatic analyzer (TBA-120FR;

ing serum FGF-23 in normal rats [22]. However, administration Toshiba Medical Systems, Tochigi, Japan). PTH in plasma was mea-

of exogenous eldecalcitol or calcitriol affects the synthesis and/or sured by rat intact PTH ELISA kit (Immutopics International, San

degradation of endogenous calcitriol, and exogenous eldecalcitol or Clemente, CA, USA). FGF-23 in serum was measured by FGF-23

H. Saito, S. Harada / Journal of Steroid Biochemistry & Molecular Biology 144 (2014) 189–196 191

A. B. Vehicle

12 a a 3 Eldecalcitol a Calcitriol 9

(mg/dL) 2

calcium

6 creatinine)

calcium 1 (g/g

3 Urinary Serum

0 0

WT VDR KO WT VDR KO

C. D.

3 a 3

A a a 2 2 mRN

A mRN TRPV5 calbindin-D28k

1 1 Renal Renal

0 0

WT VDR KO WT VDR KO

E. F. 10 5 a k

A a 8 4 RN a 6 m

6 A 3

PV a calbindin-D9

TR 4 mRN 2

stinal 2 1 Intestinal Inte

0 0

WT VDR KO WT VDR KO

Fig. 1. Effects of eldecalcitol or calcitriol on calcium metabolism in 9-week-old wild-type and VDRKO mice. Eldecalcitol (0.2 ␮g/kg), calcitriol (2 ␮g/kg), or vehicle was

administered daily to 9-week-old wild-type or VDRKO mice for 14 days. Blood, intestine, and kidney samples were recovered for further analyses. Serum calcium concentration

(A), urinary calcium excretion (B), TRPV5 (C) and calbindin-D28k (D) mRNA expression in the kidney, and TRPV6 (E) and calbindin-D9k (F) mRNA expression in the intestine

are shown. Values represent means + SE (n = 5 for each treatment group). (a) p < 0.05 (Compared with the vehicle control group, using Tukey’s multiple test.)

ELISA kit (Kainos Laboratories, Tokyo, Japan). Calcitriol in serum crushed in a homogenizer (Physcotron NS-310E; Microtec, Chiba,

was measured by 1,25(OH)2D RIA kit (TFB, Tokyo, Japan). Measure- Japan). Total RNA was extracted with an RNeasy Mini Kit (Qiagen,

ment of eldecalcitol in plasma was performed at the BoZo Research Hilden, Germany). cDNA was synthesized from 200 ng of total RNA

Center (Tokyo, Japan). by reverse transcription PCR using TaqMan Reverse Transcription

Reagents (Applied Biosystems, Foster City, CA, USA). The reaction

◦

2.4. Quantitative RT-PCR was performed at 37 C for 1 h. Expression of mRNA in the tis-

sues was detected using TaqMan Gene Expression Assays (Applied

◦

The right femur, intestine, and kidneys of mice, and the right Biosystems). Target cDNA was ampliﬁed by 40 cycles (1 cycle: 95 C

◦

femur, intestine, and kidneys of rats were excised and immedi- for 15 s, 60 C for 1 min) of PCR in an ABI PRISM 7000 Sequence

ately frozen in liquid nitrogen. A small portion of each of the frozen Detector System (Applied Biosystems). The TaqMan probes used in

tissues was soaked in TRIzol (Invitrogen, Carlsbad, CA, USA) and this study were TRPV5, TRPV6, calbindin-D28k, and calbindin-D9k

192 H. Saito, S. Harada / Journal of Steroid Biochemistry & Molecular Biology 144 (2014) 189–196

A. 18000 B. 5000

L) 15000 l/ on 4000 12000 (pmo

l 3000 (pmol/L) 9000 l concentrati concentration

2000 6000 calcitrio

eldecalcito

2 of 1000 2

R =0.996 Serum R =0.971 Plasma of 3000

0 0

0 0.1 0.2 0.3 0.4 0.5 0123 4 5

Dose of eldecalcitol (μg/kg) Dose of calcitriol(μg/kg) C. 400

300 a (pmol/L) l 200 concentration

calcitrio 100 of Serum

a a a 0 e 0.5 0.1 0.25 0.05 0.025

vehicl Dose of

eldecal citol (μg/kg) D. E. 1.5 Vehicle 120

a Eldecalcit ol A A Calcitriol mRN mRN

1 80 a a a a CYP27B1 CYP24A1

0.5 40 a

a a a Renal a Renal a a a a a a

0 0

100 1000 10000 100 1000 10000

Conc entration in blood (pmol/L) Concentration in blood (pmol/L)

Fig. 2. Effects of eldecalcitol or calcitriol on the feedback loop for endogenous calcitriol in normal rats. Six-week-old male rats were administered either eldecalcitol

(0.025, 0.05, 0.1, 0.25, 0.5 ␮g/kg) or calcitriol (0.25, 0.5, 1, 2.5, 5 ␮g/kg) for 14 days by oral gavage. Blood and kidney samples were recovered for further analyses. Plasma

concentration of eldecalcitol in the eldecalcitol-treated rats (A), serum concentration of calcitriol in the calcitriol-treated rats (B), serum concentration of endogenous calcitriol

in the eldecalcitol-treated rats (C), and renal mRNA expression of CYP27B1 (D) and CYP24A1 (E) are shown. Values represent means ± SE (n = 6 for each treatment group). (a)

p < 0.05 (Compared with the normal control group using Dunnett’s multiple test.)

of mice and TRPV5, TRPV6, calbindin-D28k, calbindin-D9k, recep- by Tukey’s test in Section 2.2.1, and by Dunnett’s test in Section

tor activator of NF-␬B ligand (RANKL), FGF-23, CYP27B1, CYP24A1, 2.2.2.

and VDR of rats. 18S rRNA was used as a control. Data are repre-

sented as expression relative to that in rats treated with vehicle

3. Results

control.

3.1. Effects of eldecalcitol on calcium metabolism in VDRKO mice

2.5. Statistical analysis

A dose of 2 ␮g/kg calcitriol or 0.2 ␮g/kg eldecalcitol admin-

Data are presented as mean ± standard error (SE). Statistical istered daily by oral gavage for 14 days signiﬁcantly increased

analysis was performed using the SAS System for Windows (SAS serum calcium and urinary calcium excretion compared with vehi-

Institute, Cary, NC, USA). Statistical signiﬁcance was determined cle administration in WT mice. However, neither eldecalcitol nor

H. Saito, S. Harada / Journal of Steroid Biochemistry & Molecular Biology 144 (2014) 189–196 193

A. B. 10 6

9 5 a (mg/dL) a a 4 8

orus 3 a

7 Vehicle phosphorus creatinine)

a 2 Eldecalcitol phosph (g/g 6 Calcitriol 1 Urinary

5 0

Serum

100 1000 10000 100 1000 10000

Conc entration in blood (pmol/L)

Concentr ation in blood (pmol/L)

C. 16 D. 2.4 a 2

14 a a 1.6 (mg/dL)

a a a

calcium a 12 a 1.2 creatinine) a 0.8 calcium a

10 (g/g Urinary 0.4 a

Serum 8 0

100 1000 10000 100 1000 10000

Conc entration in blood (pmol/L)

Concentr ation in blood (pmol/L) E. F. 16 600 a a 500 12 a (ng/mL)

(pg/mL) 400

8 300 PTH

FGF-23 200 4

100 a Plasma a a a Serum a a

0 0

100 1000 10000 100 1000 10000

Concentr ation in blood (pmol/L) Concentration in blood (pmol/L)

Fig. 3. Effects of eldecalcitol or calcitriol on calcium and phosphorus metabolism in normal rats. Six-week-old male rats were administered either eldecalcitol or calcitriol for

14 days by oral gavage. Blood and urine samples were recovered for further analyses. Serum phosphorus (A), urinary phosphorus excretion (B), serum calcium (C), urinary

calcium excretion (D), serum FGF-23 (E), and plasma PTH (F) are shown. Values represent means ± SE (n = 6 for each treatment group). (a) p < 0.05 (Compared with the normal

control group using Dunnett’s multiple test.)

calcitriol affected serum or urinary calcium in the VDRKO mice result indicates that in order to reach the same concentration in the

(Fig. 1A and B). Calcitriol and eldecalcitol signiﬁcantly increased blood, the amount of eldecalcitol required is approximately 1/40

the expression of renal TRPV5 and calbindin-D28k mRNA and the that of calcitriol. In the eldecalcitol-treated rats, serum concentra-

expression of intestinal TRPV6 and calbindin-D9k mRNA in the WT tion of calcitriol dose-dependently decreased and fell to below the

mice. On the other hand, the expression of these genes in the VDRKO limit of detection at 0.1 ␮g/kg (Fig. 2C). Treatment with eldecalcitol

mice was not altered by the treatment (Fig. 1C–F). These results and calcitriol signiﬁcantly reduced renal CYP27B1 gene expression

indicate that the calcemic actions of calcitriol and eldecalcitol are and dose-dependently increased renal CYP24A1 gene expression

mediated by VDR. (Fig. 2D and E). These results suggest that the administration of

eldecalcitol and calcitriol reduces endogenous production of cal-

3.2. Effects of eldecalcitol on calcium and phosphorus metabolism citriol and stimulates degradation of calcitriol in the kidneys.

in normal rats Blood concentrations of eldecalcitol and calcitriol correlated

with urinary phosphorus excretion. Serum phosphorus slightly

Eldecalcitol (0.025, 0.05, 0.1, 0.25, and 0.5 ␮g/kg) or cal- decreased along with the increase in eldecalcitol concentration in

citriol (0.25, 0.5, 1, 2.5, and 5 ␮g/kg) administered daily by oral blood, whereas calcitriol concentration did not alter serum phos-

gavage for 14 days dose-dependently increased the blood con- phorus (Fig. 3A and B). Serum calcium was signiﬁcantly elevated

≥

centration of each compound. The blood concentration of each at higher blood concentrations of eldecalcitol ( 7520 pmol/L) and

≥

compound correlated well with the administered dosage (eldecal- calcitriol ( 1170 pmol/L) (Fig. 3C). Urinary calcium excretion cor-

citol: y (pmol/L) = 29,834x (␮g/kg) + 646.3, R = 0.996; calcitriol: y related with blood concentrations of calcitriol and eldecalcitol

(pmol/L) = 681.81x (␮g/kg) + 402.1, R = 0.971) (Fig. 2A and B). This (Fig. 3D). Serum FGF-23 increased at 15,800 pmol/L of eldecalcitol

194 H. Saito, S. Harada / Journal of Steroid Biochemistry & Molecular Biology 144 (2014) 189–196

A. B. 2.5 a 2 a a a k 2 a 1.5

mRNA a 1.5 a 1 calbindin-D9 TRPV6

1 Vehicle 0.5 0.5 Eldecalcitol Calcitriol Intestinal Intestinal 0 0 100 1000 10000 100 1000 10000 Concentration in blood (pmol/L) Concentration in blood (pmol/L) C. D. 12 A 4 a A

mRN a

9 k 3 mRN

a a a 6 2 a a a TRPV5 a 3 1 calbindin-D28

Renal 0 0 100 1000 10000 Renal 100 1000 10000 Concentration in blood (pmol/L) Concentration in blood (pmol/L) E. F. 4 25 a A a 20 3 a mRNA mRN a L 15 2 a a 10 RANK FGF-23

1 5 Bone Bone 0 0 100 1000 10000 100 1000 10000

Concentration in blood (pmol/L) Concentration in blood (pmol/L)

Fig. 4. Effects of eldecalcitol or calcitriol on target genes in normal rats. Six-week-old male rats were administered either eldecalcitol or calcitriol for 14 days by oral gavage.

Intestine, kidney, and bone samples were recovered for further analyses. mRNA expressions of TRPV6 (A) and calbindin-D9k (B) in the intestine, TRPV5 (C) and calbindin-D28k

(D) in the kidneys, and RANKL (E) and FGF-23 (F) in bone are shown. Values represent means ± SE (n = 6 for each treatment group). (a) p < 0.05 (Compared with the normal

control group using Dunnett’s multiple test.)

and at ≥2480 pmol/L of calcitriol in blood (Fig. 3E). High concen- eldecalcitol (≥7520 pmol/L) and calcitriol (≥1170 pmol/L) (Fig. 4D).

trations of eldecalcitol in the blood (≥7520 pmol/L) suppressed In bone, blood concentrations of calcitriol correlated with RANKL

plasma PTH concentration, whereas plasma PTH concentration was and FGF-23 gene expression; however, only the highest concen-

reduced from low blood calcitriol concentrations (≥590 pmol/L) tration of eldecalcitol (15,800 pmol/L) induced RANKL and FGF-23

(Fig. 3F). gene expression (Fig. 4E).

Blood concentration of calcitriol correlated with VDR gene

3.3. Effects of eldecalcitol on target gene expression in normal rats expression in the kidneys and bone (Fig. 5B and C), but calcitriol did

not affect VDR gene expression in the intestine (Fig. 5A). Induction

In the intestine, TRPV6 gene expression was induced from rel- of VDR gene expression in the intestine and kidneys were asso-

atively low blood concentrations of eldecalcitol (≥1220 pmol/L) ciated with increasing concentration of eldecalcitol in the blood

(Fig. 4A). Similarly, induction of TRPV6 gene expression was (Fig. 5A and B). In bone, signiﬁcant induction of VDR gene expres-

observed from low concentrations of calcitriol (590 pmol/L and sion was observed only at the highest concentration of eldecalcitol

≥

2480 pmol/L). Calbindin-D9k gene expression was unchanged by (Fig. 5C).

the administration of calcitriol or eldecalcitol (Fig. 4B). In the kid- Taken together, these results show that, in comparison to cal-

neys, TRPV5 mRNA expression was signiﬁcantly elevated at the citriol, relatively higher concentrations of eldecalcitol in the blood

highest concentration of eldecalcitol (15,800 pmol/L) and at high were required to stimulate expression of vitamin D target genes

concentrations of calcitriol (≥1170 pmol/L) (Fig. 4C). Calbindin- in the kidneys (VDR, TRPV5, and calbindin-D28k) and bone (VDR,

D28k mRNA was increased at the higher blood concentrations of RANKL, and FGF-23).

H. Saito, S. Harada / Journal of Steroid Biochemistry & Molecular Biology 144 (2014) 189–196 195

A. Vehicle B.

2 a 8 Eldecalcit ol a A Calcitriol a A

RN 1.5 6 a m mRN a a DR 1 4 V VDR a

stinal 0.5 2

Renal a Inte

0 0

100 1000 10000 100 1000 10000

Concentr ation in blood (pmol/L) Concentration in blood (pmol/L)

C. 3 a a a A

2 a mRN

VDR 1 Bone

100 1000 10000

Concentr ation in blood (pmol/L)

Fig. 5. Effects of eldecalcitol or calcitriol on VDR mRNA expression in normal rats. Six-week-old male rats were administered either eldecalcitol or calcitriol for 14 days by

oral gavage. Intestine, kidney, and bone samples were recovered for further analyses. mRNA expressions of intestinal VDR (A), renal VDR (B), and bone VDR (C) are shown.

Values represent means ± SE (n = 6 for each treatment group). (a) p < 0.05 (Compared with the normal control group using Dunnett’s multiple test.)

3.4. Comparison of the ‘true in vivo biological activity’ of intestinal TRPV6 and VDR gene expression were comparable to

eldecalcitol and calcitriol those of calcitriol (Figs. 4A and 5A).

In order to compare the true biological activity of calcitriol and 4. Discussion

eldecalcitol in vivo, the blood concentration required to elicit a 50%

response in each activity was calculated from the raw data above. The half-life of eldecalcitol in the blood is much longer than

The ratio of biological activity was obtained by dividing the 50% it is for calcitriol [23]. Although eldecalcitol strongly induces

response concentration of calcitriol by that of eldecalcitol. Based CYP24A1 in the intestine and kidneys [24], eldecalcitol itself is

on these calculations, eldecalcitol was approximately 1/4 to 1/7 hardly degraded by CYP24A1 [25]. At the same time, eldecalcitol

as active as calcitriol in increasing serum calcium and FGF-23, in strongly suppresses CYP27B1 in the kidneys. Blood concentration

stimulating urinary calcium excretion, and in suppressing plasma of eldecalcitol during treatment in clinical trials was reported to

PTH (Table 1). Eldecalcitol was approximately 1/3 to 1/8 as active be relatively high (200–250 pg/mL) in comparison to the normal

as calcitriol in stimulating expression of target genes in the kid- range of calcitriol (30–60 pg/mL) in humans [15]. An approximately

neys (VDR, TRPV5, and calbindin-D28k) and bone (VDR, FGF-23, 50% reduction in blood calcitriol was observed during eldecalcitol

and RANKL). The biological activities of eldecalcitol in increasing treatment in the clinical trial.

Table 1

Relative biological activity of eldecalcitol compared to that of calcitriol.

Blood concentration required for a 50% response (pmol/L) Relative activity

Eldecalcitol Calcitriol

Biochemical parameters

Serum calcium 8841 1288 0.15

Urinary calcium excretion 4856 1096 0.23

Serum FGF-23 11,232 1779 0.16

Plasma PTH 3876 563.7 0.15

Target gene

Bone

VDR 10,791 1328 0.12

FGF-23 11,506 1668 0.14

RANKL 10,711 1753 0.16

Kidney

VDR 6711 917 0.14

TRPV5 11,033 1850 0.17

Calbindin-D28k 6768 1946 0.29

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