JTT-305, an orally active calcium-sensing receptor antagonist, stimulates transient parathyroid hormone release and bone formation in ovariectomized rats

European Journal of Pharmacology 668 (2011) 331–336

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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Endocrine Pharmacology

JTT-305, an orally active calcium-sensing receptor antagonist, stimulates transient parathyroid hormone release and bone formation in ovariectomized rats Shuichi Kimura ⁎, Takashi Nakagawa, Yushi Matsuo, Yuji Ishida, Yoshihisa Okamoto, Mikio Hayashi Biological/Pharmaceutical Research Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco Inc., Osaka, Japan

a r t i c l e

i n f o

Article history: Received 9 May 2011 Received in revised form 23 June 2011 Accepted 7 July 2011 Available online 28 July 2011 Keywords: Calcium-sensing receptor Antagonist Parathyroid hormone Bone mineral density Bone formation

a b s t r a c t Intermittent administration of parathyroid hormone (PTH) has a potent anabolic effect on bone in humans and animals. Calcium-sensing receptor (CaSR) antagonists stimulate endogenous PTH secretion through CaSR on the surface of parathyroid cells and thereby may be anabolic agents for osteoporosis. JTT-305 is a potent oral shortacting CaSR antagonist and transiently stimulates endogenous PTH secretion. The objective of the present study was to investigate the effects of JTT-305 on PTH secretion and bone in ovariectomized rats. Female rats, immediately after ovariectomy (OVX), were orally administered vehicle or JTT-305 (0.3, 1, or 3 mg/kg) for 12 weeks. The serum PTH concentrations were transiently elevated with increasing doses of JTT-305. In the proximal tibia, JTT-305 prevented OVX-induced decreases in both the cancellous and total bone mineral density (BMD) except for the 0.3 mg/kg dose. At the 3 mg/kg dose, JTT-305 increased the mineralizing surface and bone formation rate in histomorphometry. The efficacy of JTT-305 at the 3 mg/kg dose on the BMD corresponded to that of exogenous rat PTH1–84 injection at doses between 3 and 10 μg/kg. In conclusion, JTT-305 stimulated endogenous transient PTH secretion and bone formation, and consequently prevented bone loss in OVX rats. These results suggest that JTT-305 is orally active and has the potential to be an anabolic agent for the treatment of osteoporosis. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Parathyroid hormone (PTH) is an attractive agent for the treatment of osteoporosis. Intermittent PTH injection stimulates new bone formation and remarkably restores ovariectomy (OVX)-induced bone loss in rats (Fox et al., 2006; Meng et al., 1996; Mitlak et al., 1996; Sato et al., 2002) and primates (Brommage et al., 1999; Fox et al., 2007; Jerome et al., 2001). In a study in humans, teriparatide (recombinant human PTH1–34) increased the vertebral, femoral, and total-body bone mineral density (BMD), and decreased the risk of vertebral and nonvertebral fractures in postmenopausal osteoporosis (Neer et al., 2001). Furthermore, teriparatide increased the vertebral and hip BMD, and decreased new vertebral fractures in glucocorticoid-induced osteoporosis (Saag et al., 2007). Preos (recombinant full-length human PTH1–84) increased the vertebral and hip BMD, and decreased new or worsening vertebral fractures in postmenopausal osteoporosis (Greenspan et al., 2007). However, PTH must be administered subcutaneously and is very expensive owing to its peptide formulation. The calcium-sensing receptor (CaSR), which was cloned from the bovine parathyroid gland in 1993, is a member of the class C family of G

⁎ Corresponding author at: Biological/Pharmaceutical Research Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco Inc., 1-1 Murasaki-cho, Takatsuki, Osaka, 569-1125, Japan. Tel.: + 81 72 681 9700; fax: + 81 72 681 9722. E-mail address: [email protected] (S. Kimura). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.07.015

protein-coupled receptors (Brown et al., 1993). CaSR is functionally expressed in the parathyroid gland and kidney, and plays a key role in calcium homeostasis (Brown and MacLeod, 2001). The function of CaSR on the parathyroid gland is to regulate endogenous PTH secretion in response to blood calcium concentrations (Portale et al., 1997; Udén et al., 1992). Several pharmacological approaches to regulate PTH secretion through CaSR have been reported. CaSR agonists, which are also called calcimimetics, suppressed endogenous PTH secretion in humans (Goodman et al., 2002; Silverberg et al., 1997) and rats (Fox et al., 1999; Nemeth et al., 2004), whereas CaSR antagonists, which are also called calcilytics, stimulated endogenous PTH secretion in rats (Arey et al., 2005; Marquis et al., 2009; Nemeth et al., 2001; Shinagawa et al., 2010). Therefore, orally active CaSR antagonists that can mimic the pharmacokinetics of intermittently injected PTH may be appropriate anabolic agents for osteoporosis. Several CaSR antagonists have been advanced to clinical trials (Fitzpatrick et al., 2008; John et al., 2011; Kumar et al., 2010; Widler et al., 2008). JTT-305 (Fig. 1) was discovered as a potent oral short-acting CaSR antagonist that stimulates endogenous pulsatile PTH secretion (Shinagawa et al., 2011), and is currently undergoing clinical trials for the treatment of postmenopausal osteoporosis (Fukumoto et al., 2009). The objective of the present study was to investigate the effects of oral administration of JTT-305 on PTH secretion and bone in ovariectomized rats. To confirm whether the efficacy of JTT-305 on BMD was caused by endogenous PTH secretion, the effect of rat PTH1–84 on OVX-induced bone loss was also evaluated.

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CH3 CO2H H OH H N

O H

CH3

H3C CH3

F

. H SO . H O 2 4 2

Cl 2

Fig. 1. Chemical structure of JTT-305.

2. Materials and methods 2.1. Cell culture, transfection, and intracellular Ca 2+ mobilization assay COS-7 cells (Riken Gene Bank and Cell Bank, Tokyo, Japan) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin (100 units/ml), and streptomycin (100 μg/ml) at 37 °C under 5% CO2. The pME18S vector was kindly provided by Dr. Maruyama (Tokyo Medical and Dental University). Transient transfections of the pME18S plasmid containing a human CaSR cDNA into COS-7 cells were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA). At 24 h after the transfection, the cells were loaded with 3 μM Fura 2-AM (Wako Pure Chemicals, Osaka, Japan) in loading buffer (0.5 mM CaCl2, 1 mg/ml D-glucose, 126 mM NaCl, 4 mM KCl, 1 mM MgCl2, 20 mM HEPES, pH 7.4) containing 0.02% Pluronic F-127 for 30 min at room temperature. The cells were washed and resuspended in loading buffer containing 0.1% bovine serum albumin. The cell suspensions were excited with dual wavelengths (340 and 380 nm), and the fluorescence emission ratio at 500 nm was recorded using an intracellular calcium analyzer (CAF-110; JASCO Corporation, Tokyo, Japan). JTT-305 was incubated with the cell suspensions for 1 min before the extracellular calcium concentration was increased from 0.5 to 2 mM. The IC50 values for JTT-305 were calculated using GraphPAD Prism 4.00 (GraphPad Prism Software, San Diego, CA) from three individual experiments performed in duplicate. 2.2. Animals Virgin female Sprague–Dawley rats were purchased from Charles River Laboratories Japan (Yokohama, Japan). The rats were maintained at 23 ± 3 °C on a 12-h/12-h light–dark cycle with ad libitum access to a standard diet (AIN93G; Oriental Yeast, Tokyo, Japan) and water. During the period of the experiment, time-restricted feeding was used, with the feeding time being approximately 7 h after dosing. All the animal procedures and protocols complied with the guidelines for animal experimentation set by the Ethics Committee for Animal Use at Japan Tobacco Inc. 2.3. Experimental design for OVX model 2.3.1. Experiment 1 Virgin female 29-week-old Sprague–Dawley rats were used. The rats were divided into one group of sham-operated animals (n = 12) and four groups of OVX animals (n = 10–12 per group) based on the BMD and body weight. The rats were subjected to either bilateral OVX or a sham operation. After a 2-day recovery period, the OVX rats were given vehicle (0.5% methyl cellulose) or JTT-305 (0.3, 1, or 3 mg/kg) suspended in vehicle orally once daily for 12 weeks. The shamoperated rats were given vehicle orally once daily for 12 weeks. Blood samples were collected predose and at 15, 30, 60, 120, and 240 min after the administration of vehicle or JTT-305 on the first day of dosing for assays of the serum PTH concentrations and the plasma concentrations of JTT-305. For dynamic bone histomorphometry, the

rats were injected subcutaneously with calcein (8 mg/kg) at 12 and 5 days before necropsy. On the day following the last administration, the rats were killed by exsanguination from the abdominal aorta under anesthesia by inhalation of diethyl ether. The left tibia was excised, cleaned of excess soft tissue, and fixed in 70% ethanol for histomorphometric analysis. 2.3.2. Experiment 2 Virgin female 36-week-old Sprague–Dawley rats were used. The rats were divided into one group of sham-operated animals (n = 8) and four groups of OVX animals (n = 7–8 per group) based on the BMD and body weight. The rats were subjected to either bilateral OVX or a sham operation. After a 3-day recovery period, the OVX rats were injected subcutaneously with either vehicle (0.1% rat serum albumin and 0.001 N HCl) or rat PTH1–84 (Bachem, Bubendorf, Switzerland) (3, 10, or 30 μg/kg) dissolved in vehicle once daily for 12 weeks. The sham-operated rats were injected subcutaneously with the vehicle once daily for 12 weeks. Blood samples were collected for assays of the serum PTH concentrations predose and at 15, 30, 60, 120, and 240 min after the injection of vehicle or rat PTH1–84 on the first day of dosing. The necropsy procedures were the same as those in Experiment 1, but a histomorphometric analysis was not performed. 2.4. Pharmacokinetics and blood chemistry Blood samples were collected from the tail vein. The plasma concentrations of JTT-305 (free base) were measured by liquid chromatography tandem mass spectrometry (LC/MS/MS). The time to the maximum plasma concentration (Tmax) was obtained directly from the data. The elimination rate constant (ke) was determined by linear regression on the logarithm of the plasma concentration–time curve from Tmax to 4 h. The elimination half-life (T1/2) was calculated using the equation T1/2 = ln2 / ke. Serum PTH was measured using a commercial ELISA kit (Rat Intact PTH ELISA; Immutopics, San Clemente, CA). 2.5. Bone mineral densitometry The cancellous and total BMD of the right tibia was measured preoperatively and at 12 weeks postoperatively by quantitative computed tomography (QCT) using a LaTheta LCT-100A (Aloka, Tokyo, Japan) with a pixel size of 170 × 170 μm and a slice thickness of 1 mm. The tube voltage of the X-ray generator was 50 kV (1 mA). The scan area was positioned at 3 mm distal to the proximal epiphysis of the tibia (approximately 1 mm distal to the growth plate). The rats were anesthetized with pentobarbital (40 mg/kg) during the measurement. 2.6. Histomorphometry The fixed left tibia was embedded in methyl methacrylate and sectioned into 3-μm slices. The sections were stained with toluidine blue. The sections were subjected to histomorphometric analyses under a light microscope using an image analyzer system (Measure6; System Supply, Nagano, Japan). The measurement area was 2.15 mm in length from 0.1 mm below the growth plate. The following parameters were measured: osteoblast surface (Ob.S/BS); mineralizing surface (MS/BS); mineral apposition rate (MAR); bone formation rate (BFR/BS); osteoclast surface (Oc.S/BS); and eroded surface (ES/BS). The measurement parameters complied with the standard nomenclature approved by the American Society of Bone and Mineral Research (Parfitt et al., 1987). 2.7. Statistical analysis All data are presented as means ± S.E.M. Statistical analyses were performed using SAS System Version 8.2 and SAS Preclinical Package

S. Kimura et al. / European Journal of Pharmacology 668 (2011) 331–336

3. Results

A Plasma JTT-305 (nM)

Version 5.0 (SAS Institute Japan, Tokyo, Japan). An F-test was used to test for variances between two groups. A Student's t-test was used for data with equal variances and a Welch's t-test was used for data with unequal variances. Differences among three or more groups were tested by the following method. A Bartlett's homogeneity of variance test was performed, followed by a Dunnett's multiple comparison test for data with equal variances and a Steel's multiple comparison test for data with unequal variances. Differences were considered significant for values of P b 0.05 (two-sided).

333

8000

6000

4000

2000

3.1. Effect of JTT-305 on CaSR in vitro 0

3.2. Pharmacokinetic and pharmacodynamic profile of JTT-305 To evaluate the effect of JTT-305 on bone loss in OVX rats, administration of JTT-305 was performed for 12 weeks. The effect of rat PTH1–84 on OVX-induced bone loss was also evaluated to confirm whether the efficacy of JTT-305 on bone was caused by endogenous PTH secretion. On the first day of dosing, the plasma concentrations of JTT-305 after oral administration were rapidly elevated in a dosedependent manner in Experiment 1 (Fig. 3A). The peak plasma concentrations of JTT-305 were observed at 15–30 min after administration and were 374 nM (0.3 mg/kg), 2696 nM (1 mg/kg), and 5142 nM (3 mg/kg). At the doses of 1 and 3 mg/kg, the Tmax values were 0.5 h (1 mg/kg) and 0.3 h (3 mg/kg), and the T1/2 values were 1.9 h (1 mg/kg) and 2.9 h (3 mg/kg). Consistent with the pharmacokinetic data, the serum PTH concentrations were transiently elevated in a dose-dependent manner (Fig. 3B). The peak serum concentrations of PTH were observed at 15 min after administration and were 1.3fold (0.3 mg/kg), 6.0-fold (1 mg/kg), and 10.8-fold (3 mg/kg) higher than the baseline values (Fig. 3B). On the first day of dosing, the serum PTH concentrations were elevated after injection of rat PTH1–84 in a dose-dependent manner in Experiment 2 (Fig. 4). The peak serum concentrations of PTH were

B

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700

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100 0

0

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240

Time (min) Fig. 3. Serum PTH concentrations in OVX rats after oral administration of JTT-305. Plasma concentrations of JTT-305 were measured on the first day of dosing. Data represent mean ± S.E.M.; n = 4 per group (A). Serum PTH concentrations were measured on the first day of dosing. Data represent means ± S.E.M.; n = 10–12 per group (B). Symbols: OVX-vehicle (open circle), JTT-305 0.3 mg/kg (closed triangle), 1 mg/kg (closed diamond), and 3 mg/kg (closed square). ††P b 0.01 vs. 0 h time point by Steel's t-test.

4-fold (3 μg/kg), 20-fold (10 μg/kg), and 36-fold (30 μg/kg) higher than the baseline values (Fig. 4). The changes in the serum PTH concentration for JTT-305 at the 3 mg/kg dose corresponded to those for rat PTH1–84 at the doses between 3 and 10 μg/kg.

††

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Serum PTH (pg/ml)

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* ** 0

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Ca2+ mobilization (% of 2 mM calcium response)

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Time (min)

Serum PTH (pg/ml)

The potency of JTT-305 for antagonizing CaSR was evaluated using an intracellular Ca 2+ mobilization assay. JTT-305 dose-dependently inhibited the increases in the intracellular Ca 2+ concentrations induced by 2 mM extracellular calcium in COS-7 cells transiently transfected with human CaSR (Fig. 2). Analysis of the dose–response curve produced an IC50 for JTT-305 of 86 nM. These data indicated that JTT-305 is a potent antagonist of human CaSR.

1

10

100

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10000

JTT-305 (nM) Fig. 2. Effect of JTT-305 on extracellular calcium-induced intracellular Ca2+ mobilization in COS-7 cells transiently transfected with human CaSR. JTT-305 inhibited extracellular calcium (2 mM) induced intracellular Ca2+ mobilization with an IC50 of 86 ± 6 nM (n = 3). Data represent means ± S.E.M. from three individual experiments in duplicate.

0

0

†

60

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240

Time (min) Fig. 4. Serum PTH concentrations in OVX rats after subcutaneous injection of rat PTH1–84. Serum PTH concentrations were measured on the first day of dosing. Symbols: OVX-vehicle (open circle), rat PTH1–84 3 μg/kg (open triangle), 10 μg/kg (open diamond), and 30 μg/kg (open square). Data represent means±S.E.M.; n= 7–8 per group. *P b 0.05 and **P b 0.01 vs. 0 h time point by Dunnett's test. †P b 0.05 and ††P b 0.01 vs. 0 h time point by Steel's t-test.

S. Kimura et al. / European Journal of Pharmacology 668 (2011) 331–336

3.3. Effect of JTT-305 or rat PTH1–84 on BMD

A

450

The cancellous and total BMD of the proximal tibia was measured by QCT. The cancellous and total BMD was significantly decreased in the OVX-vehicle group compared with the sham group at 12 weeks after OVX in Experiment 1 (Figs. 5A and B). At the doses of 1 and 3 mg/ kg, JTT-305 dose-dependently and significantly inhibited the OVXinduced decreases in the cancellous BMD (+ 22% and + 24%, respectively) and total BMD (+8% and + 9%, respectively) (Figs. 5A and B). The cancellous and total BMD was also significantly decreased in the OVX-vehicle group compared with the sham group at 12 weeks after OVX in Experiment 2 (Figs. 6A and B). At the 3 μg/kg dose, rat PTH1–84 did not prevent the OVX-induced decreases in the cancellous or total BMD. At the doses of 10 and 30 μg/kg, rat PTH1–84 dose-dependently and significantly prevented the OVX-induced decreases in the cancellous BMD (+39% and +91%, respectively) and total BMD (+17% and + 31%, respectively).

Cancellous BMD (mg/cm3)

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Rat PTH1-84 Rat PTH1-84 Rat PTH1-84 3 µg/kg 10 µg/kg 30 µg/kg OVX

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595

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450

Cancellous BMD (mg/cm3)

To evaluate whether JTT-305 stimulated bone turnover, histomorphometric analyses of the proximal tibia were performed in Experiment 1. The bone formation parameters, assessed by Ob.S/BS, MS/BS, MAR, and BFR/BS, were significantly increased in the OVX-vehicle group

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Vehicle Rat PTH1-84 Rat PTH1-84 Rat PTH1-84 3 µg/kg 10 µg/kg 30 µg/kg

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* Fig. 6. Effect of rat PTH1–84 for 12 weeks on cancellous and total BMD of the proximal tibia in OVX rats. Cancellous BMD (A) and total BMD (B) of the proximal tibia was measured by QCT. Data represent means± S.E.M.; n = 7–8 per group. ##P b 0.01 vs. sham group by Student's t-test, **P b 0.01 vs. OVX-vehicle group by Dunnett's test.

300 ##

250 200 0 Sham

Vehicle

JTT-305 0.3 mg/kg

JTT-305 1 mg/kg

JTT-305 3 mg/kg

OVX

B

Total BMD (mg/cm3)

3.4. Effect of JTT-305 on histomorphometric bone turnover

620

compared with the sham group at 12 weeks after OVX (Table 1). The bone resorption parameters, assessed by Oc.S/BS and ES/BS, were also significantly increased in the OVX-vehicle group compared with the sham group at 12 weeks after OVX. At the 3 mg/kg dose, JTT-305 significantly increased MS/BS (+36%) and BFR/BS (+41%) without affecting ES/BS and Oc.S/BS. Ob.S/BS and MAR were not affected by JTT305 (Table 1).

Total BMD (mg/cm3)

595 570 545

**

520 495

**

##

Parameter

470 445 0 Sham

Vehicle

Table 1 Effect of JTT-305 for 12 weeks on histomorphometric bone turnover of the proximal tibia in OVX rats.

JTT-305 0.3 mg/kg

JTT-305 1 mg/kg

JTT-305 3 mg/kg

OVX Fig. 5. Effect of JTT-305 for 12 weeks on cancellous and total BMD of the proximal tibia in OVX rats. Cancellous BMD (A) and total BMD (B) of the proximal tibia was measured by QCT. Data represent means ± S.E.M.; n = 10–12 per group. ##P b 0.01 vs. sham group by Student's t-test, *P b 0.05 and **P b 0.01 vs. OVX-vehicle group by Dunnett's test.

Ob.S/BS (%) MS/BS (%) MAR (μm/day) BFR/BS (μm3/μm2/year) ES/BS (%) Oc.S/BS (%)

Sham 1.4 ± 0.3 8.3 ± 0.9 0.73 ± 0.03 0.022 ± 0.003 2.7 ± 0.3 0.7 ± 0.1

OVX-vehicle a

4.7 ± 1.2 22.1 ± 1.1b 0.89 ± 0.05d 0.073 ± 0.008b 6.3 ± 0.4b 2.5 ± 0.3e

Data represent mean ± S.E.M.; n = 8 per group. a P b 0.05 vs. the sham group by Welch's t-test. b P b 0.01 vs. the sham group by Student's t-test. c P b 0.01 vs. the OVX-vehicle group by Student's t-test. d P b 0.05 vs. the sham group by Student's t-test. e P b 0.01 vs. the sham group by Welch's t-test.

JTT-305 3 mg/kg 8.8 ± 2.1 30.1 ± 1.3c 0.94 ± 0.03 0.104 ± 0.005c 5.2 ± 0.8 2.3 ± 0.4

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4. Discussion

Acknowledgments

This study has shown that JTT-305 is a potent oral short-acting CaSR antagonist leading to endogenous pulsatile PTH secretion. A 12-week treatment of JTT-305 stimulated bone formation without affecting bone resorption, and consequently prevented decreases in both the cancellous and total BMD in OVX rats. JTT-305 dose-dependently elevated the serum PTH concentrations and the changes in the serum PTH concentration for the 3 mg/kg dose corresponded to those for exogenous rat PTH1–84, full-length rat PTH, injection at the doses between 3 and 10 μg/kg. The efficacy of the 3 mg/ kg dose on the BMD also corresponded to that of exogenous rat PTH1–84 injection at doses between 3 and 10 μg/kg. These data suggest that the efficacy of JTT-305 on bone is defined by the endogenous PTH secretion pattern. OVX rats at different ages were used in Experiments 1 and 2. In a preliminary study using OVX rats at similar ages to those in Experiments 1 and 2, we confirmed that there was no difference in the efficacy of PTH for increasing bone mass among rats at different ages. Therefore, it is considered that the different ages had no influence on the efficacy of PTH for preventing bone loss in this study. At the doses of 1 and 3 mg/kg, JTT-305 had a short Tmax (≤0.5 h) and T1/2 (b 3 h) compared with NPS 2143 (Tmax =1–2 h and T1/2 N 8 h) (Gowen et al., 2000). Consistent with the pharmacokinetic profile, JTT305 transiently elevated the serum PTH concentrations and prevented bone loss in OVX rats. SB-423557, which was reported to be a short-acting CaSR antagonist, also transiently elevated the serum PTH concentrations and improved bone in OVX rats (Kumar et al., 2010). Since the PTH secretion pattern of SB-423557 at the 50 mg/kg dose closely resembled that of JTT-305 at the 3 mg/kg dose, it is speculated that SB-423557 has a similar pharmacokinetic profile to JTT-305. On the other hand, NPS 2143 elevated the serum PTH concentrations and maintained the peak values for 4 h after administration. The continuous PTH elevation accelerated both bone formation and bone resorption without net bone gain in OVX rats (Gowen et al., 2000). These data suggest that CaSR antagonists require a short Tmax (≤0.5 h) and T1/2 (b3 h) that lead to endogenous pulsatile PTH secretion for bone anabolism. The histomorphometric analyses showed that JTT-305 increased MS/ BS and BFR/BS without affecting ES/BS and Oc.S/BS. This result indicates that JTT-305 prevents OVX-induced bone loss through stimulation of bone formation. However, in contrast to MS/BS and BFR/BS, JTT-305 did not affect MAR and Ob.S/BS. It was reported that PTH treatment robustly increased MAR and Ob.S/BS at 5 weeks, but these parameters decreased to the control levels within 10–15 weeks in OVX rats (Wronski et al., 1993). Ma et al. (1995) showed that PTH increased bone formation in rats after both 15 and 75 days of treatment, although the bone formation parameters after the 75-day treatment were apparently lower than those after the 15-day treatment. Therefore, JTT-305 may have transiently increased MAR and Ob.S/BS at an early stage of this study, which we would not have detected. Although it has been reported that CaSR is expressed in osteoblasts and osteoclasts (Dvorak et al., 2004; Kameda et al., 1998; Yamaguchi et al., 1998, 2001), a possible direct effect of CaSR on bone remains controversial. In our preliminary study, CaSR was hardly expressed in human osteoblasts and osteoclasts (data not shown). In addition, CaSR-deficient mice showed growth retardation and abnormalities of mineralization in bone and cartilage (Garner et al., 2001). However, these abnormalities were mainly caused by severe hyperparathyroidism, and CaSR-deficient mice with correction of the hyperparathyroidism showed normal growth and a normal skeletal phenotype (Tu et al., 2003). These data suggest that JTT-305 is unlikely to have a significant direct effect on bone. In conclusion, JTT-305 is a potent oral short-acting CaSR antagonist that stimulates endogenous pulsatile PTH secretion and prevents bone loss in OVX rats through stimulation of bone formation. These results suggest that JTT-305 is orally active and has the potential to be an anabolic agent for the treatment of osteoporosis.

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