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An orally active calcium-sensing receptor antagonist that transiently increases plasma concentrations of PTH and stimulates bone formation
Bone 46 (2010) 534–542
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Bone j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b o n e
An orally active calcium-sensing receptor antagonist that transiently increases plasma concentrations of PTH and stimulates bone formation Sanjay Kumar a,⁎, Christopher J. Matheny a, Sandra J. Hoffman b, Robert W. Marquis b, Maggie Schultz b, Xiaoguang Liang b, Janice A. Vasko b, George B. Stroup a, Vernal R. Vaden a, Hyking Haley a, John Fox c, Eric G. DelMar c, Edward F. Nemeth c, Amparo M. Lago a, James F. Callahan a, Pradip Bhatnagar a, William F. Huffman b, Maxine Gowen a, Bingming Yi b, Theodore M. Danoff b, Lorraine A. Fitzpatrick a a b c
GlaxoSmithKline, UM 2230, 709 Swedeland Road, King of Prussia, PA 19406-2711, USA GlaxoSmithKline, Collegeville, PA, USA NPS Pharmaceuticals, Salt Lake City, USA
a r t i c l e
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Article history: Received 19 May 2009 Revised 24 August 2009 Accepted 22 September 2009 Available online 26 September 2009 Edited by: R. Baron Keywords: Parathyroid hormone Parathyroid gland Calcium-sensing receptor Bone formation Osteoporosis
a b s t r a c t Daily subcutaneous administration of exogenous parathyroid hormone (PTH) promotes bone formation in patients with osteoporosis. Here we describe two novel, short-acting calcium-sensing receptor antagonists (SB-423562 and its orally bioavailable precursor, SB-423557) that elicit transient PTH release from the parathyroid gland in several preclinical species and in humans. In an ovariectomized rat model of bone loss, daily oral administration of SB-423557 promoted bone formation and improved parameters of bone strength at lumbar spine, proximal tibia and midshaft femur. Chronic administration of SB-423557 did not increase parathyroid cell proliferation in rats. In healthy human volunteers, single doses of intravenous SB-423562 and oral SB-423557 elicited transient elevations of endogenous PTH concentrations in a profile similar to that observed with subcutaneously administered PTH. Both agents were well tolerated in humans. Transient increases in serum calcium, an expected effect of increased parathyroid hormone concentrations, were observed post-dose at the higher doses of SB-423557 studied. These data constitute an early proof of principle in humans and provide the basis for further development of this class of compound as a novel, orally administered bone-forming treatment for osteoporosis. © 2009 Elsevier Inc. All rights reserved.
Introduction Osteoporosis is prevalent in postmenopausal women, with approximately one in three women aged over 50 years affected [1], and is increasing in incidence due, in part, to an aging population [2]. Furthermore, osteoporosis affects around 200 million people worldwide, and thus represents a substantial financial burden [1]. In 2002, the combined annual cost of osteoporosis-related fractures was estimated at US$20 billion in the US and US$30 billion in Europe [3]. The pathogenesis of osteoporosis results from an imbalance in bone turnover and a net increase in bone resorption leading to reduced bone mass and an increased risk of fracture. Current treatment options for osteoporosis either inhibit bone resorption through the use of antiresorptive agents (e.g., bisphosphonates, estradiol, calcitonin and raloxifene) and promote bone formation with anabolic agents (e.g., intact parathyroid hormone [PTH(1–84)] and the 34-amino acid peptide [PTH(1–34)]) or a combination of both approaches as suggested for strontium ranelate [4]. However, controversy surrounding the bone-forming effect of strontium ranelate has recently arisen [5]. ⁎ Corresponding author. Fax: +1 610 270 5580. E-mail address: [email protected] (S. Kumar). 8756-3282/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2009.09.028
Net bone formation is characterized by an increase in bone mineral density (BMD) and increased trabecular and cortical areas, resulting in a bone architecture with increased strength that is more resistant to fracture [6]. The bone-forming effects and increased bone strength following transient exposure to PTH through intermittent, subcutaneous administration of PTH(1–84) or PTH(1–34) in animal models and healthy human volunteers, as well as in patients with osteoporosis, have been well documented [7-13]. In contrast, continuous infusion of PTH leads to increased bone turnover, but without net formation, resulting in overall bone loss [14]. PTH plays a central role in calcium homeostasis, through its effects on renal calcium excretion, bone resorption, and, indirectly, intestinal calcium absorption. PTH secretion from the parathyroid gland is negatively regulated by ionized serum calcium (Ca2+), mediated through the calcium-sensing receptor (CaR). The CaR is a G-proteincoupled receptor present on cells of the parathyroid gland, thyroid C-cells, bone, kidney and gastrointestinal tissues, among others, and is activated by increased concentrations of extracellular Ca2+ [15]. As the concentration of Ca2+ in the blood increases, activation of CaRs on parathyroid cells inhibits PTH secretion. Conversely, a decrease in Ca2+ triggers PTH release. Targeting this regulatory mechanism, we have previously shown that small-molecule CaR antagonists stimulate
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significant but sustained increases in PTH concentrations in animal models, resulting in an increase in both bone formation and bone resorption [16-19]. The lack of favorable effects on bone remodeling was circumvented by co-treatment with estradiol, which reduced bone resorption leading to a net increase in bone formation and BMD [16]. In this report, we describe the development of a short-acting CaR antagonist (SB-423562) and its orally active precursor (SB-423557), demonstrating bone-forming effects in animal models and transient increased concentrations of plasma PTH in humans as a clinical proof of principle. Methods Synthesis of compounds NPS2143 was synthesized by NPS Pharmaceuticals [16]; SB423562 and SB-423557 were synthesized by GSK (details to be published elsewhere). PTH release in the rat, dog, and monkey following oral administration of SB-423557 All procedures were performed in accordance with protocols approved by the GlaxoSmithKline Institutional Animal Care and Use Committee, and met or exceeded the standards of the American Association for the Accreditation of Laboratory Animal Care (AAALAC), the US Department of Health and Human Services and all local and federal animal welfare laws. To determine the PTH(1–84) release in the plasma of the various species, SB-423557 was orally administered in a formulation of 1% DMSO, 20% Cavitron (Cargill, Inc., Cedar Rapids, IA). Blood was collected just prior to dosing and at various time points post-dosing, placed into heparinized tubes and centrifuged, and the plasma was then removed and frozen. The concentration of PTH(1–84) in the plasma was determined using a immunoradiometric assay for the rat and an ELISA for both the dog and monkey (Immutopics International, San Clemente, CA). Bone formation oral SB-423557 in ovariectomized rats To examine the bone-forming effect of SB-423557, 6-month-old virgin Sprague–Dawley female rats underwent ovariectomy (OVX) or sham surgery and were left untreated for 6 weeks to allow bone loss to occur. Rats were randomized into groups (n = 8–12 per group) and were treated for an additional 12 weeks. The sham and OVX animals received daily oral treatment with vehicle or SB-423557 (50 mg/kg). A further group of animals was treated with subcutaneous (s.c.) rat PTH(1–34) (5 μg/kg) (Bachem Americas, Torrance, CA) as a positive control for comparison purposes. To measure dynamic histomorphometric changes of lumbar vertebra (L3) and distal tibia, rats from all groups were given fluorochrome labels (calcein 10 mg/kg) twice daily, 6 and 13 days prior to sacrifice. In vivo BMD measurements of the lumbar vertebrae (L3 to L6) were made at baseline (week -1) and at weeks 6, 10, 14 and 18 using dual energy X-ray absorptiometry equipped with high-resolution scanning software (Hologic QDR-4500A, Hologic, Bedford, MA). Individual animals were anesthetized with isoflurane and placed prone on the table. BMD was determined using a region of interest of 55 lines wide. Volumetric trabecular BMD (eight animals per group) of the left proximal tibia was determined at the same time points by peripheral quantitative computed tomography using the Stratec/Norland Research M (Norland Medical Systems, Fort Atkinson, WI). Individual animals were anesthetized with isoflurane and placed on their left side. The left leg was secured and a three-dimensional 0.5 mm slice was taken at a point distal to the growth plate that was 15% of the
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length between the growth plate and the tibia–fibula junction. Settings for the mask were as follows: object length, 200 mm; voxel size, 0.1 mm; diameter, 40 mm; speed, 3 mm/s; number of blocks, 2; scout view (SV) speed, 30 mm/s; and SV distance between lines, 0.5 mm. Rats were housed in metabolic cages for 24 h for the collection of urine during weeks 5 and 12, to determine deoxypyridinoline excretion. Blood was also collected at weeks 5 and 12 and serum was stored frozen for analysis of osteocalcin. The distal tibia and L3 vertebrae were fixed in ethanol and stained with Villanueva Bone Stain (Polysciences, Inc., Warrington, PA). After destaining and dehydration, the bones were embedded in 90% methyl methacrylate/10% dibutyl phthalate/benzoyl peroxide (all from Sigma-Aldrich, St. Louis, MO). A 30 μm tibia–fibula junction cross section of the distal tibia was collected with a SP1600 microtome (Leica Instruments GmbH, Nusseloch, Germany) for analysis of cortical bone. A 5 μm crown section of the L3 vertebrae was cut using a Leica SM2500S microtome for analysis of trabecular bone. All measurements were performed using standard histomorphometric methods [20]. Undecalcified sections were analyzed using Osteomeasure software version 3.02 (Osteometrics, Atlanta, GA). Bone measurements were assessed using bright field fluorescence and polarized light microscopy for the dynamic and static parameters, respectively. A single observer who was blinded to the specimen identity made all measurements. Biomechanical testing was performed on the right midshaft femur and L5 vertebrae. For the compression test of the L5 vertebrae, the posterior pedicle arch, spinous process and the cranial and caudal ends of each vertebral body were removed to obtain a vertebral body specimen with parallel surfaces and a height of approximately 4 mm. The specimens were then placed between two plates of an Instron Mechanical Testing Machine (Instron 4465 retrofitted to 5500, Norwood, MA), and a load was applied at a constant displacement rate of 6 mm/min until failure. The locations for maximum load at failure, stiffness and energy absorbed were selected manually from the load and displacement curve and then calculated by Merlin II software (Instron). The intrinsic properties, stress, elastic modulus and toughness were calculated from maximum load, stiffness, energy absorbed, cross-sectional area and height. For the three-point bending test of the midshaft femur, the whole femur was placed on the lower supports of a three-point bending fixture with the anterior side facing downward in an Instron Mechanical Testing Machine. The span between the two lower supports was set at 14 mm. The upper loading device was aligned to the center of the femoral shaft. The load was applied at a constant displacement rate of 6 mm/min until failure. The locations of maximal load, stiffness and energy absorbed were selected manually and values were calculated by Merlin II software. Statistical analyses All data are presented as mean ± the standard error of the mean (SEM). Statistical comparisons for PTH release in the different species utilized one-way analysis of variance (ANOVA) repeated measures analysis. Statistical comparisons between the OVX control and the different treatments were performed by one-way ANOVA followed by Tukey–Kramer multiple comparison test. The data analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA). p ≤ 0.05 was considered statistically significant. Parathyroid cell proliferation Parathyroid cell proliferation was examined in sham and OVX rats which received SB-423557 at 10, 30 or 100 μmol/kg (5, 15, and 50 mg/kg, respectively) by daily oral gavage for 12 weeks. In addition, one OVX group received rat PTH(1–34) (1 μg/kg) by daily s.c.
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injection. To label proliferating cells, eight animals from each group were infused s.c. with 5-bromo-2′-deoxyuridine (BrdU), a thymidine analog, during the final 4 days of the study. This infusion was achieved using a model 2001 osmotic minipump (Alza, Palo Alto, CA). Each animal was anesthetized with isoflurane and the pump was implanted s.c. between the shoulder blades. The pump infused BrdU at a rate of ~6 mg/rat/day (24 μL/day). The thyroid/parathyroid complex with a portion of adjoining trachea was collected at necropsy and fixed for 24 h at 4 °C in 10% phosphate buffered formalin. After sequential dehydration through ethanol and xylene, the tissues were embedded in Surgipath EM-400 (Surgipath Medical Industries, Richmond, IL). The thyroid/parathyroid complex was cut into 3 μm sagittal sections taken from the most central area of the parathyroid gland. The area of the gland(s) in each section was measured on a digitizer (Nikon Microscope, Summagraphics Tablet, and Histomorphometry Software from KSS Scientific) at ×100 total magnification. To obtain the nuclei profile density of the gland, four to six areas were randomly selected and all of the nuclei within these defined areas were counted. The areas were defined by an eyepiece ocular grid at a total magnification of ×200 with each area measuring 1112 μm2. The total number of nuclei in each gland profile was calculated from the number of nuclei per area and the total gland profile area. The total number of BrdU-labeled cells was counted in the total gland profile. A cell was defined as “labeled” if the BrdU staining intensity was greater than that observed in non-proliferating cells in adjacent tissues (e.g., thyroid and connective tissues) but similar staining intensity to the proliferating epithelial cells in tracheal tissues. The data from one to six sections from both glands, if available, were tabulated. Analysis of proliferating cells was performed in a blinded fashion. Proliferating cells were detected using a BrdU immunostaining kit (Zymed Labs, San Francisco, CA) according to the supplier's protocol. Statistical analyses All data are presented as mean ± the standard error of the mean (SEM). A significant difference between groups was assessed by ANOVA followed by the Tukey–Kramer multiple comparison test using commercial software (StatView version 5.0, SAS Institute, Cary, NC and GraphPad Prism version 5.0, GraphPad Software, La Jolla, CA) unless stated otherwise. Intravenous SB-423562 administration to humans All clinical studies performed were conducted in accordance with the ethical considerations detailed in the Declaration of Helsinki and applicable national or regional Good Clinical Practice guidelines. Subjects signed an informed consent prior to participation. This study was a single-blind, placebo-controlled, crossover, randomized (with respect to placebo), intravenous (i.v.), dose-rising study in healthy adult men. Subjects received placebo (i.v.) in one study session and a single i.v. dose of SB-423562 via a 10-min i.v. infusion during two study sessions separated by at least 7 days. Doses of SB-423562 of 20, 75, 155, 310 and 625 μg and 1.25, 2.5 and 5 mg were administered to up to six volunteers per dose group (n = 28).
Fig. 2. Mean (±SEM) plasma parathyroid hormone (PTH) concentrations following oral administration of SB-423557 in the rat (A). Mean (±SEM) plasma PTH concentrations following oral administration of SB-423557 in the dog and monkey (B). ⁎p b 0.001 vs. vehicle (A) and ⁎p b 0.05 vs. vehicle (B) by repeated measure one-way ANOVA.
Oral SB-423557 administration to humans This was a single-blind, placebo-controlled, crossover, randomized (with respect to placebo), two-period, period-balanced (with respect to allocation of placebo), dose-rising study in healthy adult men (n = 54). Each subject participated in two sessions separated by at least 7 days. Doses of SB-423557 of 5, 10, 20, 50, 100, 200, 400 and 500 mg were administered to up to 6 volunteers per dose group, except in the 50 mg group, where 12 volunteers were dosed. In both studies, multiple blood samples for pharmacokinetic and pharmacodynamic analyses were obtained over 24 h following dosing. SB-423557 and SB-423562 were measured by a validated HPLC/MS/MS assay. In vitro conversion of SB-423557 to SB-423562 was prevented by addition of equal volume of 10% sodium dodecyl sulfate. PTH was measured using an intact PTH immunoradiometric assay (Coat-A-Count, Diagnostic Products Corporation, Inc., Los Angeles, CA). Coat-A-Count Intact PTH IRMA is a solid-phase immunoradiometric assay employing 125I-labeled affinity-purified
Fig. 1. The structure of the calcium-sensing receptor (CaR) antagonist NPS2143, the carboxylic-acid-containing analog SB-423562 and its ethyl ester precursor SB-423557.
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polyclonal anti-PTH (1–34) antibody in liquid phase, in conjunction with affinity-purified polyclonal anti-PTH (44–84) antibodies immobilized to the wall of a polystyrene tube. By requiring binding to both the N-terminal and C-terminal fragment of PTH, this assay is designed for the quantitative measurement of intact parathyroid hormone. Statistical analyses Descriptive statistics were performed on all data. The pharmacokinetic and pharmacodynamic data were analyzed using mixed-effect models by including appropriate terms. Unless specifically mentioned, statistical significance was based on a type I error of 0.05. All statistical analyses were carried out using SAS™ for Windows version 6.12 and 8.1 (SAS Institute, Inc. Cary, NC). Results Development of CaR antagonists The small-molecule NPS2143 (Fig. 1) was identified as the first orally active CaR antagonist with an effect on bone remodeling in the OVX rat model of postmenopausal osteoporosis [16]. However, due to the prolonged compound exposure and associated plasma PTH profile, there was no evidence of a net gain in BMD [16-19]. An extensive medicinal chemistry effort (details to be published elsewhere) focused on optimizing the antagonist potency and the pharmacokinetic parameters of this compound. It was reasoned that a shorter compound half-life and resulting transient plasma PTH profile would eliminate the excessive bone resorption response associated
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with sustained increases in circulating concentrations of PTH. This work led to identification of the carboxylic acid-containing analog, SB423562 (Fig. 1), which is a potent antagonist of the CaR (IC50 = 73 nM, in vitro inhibition of CaR mediated signaling in HEK-293 cells transfected with human CaR). Following i.v. administration to rats, SB-423562 was cleared rapidly (89 mL/min/kg) and had a short half-life (0.23 h) with a significantly lower volume of distribution compared with NPS2143 (0.8 vs. 11 L/kg) resulting in a transient increase in plasma concentrations of PTH (data not shown). Unfortunately, the systemic exposure of SB-423562 following oral administration was not sufficient to elicit robust release of PTH in preclinical animal models. The lack of oral bioavailability of SB-423562 was attributed to poor intestinal absorption and high first-pass hepatic extraction. To circumvent the poor absorption associated with the zwitterion SB-423562, the ethyl ester precursor, SB-423557, was developed (Fig. 1). SB-423557 also exhibited CaR antagonist activity in vitro (IC50 = 520 nM). Oral administration of SB-423557 resulted in dose-dependent systemic exposure of its corresponding acid SB-423562, which in turn elicited a dose-dependent and transient increase in circulating concentrations of endogenous PTH in the rat (Fig. 2A). Transient and statistically significant dose-dependent rise (∼10%) in serum Ca2+ were observed between 1 and 2 h post-dose time points (data not shown). A similar transient increase in PTH was also seen in dogs and monkeys following oral administration of SB-423557 (Fig. 2B). In all species, the ethyl ester was generally not detected systemically, indicating rapid conversion to SB-423562 in vivo.
Fig. 3. The mean (+SEM) week 18 bone mineral density (BMD) at the lumbar spine (A), bone formation rate per total volume (BFR/TV) reference of the third lumbar vertebrae (B), ultimate strength of the fifth lumbar vertebrae (C), percentage of cortical area (D) and endocortical bone formation rate per bone surface (BFR/BS) of the distal tibia (E), and ultimate strength (F) of the femoral diaphysis in the rat. ⁎p b 0.05 vs. ovariectomy determined by repeated measures one-way ANOVA followed by Tukey–Kramer test.
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Bone-forming effect of SB-423557 in ovariectomized rats Oral dosing of SB-423557 led to high blood concentrations (2– 4 μM) of SB-423562 after 20–60 min. PTH concentrations in plasma peaked between 10 and 35 pM within 20 min and returned to baseline concentrations 2–3 h after SB-423557 administration (data not shown). Significant bone loss was observed by week 6 post-OVX at both the lumbar spine and proximal tibia. Both sites continued to lose additional bone mass reaching a nadir by week 14–18. Administration of SB-423557 or PTH(1–34), which began at week 6 post-OVX and continued for 12 additional weeks, elicited significant increases in BMD in the lumbar spine compared with OVX controls at week 18 (Fig. 3A). These increases due to treatment were also observed at weeks 10 and 14 (data not shown). Following administration of PTH(1–34), trabecular BMD of the proximal tibia significantly increased compared with OVX controls (Table 1). An increase in trabecular BMD in the proximal tibia was also seen with SB-423557 at all time points compared with OVX controls, although this was not statistically significant. In rats treated with SB-423557, histomorphometric analysis indicated a greater cortical area (∼83%; Fig. 3D) and endocortical bone formation rate (2.2-fold increase; Fig. 3E) of the distal tibia compared with vehicle-treated OVX animals, with no effect on eroded perimeter (Table 1). In the spine, SB-423557 treatment resulted in an increase in trabecular bone area and bone formation rate, although this increase was not statistically significant (Table 1 and Fig. 3B, respectively). More pertinently, the gains in both cortical and trabecular bone resulted in statistically significant increased parameters of strength in the lumbar spine (Fig. 3C) and femoral midshaft (Fig. 3F) for animals treated with either SB-423557 or PTH(1–34) (Table 1). Urine concentrations of deoxypyridinoline for all OVX groups were significantly elevated 5 weeks after surgery. Treatment with PTH(1–34) significantly lowered deoxypyridinoline concentrations compared to the OVX control (p b 0.05), while animals treated with SB-423557 were not different from the controls (data not shown). Following OVX surgery, osteocalcin concentrations were significantly elevated at week 5 (p b 0.01); however, no effect on osteocalcin concentrations was observed following administration of SB-423557 or PTH(1–34) (data not shown). Thus, oral administration of SB-423557 to OVX rats elicited transient increases in plasma concentrations of PTH, which led to advantageous effects on bone turnover by enhancing both trabecular and cortical bone formation, resulting in increased bone mass and strength in both vertebrae and long bones compared to that of OVX controls.
Fig. 4. Mean effect (±SEM) of daily oral administration of SB-423557 at 10, 30 and 100 μmol/kg (5, 15 or 50 mg/kg) or s.c. injection of rat PTH(1–34) (1 μg/kg) for 12 weeks on parathyroid cell proliferation in ovariectomized (OVX) rats.
Parathyroid gland proliferation One possible consequence of repeated stimulation of the parathyroid gland is increased parathyroid cell proliferation and the induction of secondary hyperparathyroidism. To address this theoretical concern, parathyroid gland proliferation in response to SB-423557 was evaluated at three dose levels (10, 30 and 100 μmol/kg or 5, 15 and 50 mg/kg) after 12 weeks of treatment. There were no significant differences in any treated group compared with controls in the number of labeled cells quantified (p = 0.12), area quantified (p = 0.41) or the total number of cells counted in the parathyroid gland (p = 0.65). There were no significant differences in the incorporation of the BrdU label in any treated group when compared with sham or OVX vehicle treated controls (Fig. 4). Proof of principle in humans We sought to provide proof of principle for CaR antagonism as a means of eliciting endogenous PTH release in humans and also to generate data regarding the safety of this approach. Single doses, ranging from 20 μg to 5 mg, of SB-423562 were administered i.v. to healthy male volunteers. The SB-423562 area under the curve (AUC) and maximal concentration (Cmax) increased in an approximately dose-proportional manner. Systemic clearance of SB-423562 was approximately 500 mL/min and the volume of distribution was approximately 20 L with a half-life of ≤ 1 h.
Table 1 Bone mass, histomorphometric and strength analysis of both trabecular and cortical bone in rats following administration of SB-423557 or PTH(1–34). Parameter
Unit
Sham
OVX
OVX + SB-423557 (50 mg/kg)
OVX + PTH(1–34) (5 μg/kg)
Proximal tibia trabecular BMD LV3—trabecular area LV3—eroded perimeter LV5—maximum load LV5—stiffness LV5—energy LV5—elastic modulus LV5—toughness DT—endocortical eroded perimeter Mid-femur—maximum load Mid-femur—stiffness Mid-femur—energy Mid-femur—elastic modulus Mid-femur—toughness
% of week 6 % % N N/mm mJ MPa mJ/m3 % N N/mm mJ MPa mJ/m3
92.5 ± 2.9⁎ 25.1 ± 2.2⁎ 0.30 ± 0.08⁎ 331.3 ± 18.2 2080 ± 177⁎
65.1 ± 3.3 14.4 ± 1.7 1.27 ± 0.36 268.3 ± 24.5 1145 ± 183 31.9 ± 4.6 372.4 ± 63.3 0.65 ± 0.09 2.0 ± 0.4 212.9 ± 8.3 445.0 ± 20.5 66.3 ± 3.1 3646 ± 242 4.94 ± 0.28
75.7 ± 5.1 18.3 ± 1.7 1.44 ± 0.17 317.1 ± 17.4 2222 ± 225⁎
96.3 ± 3.4⁎ 31.3 ± 2.3⁎ 0.45 ± 0.08⁎ 357.8 ± 16.1⁎ 2416 ± 265⁎ 51.4 ± 4.9⁎
40.3 ± 3.0 794.3 ± 69.2⁎ 0.92 ± 0.05 0.6 ± 0.2 240.7 ± 10.2 812.9 ± 47.8⁎ 86.2 ± 7.2 6288 ± 301⁎ 6.86 ± 0.45⁎
38.7 ± 3.0 800.5 ± 77.1⁎ 0.86 ± 0.05 1.01 ± 0.2 223.8 ± 8.5 732.1 ± 24.5⁎
950.4 ± 120.0⁎ 1.22 ± 0.11⁎ 1.2 ± 0.6 238.0 ± 7.6 785.3 ± 25.4⁎
86.2 ± 7.4 6055 ± 321⁎ 6.89 ± 0.45⁎
78.3 ± 6.7 6495 ± 297⁎ 6.36 ± 0.45
Mean ± standard error mean (⁎p b 0.05 vs. ovariectomy [OVX] determined by repeated measures one-way ANOVA followed by Tukey–Kramer test). Bone mineral density (BMD) of the trabecular bone of the proximal tibia determined by peripheral quantitative computed tomography. Lumbar spine BMD determined by dual X-ray absorptiometry. Mechanical properties of the LV5 determined using vertebral compression. Mechanical properties of the mid-femur determined using three-point bending test. LV3 = third lumbar vertebrae; LV5 = fifth lumbar vertebrae; DT = distal tibia.
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Fig. 5. Mean (±SEM) serum parathyroid hormone (PTH) concentrations following i.v. SB-423562 administration in healthy human subjects. m: minutes; h: hours.
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100 mg and less. However, up to ∼2-fold increase in AUC (0–24 h) was observed at doses of 200 mg and higher (Table 2). Transient serum Ca2+ elevations were reported with administration of SB423557, although these remained within the normal range. For illustration, the mean serum ionized serum calcium in the placebo and 400 mg dose groups is shown in Fig. 8. No change in urinary calcium excretion was noted in any dose group. Oral administration of SB-423557 was generally well tolerated. No deaths, serious AEs or withdrawals due to AEs were reported during the study. A total of 38 AEs were reported, 14 subjects reporting AEs with SB-423557 and 12 subjects with placebo. The most frequently reported AE was headache, which occurred in 8 of 53 subjects (15%) receiving SB-423557 and 8 of 54 subjects (15%) in the placebo group. Only a mild headache in one subject given a dose of 10 mg SB-423557 was considered to be related to the study medication. Discussion
Predose serum PTH ranged from ∼25 to 75 pg/mL. A transient and dose-dependent increase in serum PTH was observed with increasing doses of SB-423562 (Fig. 5). Maximal PTH concentrations were achieved 10–15 min following the start of the 10-min infusion, and elevations of PTH typically lasted b 1 h. Maximum PTH concentrations were increased 3- to 5.5-fold on average relative to baseline at doses of 1.25–5 mg. No effect on serum ionized calcium (Fig. 6) or urinary calcium excretion was noted. Administration (i.v.) of SB-423562 was generally well tolerated. No deaths, serious adverse events (AEs) or withdrawals due to AEs were reported during the study. Reported changes in laboratory parameters and vital signs were not considered to be related to the study medication and were generally asymptomatic. In preclinical species, the oral bioavailability of SB-423562 was low, whereas administration of SB-423557, an ethyl ester precursor of SB-423562, resulted in improved systemic delivery of SB-423562. Thus, a subsequent clinical study was conducted to evaluate SB423557 as an orally active CaR antagonist in humans. Single oral doses ranging from 5 to 500 mg of SB-423557 were administered to healthy male volunteers. SB-423557 resulted in a transient SB-423562 profile in the blood, whereas SB-423557 generally was not detectable, suggesting rapid conversion to SB-423562 in vivo. SB-423562 AUC and Cmax increased with the dose of SB-423557 in a dose-proportional manner, except at the 500 mg dose, which demonstrated high between-subject variability and slightly lower mean Cmax compared to the 400 mg dose level. The terminal half-life of SB-423562 increased with increasing dose of SB-423557 and ranged from 1.4 to 3.7 h; tmax (time to maximal concentration) ranged from 2 to 3 h across all dose ranges. A summary of SB-423562 pharmacokinetic parameters is presented in Table 2. Compared with i.v. administered SB-423562, the delayed tmax and longer half-life following orally administered SB423557 are consistent with absorption-rate limited disposition (so called ‘flip-flop’ pharmacokinetics). The plasma profile of PTH closely resembled the pattern of SB423562 concentrations in the blood (Fig. 7). Consistent increases in plasma PTH concentrations were observed following SB-423557 doses of 50 mg and greater. No significant change in plasma PTH was observed at 5, 10 and 20 mg doses. The duration of PTH elevation (defined as a PTH level at least twice that of pre-dose concentrations) was less than 8 h and median PTH tmax ranged from 1.0 to 2.3 h for doses 50 mg or higher. A summary of selected PTH pharmacodynamic parameters is presented in Table 2. For doses of 50 mg and greater, a 1.8- to 4.2-fold increase in PTH concentrations relative to baseline was noted at tmax. The most robust mean PTH response was observed in the 400 mg dose group with Cmax being as much as 423% higher compared to placebo. PTH area under the curve over 24 h post-dose (AUC(0–24 h)) was not significantly different from placebo at doses of
The data presented here demonstrate a transient increase in plasma concentrations of PTH in animal models and humans following both intravenous administration of SB-423562 and oral administration of its precursor, SB-423557. In addition, the boneforming effects of CaR antagonist was demonstrated in a rat model of bone loss, with increased BMD and trabecular and cortical bone formation, coupled with improved bone strength in the spine and long bones. Current treatments to address bone loss associated with osteoporosis either reduce bone resorption or promote bone formation. In addition, strontium ranelate has been proposed to act on both pathways [4], although its influence on bone formation remains controversial [5]. Subcutaneous administration of PTH(1–34) or PTH (1–84) improves cortical and trabecular BMD and cortical thickness associated with both endosteal and periosteal bone formation, and leads to improved bone strength in animal models and reduced fracture risk in patients with osteoporosis [7,9,21-23]. Thus PTHmediated stimulation of bone formation is of key importance in the treatment of osteoporosis. The development of orally active CaR antagonists which stimulate endogenous PTH secretion from the parathyroid gland provides an alternative to exogenous subcutaneously administered PTH for increasing bone formation. However, first-generation CaR antagonists were unable to produce a sufficiently transient PTH profile required for bone formation [16-19,24]. We have now developed a secondgeneration CaR antagonist, SB-423562, and its orally active precursor, SB-423557, which elicits transient PTH profiles in animal models and healthy male volunteers. In addition, in an OVX rat model of bone loss, daily oral SB-423557 administration led to increased bone formation
Fig. 6. Mean (±SEM) serum ionized calcium concentrations following i.v. SB-423562 administration in healthy human subjects. m: minutes; h: hours.
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Table 2 Summary of selected blood SB-423562 pharmacokinetic and plasma PTH pharmacodynamic parameters following oral administration of SB-423557. Dose (mg) Placebo 5 10 20 50 100 200 400 500
SB-423562 parameters
PTH parameters
AUC(0–t)a (ng⁎h/mL)
Cmaxa (ng/mL)
tmaxb (h)
t1/2a (h)
AUC(0-24 h)a (pg.h/mL)
Cmaxa (pg/mL)
tmaxb (h)
Max % changec
NA ND 19 (72) 36 (72) 157 (72) 362 (55) 898 (25) 1865 (30) 1808 (80)
NA ND 19 (38) 20 (35) 51 (69) 96 (62) 215 (34) 402 (31) 327 (78)
NA ND 2.0 (1.0, 2.0 (0.8, 2.8 (1.0, 2.3 (1.3, 2.0 (1.3, 3.0 (1.0, 2.8 (1.3,
NA ND ND ND 1.4 (22) 1.7 (22) 2.1 (33) 2.4 (21) 3.7 (26)
776 (33) 529 (22) 732 (51) 743 (29) 779 (33) 878 (40) 1073 (37) 1507 (35) 1003 (35)
42 (33) 29 (25) 42 (33) 44 (40) 54 (53) 69 (36) 131 (57) 191 (43) 113 (31)
8.0 8.0 2.5 8.0 2.3 1.0 1.4 2.0 1.5
NA 4.2 (−62.6, 71.0) 2.5 (−62.3, 67.3) 0.6 (−70.5, 71.6) 81.2 (32.2, 130) 97.7 (32.8, 163) 262 (196, 328) 423 (357, 488) 301 (236, 365)
3.0) 3.5) 4.0) 4.0) 3.5) 4.0) 3.0)
(0.3, 23.9) (1.8, 12.0) (0.5, 12.0) (1.8, 12.0) (0.5, 12.0) (0.5, 3.5) (0.8, 2.5) (0.5, 2.5) (0.7, 3.0)
NA = not applicable, ND = not determined, AUC(0–t) = area under the concentration-time curve from time zero (pre-dose) to time of last quantifiable concentration, Cmax = maximum plasma concentration, tmax = time of maximum observed concentration, t1/2 = terminal phase half-life, PTH Cmax = maximum PTH concentration post-dose, AUC (0–24 h) = area under the concentration-time curve from time zero to 24 h post-dose determined by the trapezoidal method. a Geometric mean (CV%) reported for AUC and Cmax. b Median (range) reported for tmax. c Maximum percent change defined as 100 ⁎ ((PTH Cmax − baseline)/baseline)); baseline defined as pre-dose concentration. Reported as point estimate (95% confidence interval). Parameter point estimate is the difference of adjusted LSmeans between active and placebo regimens.
and bone strength, demonstrating a preclinical proof of concept for this new class of compound. Overall, the bone-forming effect of SB-423557 in the rat was of a somewhat lesser magnitude compared with subcutaneously admin-
Fig. 7. Mean (±SEM) SB-423562 concentrations in blood (A) and parathyroid hormone (PTH) concentration in plasma (B) following oral administration of SB-423557 to healthy human subjects. Doses b 50 mg are not shown for clarity.
istered PTH(1–34). Although the OVX rat model is well established to study the effect of PTH on bone, concentrations of PTH several-fold higher are required compared with doses of PTH that are used in clinical practice [7,25,26]. It may not be possible to achieve this level of PTH release from the parathyroid gland in the rat via CaR antagonism, presumably due to either a physiologic limit in the amount of endogenous PTH available for immediate release from the parathyroid or due to feedback mechanisms. While caution should be applied when directly comparing PTH concentrations due to differences in the immunoassays for PTH(1–34) and PTH(1–84), on a molar basis, peak concentrations of PTH in the rat were ∼4-fold lower following SB-423557 compared to s.c. PTH(1–34). Accepting this difference, the lower PTH exposure following SB-423557 likely contributed to the somewhat lower bone-forming effects of this compound compared to s.c. PTH(1–34). Repeated antagonism of the CaR could theoretically lead to parathyroid gland hyperplasia. However, whether chronic stimulation of the parathyroid gland by, for example, dietary calcium deficiency increases parathyroid cell proliferation is controversial. Increased proliferation has been seen in weanling [27], but not in older rats fed low calcium diets [28]. Similarly, secondary or tertiary hyperparathyroidism is seen in patients with hypophosphatemic osteomalacia or Xlinked dominant hypophosphatamic rickets, respectively, on longterm oral phosphate therapy [29,30]. However, these may or may not
Fig. 8. Mean (±SEM) serum calcium concentrations in healthy human subjects following oral administration with either 400 mg of SB-423557 or placebo.
S. Kumar et al. / Bone 46 (2010) 534–542
involve CaR. Therefore, we examined the effect of SB-423557 on the parathyroid gland in the rat model. This study showed that daily administration of SB-423557 to OVX rats for 12 weeks did not increase parathyroid cell proliferation, confirming previous results with the longer acting compound NPS2143 [16]. While longer duration studies will be required to confirm the effect of long-term treatment, the current data indicate that repeated antagonism of parathyroid CaR by a calcilytic for up to 12 weeks does not lead to parathyroid hyperplasia. The duration of PTH exposure is an important determinant of the relative anabolic versus catabolic effects on bone. In the rat, a transient increase in plasma PTH concentrations over l–2 h has been shown to be optimal for bone formation [31,32]. In humans, continuous elevation of PTH, as seen in moderate-to-severe hyperparathyroidism, is associated with decreased BMD [33]. In contrast, following s.c. administration of 20 μg PTH(1–34), a tmax of approximately 30 min and a duration of elevated PTH of up to 3 h have been reported, which led to improved BMD and decreased fracture risk in patients with osteoporosis [34]. The PTH response following oral administration of a range of doses of SB423557 to humans correlates with that seen following exogenous s.c. PTH therapy, with a tmax ranging from 1 to 2 h and a duration of elevated PTH from 0.5 to 6.5 h for doses of 50 to 500 mg. The duration of PTH elevation appears directly proportional to the systemic exposure of SB423562. Thus i.v. administration of SB-423562 resulted in a SB-423562 and PTH exposure that was shorter in duration whereas oral administration of SB-423557 led to a more extended exposure. In the study with i.v. administered SB-423562, no significant increases in serum ionized calcium was noted. It is important to note that calcemic response is a function of the duration and magnitude of the PTH exposure. Thus, a lack of serum Ca2+ elevation in the i.v. SB423562 study is likely due to shorter duration of PTH exposure in this study. Such transient ∼3- to 5-fold increase in endogenous PTH of a duration lasting b 30 min is unlikely to produce Ca2+ changes from either skeleton or kidney. In contrast, higher doses of orally administered SB-423557 resulted in comparable maximal PTH concentrations but with a duration lasting several hours, which were associated with modest increases in serum Ca2+. Although we did not measure renal function in these studies, no change in urinary calcium excretion was noted. Overall, single administration of SB-423562 and SB-423557 in humans elicited PTH release in a profile consistent with bone-forming activity. In addition, administration of these compounds to humans demonstrated excellent tolerability in this short-term study. However, repeat dose studies will be required to evaluate longer term safety and impact on renal function and calcium homeostasis in humans. In summary, we have designed SB-423562, a CaR antagonist, and SB-423557, an orally active ethyl ester precursor of SB-423562. These compounds elicit a transient PTH profile in animal models and humans resulting in a bone-forming effect in the OVX rat model of bone loss. Further assessment of CaR antagonists is ongoing in longterm studies to evaluate safety and efficacy parameters, including PK– PD relationships, bone biomarker response and BMD in patients with osteoporosis. Acknowledgments We thank Dr. Scott Miller and Mary Beth Bowman of the Department of Radiobiology at the University of Utah for analysis of proliferating cells in the parathyroid glands. We thank Drs. Larry Suva and Dominique Ethgen for their contributions. We also acknowledge the editorial assistance provided by Lucy Richardson during the preparation of this manuscript. References [1] Tarantino U, Cannata G, Lecce D, Celi M, Cerocchi I, Iundusi R. Incidence of fragility fractures. Aging Clin Exp Res 2007;19:7–11.
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[2] Ettinger M. Aging bone and osteoporosis: strategies for preventing fractures in the elderly. Arch Intern Med 2003;163:2237–46. [3] Cummings S, Melton L. Epidemiology and outcomes of osteoporotic fractures. Lancet 2002;359:1761–7. [4] Bonnelye E, Chabadel A, Saltel F, Jurdic P. Dual effect of strontium ranelate: stimulation of osteoblast differentiation and inhibition of osteoclast formation and resorption in vitro. Bone 2008;42:129–38. [5] Anastasilakis AD, Bhansali A, Ahluwalia J, Chanukya GV, Behera A, Dutta P. No difference between strontium ranelate (SR) and calcium/vitamin D on bone turnover markers in women with established osteoporosis previously treated with teriparatide: a randomized controlled trial. Clin Endocrinol (Oxf) 2009;70: 522–6. [6] Davison K, Siminoski K, Adachi J, Hanley D, Goltzman D, Hodsman A, et al. The effects of antifracture therapies on the components of bone strength: assessment of fracture risk today and in the future. Semin Arthritis Rheum 2006;36:10–21. [7] Neer RM, Arnaud CD, Zanchetta JR, Prince R, Gaich GA, Reginster JY, et al. Effect of parathyroid hormone (1–34) on fractures and bone mineral density in postmenopausal women with osteoporosis. N Engl J Med 2001;344: 1434–41. [8] Liu C, Kalu D. Human parathyroid hormone-(1–34) prevents bone loss and augments bone formation in sexually mature ovariectomized rats. J Bone Miner Res 1990;5:973–82. [9] Black D, Bouxsein M, Palermo L, McGowan J, Newitt D, Rosen E, et al. Randomized trial of once-weekly PTH(1–84) on bone mineral density and remodeling. J Clin Endocrinol Metab 2008;93:2166–72. [10] Zhang K, Chen J, Li Q, Li G, Tian X, Huang L, et al. Effect of intermittent injection of recombinant human parathyroid hormone on bone histomorphometry of ovariectomized rats. Acta Pharmacol Sin 2002;23:659–62. [11] Tam CS, Heersche JN, Murray TM, Parsons JA. Parathyroid hormone stimulates the bone apposition rate independently of its resorptive action: differential effects of intermittent and continuous administration. Endocrinology 1982;110: 506–12. [12] Reeve J, Meunier PJ, Parsons JA, Bernat M, Bijvoet OL, Courpron P, et al. Anabolic effect of human parathyroid hormone fragment on trabecular bone in involutional osteoporosis: a multicentre trial. Br Med J 1980;280:1340–4. [13] Whitfield JF, Morley P, Willick GE. The bone-building action of the parathyroid hormone: implications for the treatment of osteoporosis. Drugs Aging 1999;15: 117–29. [14] Lotinun S, Sibonga J, Turner R. Differential effects of intermittent and continuous administration of parathyroid hormone on bone histomorphometry and gene expression. Endocrine 2002;17:29–36. [15] Brown E. Physiology and pathophysiology of the extracellular calcium-sensing receptor. Amer J Med 1999;106:238–53. [16] Gowen M, Stroup GB, Dodds RA, James IE, Votta BJ, Smith BR, et al. Antagonizing the parathyroid calcium receptor stimulates parathyroid hormone secretion and bone formation in osteopenic rats. J Clin Invest 2000;105:1595–604. [17] Nemeth EF. The search for calcium receptor antagonists (calcilytics). J Mol Endocrinol 2002;29:15–21. [18] Nemeth EF. Pharmacological regulation of parathyroid hormone secretion. Curr Pharm Des 2002;8:2077–87. [19] Nemeth EF, Delmar EG, Heaton WL, Miller MA, Lambert LD, Conklin RL, et al. Calcilytic compounds: potent and selective Ca2+ receptor antagonists that stimulate secretion of parathyroid hormone. J Pharmacol Exp Ther 2001;299: 323–31. [20] Parfitt A, Glorieux F, Kanis J, Malluche H, Meunier P, Ott S, et al. Bone histomorphometry nomenclature, symbols and units: report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 1987;2:595–610. [21] Arita S, Ikeda S, Sakai A, Okimoto N, Akahoshi S, Nagashima M, et al. Human parathyroid hormone (1–34) increases mass and structure of the cortical shell, with resultant increase in lumbar bone strength, in ovariectomized rats. J Bone Miner Metab 2004;22:530–40. [22] Hodsman A, Kisiel M, Adachi J, Fraher L, Watson P. Histomorphometric evidence for increased bone turnover without change in cortical thickness or porosity after 2 years of cyclical hPTH(1–34) therapy in women with severe osteoporosis. Bone 2000;27:311–8. [23] Nozaka K, Miyakoshi N, Kasukawa Y, Maekawa S, Noguchi H, Shimada Y. Intermittent administration of human parathyroid hormone enhances bone formation and union at the site of cancellous bone osteotomy in normal and ovariectomized rats. Bone 2008;42:90–7. [24] Miller M, Fox J. Daily transient decreases in plasma parathyroid hormone levels induced by the calcimimetic NPS R-568 slows the rate of bone loss but does not increase bone mass in ovariectomized rats. Bone 2000;27:511–9. [25] Sato M, Schmidt A, Ma L, Smith S, Rowley E, Cole H, et al. Dose-dependent PTH effects in young, intact, female rats after 9 months of treatment. J Bone Miner Res 2001;16:S292. [26] Sato M, Zeng G, Turner C. Biosynthetic human parathyroid hormone (1–34) effects on bone quality in aged ovariectomized rats. Endocrinology 1997;138:4330–7. [27] Naveh-Many T, Rahamimov R, Livni N, Silver J. Parathyroid cell proliferation in normal and chronic renal failure rats. The effects of calcium, phosphate, and vitamin D. J Clin Invest 1995;96:1786–93. [28] Wernerson A, Widholm S, Svensson O, Reinholt F. Parathyroid cell number and size in hypocalcemic young rats. APMIS 1991;99:1096–102. [29] Su DH, Liao KM, Chang YC, Tsai KS. Secondary hyperparathyroidism as a palpable intrathyroid parathyroid gland in a patient with hypophosphatemic osteomalacia. J Bone Miner Metab 2006;24:114–7.
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Contents lists available at ScienceDirect
Bone j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b o n e
An orally active calcium-sensing receptor antagonist that transiently increases plasma concentrations of PTH and stimulates bone formation Sanjay Kumar a,⁎, Christopher J. Matheny a, Sandra J. Hoffman b, Robert W. Marquis b, Maggie Schultz b, Xiaoguang Liang b, Janice A. Vasko b, George B. Stroup a, Vernal R. Vaden a, Hyking Haley a, John Fox c, Eric G. DelMar c, Edward F. Nemeth c, Amparo M. Lago a, James F. Callahan a, Pradip Bhatnagar a, William F. Huffman b, Maxine Gowen a, Bingming Yi b, Theodore M. Danoff b, Lorraine A. Fitzpatrick a a b c
GlaxoSmithKline, UM 2230, 709 Swedeland Road, King of Prussia, PA 19406-2711, USA GlaxoSmithKline, Collegeville, PA, USA NPS Pharmaceuticals, Salt Lake City, USA
a r t i c l e
i n f o
Article history: Received 19 May 2009 Revised 24 August 2009 Accepted 22 September 2009 Available online 26 September 2009 Edited by: R. Baron Keywords: Parathyroid hormone Parathyroid gland Calcium-sensing receptor Bone formation Osteoporosis
a b s t r a c t Daily subcutaneous administration of exogenous parathyroid hormone (PTH) promotes bone formation in patients with osteoporosis. Here we describe two novel, short-acting calcium-sensing receptor antagonists (SB-423562 and its orally bioavailable precursor, SB-423557) that elicit transient PTH release from the parathyroid gland in several preclinical species and in humans. In an ovariectomized rat model of bone loss, daily oral administration of SB-423557 promoted bone formation and improved parameters of bone strength at lumbar spine, proximal tibia and midshaft femur. Chronic administration of SB-423557 did not increase parathyroid cell proliferation in rats. In healthy human volunteers, single doses of intravenous SB-423562 and oral SB-423557 elicited transient elevations of endogenous PTH concentrations in a profile similar to that observed with subcutaneously administered PTH. Both agents were well tolerated in humans. Transient increases in serum calcium, an expected effect of increased parathyroid hormone concentrations, were observed post-dose at the higher doses of SB-423557 studied. These data constitute an early proof of principle in humans and provide the basis for further development of this class of compound as a novel, orally administered bone-forming treatment for osteoporosis. © 2009 Elsevier Inc. All rights reserved.
Introduction Osteoporosis is prevalent in postmenopausal women, with approximately one in three women aged over 50 years affected [1], and is increasing in incidence due, in part, to an aging population [2]. Furthermore, osteoporosis affects around 200 million people worldwide, and thus represents a substantial financial burden [1]. In 2002, the combined annual cost of osteoporosis-related fractures was estimated at US$20 billion in the US and US$30 billion in Europe [3]. The pathogenesis of osteoporosis results from an imbalance in bone turnover and a net increase in bone resorption leading to reduced bone mass and an increased risk of fracture. Current treatment options for osteoporosis either inhibit bone resorption through the use of antiresorptive agents (e.g., bisphosphonates, estradiol, calcitonin and raloxifene) and promote bone formation with anabolic agents (e.g., intact parathyroid hormone [PTH(1–84)] and the 34-amino acid peptide [PTH(1–34)]) or a combination of both approaches as suggested for strontium ranelate [4]. However, controversy surrounding the bone-forming effect of strontium ranelate has recently arisen [5]. ⁎ Corresponding author. Fax: +1 610 270 5580. E-mail address: [email protected] (S. Kumar). 8756-3282/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2009.09.028
Net bone formation is characterized by an increase in bone mineral density (BMD) and increased trabecular and cortical areas, resulting in a bone architecture with increased strength that is more resistant to fracture [6]. The bone-forming effects and increased bone strength following transient exposure to PTH through intermittent, subcutaneous administration of PTH(1–84) or PTH(1–34) in animal models and healthy human volunteers, as well as in patients with osteoporosis, have been well documented [7-13]. In contrast, continuous infusion of PTH leads to increased bone turnover, but without net formation, resulting in overall bone loss [14]. PTH plays a central role in calcium homeostasis, through its effects on renal calcium excretion, bone resorption, and, indirectly, intestinal calcium absorption. PTH secretion from the parathyroid gland is negatively regulated by ionized serum calcium (Ca2+), mediated through the calcium-sensing receptor (CaR). The CaR is a G-proteincoupled receptor present on cells of the parathyroid gland, thyroid C-cells, bone, kidney and gastrointestinal tissues, among others, and is activated by increased concentrations of extracellular Ca2+ [15]. As the concentration of Ca2+ in the blood increases, activation of CaRs on parathyroid cells inhibits PTH secretion. Conversely, a decrease in Ca2+ triggers PTH release. Targeting this regulatory mechanism, we have previously shown that small-molecule CaR antagonists stimulate
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significant but sustained increases in PTH concentrations in animal models, resulting in an increase in both bone formation and bone resorption [16-19]. The lack of favorable effects on bone remodeling was circumvented by co-treatment with estradiol, which reduced bone resorption leading to a net increase in bone formation and BMD [16]. In this report, we describe the development of a short-acting CaR antagonist (SB-423562) and its orally active precursor (SB-423557), demonstrating bone-forming effects in animal models and transient increased concentrations of plasma PTH in humans as a clinical proof of principle. Methods Synthesis of compounds NPS2143 was synthesized by NPS Pharmaceuticals [16]; SB423562 and SB-423557 were synthesized by GSK (details to be published elsewhere). PTH release in the rat, dog, and monkey following oral administration of SB-423557 All procedures were performed in accordance with protocols approved by the GlaxoSmithKline Institutional Animal Care and Use Committee, and met or exceeded the standards of the American Association for the Accreditation of Laboratory Animal Care (AAALAC), the US Department of Health and Human Services and all local and federal animal welfare laws. To determine the PTH(1–84) release in the plasma of the various species, SB-423557 was orally administered in a formulation of 1% DMSO, 20% Cavitron (Cargill, Inc., Cedar Rapids, IA). Blood was collected just prior to dosing and at various time points post-dosing, placed into heparinized tubes and centrifuged, and the plasma was then removed and frozen. The concentration of PTH(1–84) in the plasma was determined using a immunoradiometric assay for the rat and an ELISA for both the dog and monkey (Immutopics International, San Clemente, CA). Bone formation oral SB-423557 in ovariectomized rats To examine the bone-forming effect of SB-423557, 6-month-old virgin Sprague–Dawley female rats underwent ovariectomy (OVX) or sham surgery and were left untreated for 6 weeks to allow bone loss to occur. Rats were randomized into groups (n = 8–12 per group) and were treated for an additional 12 weeks. The sham and OVX animals received daily oral treatment with vehicle or SB-423557 (50 mg/kg). A further group of animals was treated with subcutaneous (s.c.) rat PTH(1–34) (5 μg/kg) (Bachem Americas, Torrance, CA) as a positive control for comparison purposes. To measure dynamic histomorphometric changes of lumbar vertebra (L3) and distal tibia, rats from all groups were given fluorochrome labels (calcein 10 mg/kg) twice daily, 6 and 13 days prior to sacrifice. In vivo BMD measurements of the lumbar vertebrae (L3 to L6) were made at baseline (week -1) and at weeks 6, 10, 14 and 18 using dual energy X-ray absorptiometry equipped with high-resolution scanning software (Hologic QDR-4500A, Hologic, Bedford, MA). Individual animals were anesthetized with isoflurane and placed prone on the table. BMD was determined using a region of interest of 55 lines wide. Volumetric trabecular BMD (eight animals per group) of the left proximal tibia was determined at the same time points by peripheral quantitative computed tomography using the Stratec/Norland Research M (Norland Medical Systems, Fort Atkinson, WI). Individual animals were anesthetized with isoflurane and placed on their left side. The left leg was secured and a three-dimensional 0.5 mm slice was taken at a point distal to the growth plate that was 15% of the
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length between the growth plate and the tibia–fibula junction. Settings for the mask were as follows: object length, 200 mm; voxel size, 0.1 mm; diameter, 40 mm; speed, 3 mm/s; number of blocks, 2; scout view (SV) speed, 30 mm/s; and SV distance between lines, 0.5 mm. Rats were housed in metabolic cages for 24 h for the collection of urine during weeks 5 and 12, to determine deoxypyridinoline excretion. Blood was also collected at weeks 5 and 12 and serum was stored frozen for analysis of osteocalcin. The distal tibia and L3 vertebrae were fixed in ethanol and stained with Villanueva Bone Stain (Polysciences, Inc., Warrington, PA). After destaining and dehydration, the bones were embedded in 90% methyl methacrylate/10% dibutyl phthalate/benzoyl peroxide (all from Sigma-Aldrich, St. Louis, MO). A 30 μm tibia–fibula junction cross section of the distal tibia was collected with a SP1600 microtome (Leica Instruments GmbH, Nusseloch, Germany) for analysis of cortical bone. A 5 μm crown section of the L3 vertebrae was cut using a Leica SM2500S microtome for analysis of trabecular bone. All measurements were performed using standard histomorphometric methods [20]. Undecalcified sections were analyzed using Osteomeasure software version 3.02 (Osteometrics, Atlanta, GA). Bone measurements were assessed using bright field fluorescence and polarized light microscopy for the dynamic and static parameters, respectively. A single observer who was blinded to the specimen identity made all measurements. Biomechanical testing was performed on the right midshaft femur and L5 vertebrae. For the compression test of the L5 vertebrae, the posterior pedicle arch, spinous process and the cranial and caudal ends of each vertebral body were removed to obtain a vertebral body specimen with parallel surfaces and a height of approximately 4 mm. The specimens were then placed between two plates of an Instron Mechanical Testing Machine (Instron 4465 retrofitted to 5500, Norwood, MA), and a load was applied at a constant displacement rate of 6 mm/min until failure. The locations for maximum load at failure, stiffness and energy absorbed were selected manually from the load and displacement curve and then calculated by Merlin II software (Instron). The intrinsic properties, stress, elastic modulus and toughness were calculated from maximum load, stiffness, energy absorbed, cross-sectional area and height. For the three-point bending test of the midshaft femur, the whole femur was placed on the lower supports of a three-point bending fixture with the anterior side facing downward in an Instron Mechanical Testing Machine. The span between the two lower supports was set at 14 mm. The upper loading device was aligned to the center of the femoral shaft. The load was applied at a constant displacement rate of 6 mm/min until failure. The locations of maximal load, stiffness and energy absorbed were selected manually and values were calculated by Merlin II software. Statistical analyses All data are presented as mean ± the standard error of the mean (SEM). Statistical comparisons for PTH release in the different species utilized one-way analysis of variance (ANOVA) repeated measures analysis. Statistical comparisons between the OVX control and the different treatments were performed by one-way ANOVA followed by Tukey–Kramer multiple comparison test. The data analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA). p ≤ 0.05 was considered statistically significant. Parathyroid cell proliferation Parathyroid cell proliferation was examined in sham and OVX rats which received SB-423557 at 10, 30 or 100 μmol/kg (5, 15, and 50 mg/kg, respectively) by daily oral gavage for 12 weeks. In addition, one OVX group received rat PTH(1–34) (1 μg/kg) by daily s.c.
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injection. To label proliferating cells, eight animals from each group were infused s.c. with 5-bromo-2′-deoxyuridine (BrdU), a thymidine analog, during the final 4 days of the study. This infusion was achieved using a model 2001 osmotic minipump (Alza, Palo Alto, CA). Each animal was anesthetized with isoflurane and the pump was implanted s.c. between the shoulder blades. The pump infused BrdU at a rate of ~6 mg/rat/day (24 μL/day). The thyroid/parathyroid complex with a portion of adjoining trachea was collected at necropsy and fixed for 24 h at 4 °C in 10% phosphate buffered formalin. After sequential dehydration through ethanol and xylene, the tissues were embedded in Surgipath EM-400 (Surgipath Medical Industries, Richmond, IL). The thyroid/parathyroid complex was cut into 3 μm sagittal sections taken from the most central area of the parathyroid gland. The area of the gland(s) in each section was measured on a digitizer (Nikon Microscope, Summagraphics Tablet, and Histomorphometry Software from KSS Scientific) at ×100 total magnification. To obtain the nuclei profile density of the gland, four to six areas were randomly selected and all of the nuclei within these defined areas were counted. The areas were defined by an eyepiece ocular grid at a total magnification of ×200 with each area measuring 1112 μm2. The total number of nuclei in each gland profile was calculated from the number of nuclei per area and the total gland profile area. The total number of BrdU-labeled cells was counted in the total gland profile. A cell was defined as “labeled” if the BrdU staining intensity was greater than that observed in non-proliferating cells in adjacent tissues (e.g., thyroid and connective tissues) but similar staining intensity to the proliferating epithelial cells in tracheal tissues. The data from one to six sections from both glands, if available, were tabulated. Analysis of proliferating cells was performed in a blinded fashion. Proliferating cells were detected using a BrdU immunostaining kit (Zymed Labs, San Francisco, CA) according to the supplier's protocol. Statistical analyses All data are presented as mean ± the standard error of the mean (SEM). A significant difference between groups was assessed by ANOVA followed by the Tukey–Kramer multiple comparison test using commercial software (StatView version 5.0, SAS Institute, Cary, NC and GraphPad Prism version 5.0, GraphPad Software, La Jolla, CA) unless stated otherwise. Intravenous SB-423562 administration to humans All clinical studies performed were conducted in accordance with the ethical considerations detailed in the Declaration of Helsinki and applicable national or regional Good Clinical Practice guidelines. Subjects signed an informed consent prior to participation. This study was a single-blind, placebo-controlled, crossover, randomized (with respect to placebo), intravenous (i.v.), dose-rising study in healthy adult men. Subjects received placebo (i.v.) in one study session and a single i.v. dose of SB-423562 via a 10-min i.v. infusion during two study sessions separated by at least 7 days. Doses of SB-423562 of 20, 75, 155, 310 and 625 μg and 1.25, 2.5 and 5 mg were administered to up to six volunteers per dose group (n = 28).
Fig. 2. Mean (±SEM) plasma parathyroid hormone (PTH) concentrations following oral administration of SB-423557 in the rat (A). Mean (±SEM) plasma PTH concentrations following oral administration of SB-423557 in the dog and monkey (B). ⁎p b 0.001 vs. vehicle (A) and ⁎p b 0.05 vs. vehicle (B) by repeated measure one-way ANOVA.
Oral SB-423557 administration to humans This was a single-blind, placebo-controlled, crossover, randomized (with respect to placebo), two-period, period-balanced (with respect to allocation of placebo), dose-rising study in healthy adult men (n = 54). Each subject participated in two sessions separated by at least 7 days. Doses of SB-423557 of 5, 10, 20, 50, 100, 200, 400 and 500 mg were administered to up to 6 volunteers per dose group, except in the 50 mg group, where 12 volunteers were dosed. In both studies, multiple blood samples for pharmacokinetic and pharmacodynamic analyses were obtained over 24 h following dosing. SB-423557 and SB-423562 were measured by a validated HPLC/MS/MS assay. In vitro conversion of SB-423557 to SB-423562 was prevented by addition of equal volume of 10% sodium dodecyl sulfate. PTH was measured using an intact PTH immunoradiometric assay (Coat-A-Count, Diagnostic Products Corporation, Inc., Los Angeles, CA). Coat-A-Count Intact PTH IRMA is a solid-phase immunoradiometric assay employing 125I-labeled affinity-purified
Fig. 1. The structure of the calcium-sensing receptor (CaR) antagonist NPS2143, the carboxylic-acid-containing analog SB-423562 and its ethyl ester precursor SB-423557.
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polyclonal anti-PTH (1–34) antibody in liquid phase, in conjunction with affinity-purified polyclonal anti-PTH (44–84) antibodies immobilized to the wall of a polystyrene tube. By requiring binding to both the N-terminal and C-terminal fragment of PTH, this assay is designed for the quantitative measurement of intact parathyroid hormone. Statistical analyses Descriptive statistics were performed on all data. The pharmacokinetic and pharmacodynamic data were analyzed using mixed-effect models by including appropriate terms. Unless specifically mentioned, statistical significance was based on a type I error of 0.05. All statistical analyses were carried out using SAS™ for Windows version 6.12 and 8.1 (SAS Institute, Inc. Cary, NC). Results Development of CaR antagonists The small-molecule NPS2143 (Fig. 1) was identified as the first orally active CaR antagonist with an effect on bone remodeling in the OVX rat model of postmenopausal osteoporosis [16]. However, due to the prolonged compound exposure and associated plasma PTH profile, there was no evidence of a net gain in BMD [16-19]. An extensive medicinal chemistry effort (details to be published elsewhere) focused on optimizing the antagonist potency and the pharmacokinetic parameters of this compound. It was reasoned that a shorter compound half-life and resulting transient plasma PTH profile would eliminate the excessive bone resorption response associated
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with sustained increases in circulating concentrations of PTH. This work led to identification of the carboxylic acid-containing analog, SB423562 (Fig. 1), which is a potent antagonist of the CaR (IC50 = 73 nM, in vitro inhibition of CaR mediated signaling in HEK-293 cells transfected with human CaR). Following i.v. administration to rats, SB-423562 was cleared rapidly (89 mL/min/kg) and had a short half-life (0.23 h) with a significantly lower volume of distribution compared with NPS2143 (0.8 vs. 11 L/kg) resulting in a transient increase in plasma concentrations of PTH (data not shown). Unfortunately, the systemic exposure of SB-423562 following oral administration was not sufficient to elicit robust release of PTH in preclinical animal models. The lack of oral bioavailability of SB-423562 was attributed to poor intestinal absorption and high first-pass hepatic extraction. To circumvent the poor absorption associated with the zwitterion SB-423562, the ethyl ester precursor, SB-423557, was developed (Fig. 1). SB-423557 also exhibited CaR antagonist activity in vitro (IC50 = 520 nM). Oral administration of SB-423557 resulted in dose-dependent systemic exposure of its corresponding acid SB-423562, which in turn elicited a dose-dependent and transient increase in circulating concentrations of endogenous PTH in the rat (Fig. 2A). Transient and statistically significant dose-dependent rise (∼10%) in serum Ca2+ were observed between 1 and 2 h post-dose time points (data not shown). A similar transient increase in PTH was also seen in dogs and monkeys following oral administration of SB-423557 (Fig. 2B). In all species, the ethyl ester was generally not detected systemically, indicating rapid conversion to SB-423562 in vivo.
Fig. 3. The mean (+SEM) week 18 bone mineral density (BMD) at the lumbar spine (A), bone formation rate per total volume (BFR/TV) reference of the third lumbar vertebrae (B), ultimate strength of the fifth lumbar vertebrae (C), percentage of cortical area (D) and endocortical bone formation rate per bone surface (BFR/BS) of the distal tibia (E), and ultimate strength (F) of the femoral diaphysis in the rat. ⁎p b 0.05 vs. ovariectomy determined by repeated measures one-way ANOVA followed by Tukey–Kramer test.
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Bone-forming effect of SB-423557 in ovariectomized rats Oral dosing of SB-423557 led to high blood concentrations (2– 4 μM) of SB-423562 after 20–60 min. PTH concentrations in plasma peaked between 10 and 35 pM within 20 min and returned to baseline concentrations 2–3 h after SB-423557 administration (data not shown). Significant bone loss was observed by week 6 post-OVX at both the lumbar spine and proximal tibia. Both sites continued to lose additional bone mass reaching a nadir by week 14–18. Administration of SB-423557 or PTH(1–34), which began at week 6 post-OVX and continued for 12 additional weeks, elicited significant increases in BMD in the lumbar spine compared with OVX controls at week 18 (Fig. 3A). These increases due to treatment were also observed at weeks 10 and 14 (data not shown). Following administration of PTH(1–34), trabecular BMD of the proximal tibia significantly increased compared with OVX controls (Table 1). An increase in trabecular BMD in the proximal tibia was also seen with SB-423557 at all time points compared with OVX controls, although this was not statistically significant. In rats treated with SB-423557, histomorphometric analysis indicated a greater cortical area (∼83%; Fig. 3D) and endocortical bone formation rate (2.2-fold increase; Fig. 3E) of the distal tibia compared with vehicle-treated OVX animals, with no effect on eroded perimeter (Table 1). In the spine, SB-423557 treatment resulted in an increase in trabecular bone area and bone formation rate, although this increase was not statistically significant (Table 1 and Fig. 3B, respectively). More pertinently, the gains in both cortical and trabecular bone resulted in statistically significant increased parameters of strength in the lumbar spine (Fig. 3C) and femoral midshaft (Fig. 3F) for animals treated with either SB-423557 or PTH(1–34) (Table 1). Urine concentrations of deoxypyridinoline for all OVX groups were significantly elevated 5 weeks after surgery. Treatment with PTH(1–34) significantly lowered deoxypyridinoline concentrations compared to the OVX control (p b 0.05), while animals treated with SB-423557 were not different from the controls (data not shown). Following OVX surgery, osteocalcin concentrations were significantly elevated at week 5 (p b 0.01); however, no effect on osteocalcin concentrations was observed following administration of SB-423557 or PTH(1–34) (data not shown). Thus, oral administration of SB-423557 to OVX rats elicited transient increases in plasma concentrations of PTH, which led to advantageous effects on bone turnover by enhancing both trabecular and cortical bone formation, resulting in increased bone mass and strength in both vertebrae and long bones compared to that of OVX controls.
Fig. 4. Mean effect (±SEM) of daily oral administration of SB-423557 at 10, 30 and 100 μmol/kg (5, 15 or 50 mg/kg) or s.c. injection of rat PTH(1–34) (1 μg/kg) for 12 weeks on parathyroid cell proliferation in ovariectomized (OVX) rats.
Parathyroid gland proliferation One possible consequence of repeated stimulation of the parathyroid gland is increased parathyroid cell proliferation and the induction of secondary hyperparathyroidism. To address this theoretical concern, parathyroid gland proliferation in response to SB-423557 was evaluated at three dose levels (10, 30 and 100 μmol/kg or 5, 15 and 50 mg/kg) after 12 weeks of treatment. There were no significant differences in any treated group compared with controls in the number of labeled cells quantified (p = 0.12), area quantified (p = 0.41) or the total number of cells counted in the parathyroid gland (p = 0.65). There were no significant differences in the incorporation of the BrdU label in any treated group when compared with sham or OVX vehicle treated controls (Fig. 4). Proof of principle in humans We sought to provide proof of principle for CaR antagonism as a means of eliciting endogenous PTH release in humans and also to generate data regarding the safety of this approach. Single doses, ranging from 20 μg to 5 mg, of SB-423562 were administered i.v. to healthy male volunteers. The SB-423562 area under the curve (AUC) and maximal concentration (Cmax) increased in an approximately dose-proportional manner. Systemic clearance of SB-423562 was approximately 500 mL/min and the volume of distribution was approximately 20 L with a half-life of ≤ 1 h.
Table 1 Bone mass, histomorphometric and strength analysis of both trabecular and cortical bone in rats following administration of SB-423557 or PTH(1–34). Parameter
Unit
Sham
OVX
OVX + SB-423557 (50 mg/kg)
OVX + PTH(1–34) (5 μg/kg)
Proximal tibia trabecular BMD LV3—trabecular area LV3—eroded perimeter LV5—maximum load LV5—stiffness LV5—energy LV5—elastic modulus LV5—toughness DT—endocortical eroded perimeter Mid-femur—maximum load Mid-femur—stiffness Mid-femur—energy Mid-femur—elastic modulus Mid-femur—toughness
% of week 6 % % N N/mm mJ MPa mJ/m3 % N N/mm mJ MPa mJ/m3
92.5 ± 2.9⁎ 25.1 ± 2.2⁎ 0.30 ± 0.08⁎ 331.3 ± 18.2 2080 ± 177⁎
65.1 ± 3.3 14.4 ± 1.7 1.27 ± 0.36 268.3 ± 24.5 1145 ± 183 31.9 ± 4.6 372.4 ± 63.3 0.65 ± 0.09 2.0 ± 0.4 212.9 ± 8.3 445.0 ± 20.5 66.3 ± 3.1 3646 ± 242 4.94 ± 0.28
75.7 ± 5.1 18.3 ± 1.7 1.44 ± 0.17 317.1 ± 17.4 2222 ± 225⁎
96.3 ± 3.4⁎ 31.3 ± 2.3⁎ 0.45 ± 0.08⁎ 357.8 ± 16.1⁎ 2416 ± 265⁎ 51.4 ± 4.9⁎
40.3 ± 3.0 794.3 ± 69.2⁎ 0.92 ± 0.05 0.6 ± 0.2 240.7 ± 10.2 812.9 ± 47.8⁎ 86.2 ± 7.2 6288 ± 301⁎ 6.86 ± 0.45⁎
38.7 ± 3.0 800.5 ± 77.1⁎ 0.86 ± 0.05 1.01 ± 0.2 223.8 ± 8.5 732.1 ± 24.5⁎
950.4 ± 120.0⁎ 1.22 ± 0.11⁎ 1.2 ± 0.6 238.0 ± 7.6 785.3 ± 25.4⁎
86.2 ± 7.4 6055 ± 321⁎ 6.89 ± 0.45⁎
78.3 ± 6.7 6495 ± 297⁎ 6.36 ± 0.45
Mean ± standard error mean (⁎p b 0.05 vs. ovariectomy [OVX] determined by repeated measures one-way ANOVA followed by Tukey–Kramer test). Bone mineral density (BMD) of the trabecular bone of the proximal tibia determined by peripheral quantitative computed tomography. Lumbar spine BMD determined by dual X-ray absorptiometry. Mechanical properties of the LV5 determined using vertebral compression. Mechanical properties of the mid-femur determined using three-point bending test. LV3 = third lumbar vertebrae; LV5 = fifth lumbar vertebrae; DT = distal tibia.
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Fig. 5. Mean (±SEM) serum parathyroid hormone (PTH) concentrations following i.v. SB-423562 administration in healthy human subjects. m: minutes; h: hours.
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100 mg and less. However, up to ∼2-fold increase in AUC (0–24 h) was observed at doses of 200 mg and higher (Table 2). Transient serum Ca2+ elevations were reported with administration of SB423557, although these remained within the normal range. For illustration, the mean serum ionized serum calcium in the placebo and 400 mg dose groups is shown in Fig. 8. No change in urinary calcium excretion was noted in any dose group. Oral administration of SB-423557 was generally well tolerated. No deaths, serious AEs or withdrawals due to AEs were reported during the study. A total of 38 AEs were reported, 14 subjects reporting AEs with SB-423557 and 12 subjects with placebo. The most frequently reported AE was headache, which occurred in 8 of 53 subjects (15%) receiving SB-423557 and 8 of 54 subjects (15%) in the placebo group. Only a mild headache in one subject given a dose of 10 mg SB-423557 was considered to be related to the study medication. Discussion
Predose serum PTH ranged from ∼25 to 75 pg/mL. A transient and dose-dependent increase in serum PTH was observed with increasing doses of SB-423562 (Fig. 5). Maximal PTH concentrations were achieved 10–15 min following the start of the 10-min infusion, and elevations of PTH typically lasted b 1 h. Maximum PTH concentrations were increased 3- to 5.5-fold on average relative to baseline at doses of 1.25–5 mg. No effect on serum ionized calcium (Fig. 6) or urinary calcium excretion was noted. Administration (i.v.) of SB-423562 was generally well tolerated. No deaths, serious adverse events (AEs) or withdrawals due to AEs were reported during the study. Reported changes in laboratory parameters and vital signs were not considered to be related to the study medication and were generally asymptomatic. In preclinical species, the oral bioavailability of SB-423562 was low, whereas administration of SB-423557, an ethyl ester precursor of SB-423562, resulted in improved systemic delivery of SB-423562. Thus, a subsequent clinical study was conducted to evaluate SB423557 as an orally active CaR antagonist in humans. Single oral doses ranging from 5 to 500 mg of SB-423557 were administered to healthy male volunteers. SB-423557 resulted in a transient SB-423562 profile in the blood, whereas SB-423557 generally was not detectable, suggesting rapid conversion to SB-423562 in vivo. SB-423562 AUC and Cmax increased with the dose of SB-423557 in a dose-proportional manner, except at the 500 mg dose, which demonstrated high between-subject variability and slightly lower mean Cmax compared to the 400 mg dose level. The terminal half-life of SB-423562 increased with increasing dose of SB-423557 and ranged from 1.4 to 3.7 h; tmax (time to maximal concentration) ranged from 2 to 3 h across all dose ranges. A summary of SB-423562 pharmacokinetic parameters is presented in Table 2. Compared with i.v. administered SB-423562, the delayed tmax and longer half-life following orally administered SB423557 are consistent with absorption-rate limited disposition (so called ‘flip-flop’ pharmacokinetics). The plasma profile of PTH closely resembled the pattern of SB423562 concentrations in the blood (Fig. 7). Consistent increases in plasma PTH concentrations were observed following SB-423557 doses of 50 mg and greater. No significant change in plasma PTH was observed at 5, 10 and 20 mg doses. The duration of PTH elevation (defined as a PTH level at least twice that of pre-dose concentrations) was less than 8 h and median PTH tmax ranged from 1.0 to 2.3 h for doses 50 mg or higher. A summary of selected PTH pharmacodynamic parameters is presented in Table 2. For doses of 50 mg and greater, a 1.8- to 4.2-fold increase in PTH concentrations relative to baseline was noted at tmax. The most robust mean PTH response was observed in the 400 mg dose group with Cmax being as much as 423% higher compared to placebo. PTH area under the curve over 24 h post-dose (AUC(0–24 h)) was not significantly different from placebo at doses of
The data presented here demonstrate a transient increase in plasma concentrations of PTH in animal models and humans following both intravenous administration of SB-423562 and oral administration of its precursor, SB-423557. In addition, the boneforming effects of CaR antagonist was demonstrated in a rat model of bone loss, with increased BMD and trabecular and cortical bone formation, coupled with improved bone strength in the spine and long bones. Current treatments to address bone loss associated with osteoporosis either reduce bone resorption or promote bone formation. In addition, strontium ranelate has been proposed to act on both pathways [4], although its influence on bone formation remains controversial [5]. Subcutaneous administration of PTH(1–34) or PTH (1–84) improves cortical and trabecular BMD and cortical thickness associated with both endosteal and periosteal bone formation, and leads to improved bone strength in animal models and reduced fracture risk in patients with osteoporosis [7,9,21-23]. Thus PTHmediated stimulation of bone formation is of key importance in the treatment of osteoporosis. The development of orally active CaR antagonists which stimulate endogenous PTH secretion from the parathyroid gland provides an alternative to exogenous subcutaneously administered PTH for increasing bone formation. However, first-generation CaR antagonists were unable to produce a sufficiently transient PTH profile required for bone formation [16-19,24]. We have now developed a secondgeneration CaR antagonist, SB-423562, and its orally active precursor, SB-423557, which elicits transient PTH profiles in animal models and healthy male volunteers. In addition, in an OVX rat model of bone loss, daily oral SB-423557 administration led to increased bone formation
Fig. 6. Mean (±SEM) serum ionized calcium concentrations following i.v. SB-423562 administration in healthy human subjects. m: minutes; h: hours.
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Table 2 Summary of selected blood SB-423562 pharmacokinetic and plasma PTH pharmacodynamic parameters following oral administration of SB-423557. Dose (mg) Placebo 5 10 20 50 100 200 400 500
SB-423562 parameters
PTH parameters
AUC(0–t)a (ng⁎h/mL)
Cmaxa (ng/mL)
tmaxb (h)
t1/2a (h)
AUC(0-24 h)a (pg.h/mL)
Cmaxa (pg/mL)
tmaxb (h)
Max % changec
NA ND 19 (72) 36 (72) 157 (72) 362 (55) 898 (25) 1865 (30) 1808 (80)
NA ND 19 (38) 20 (35) 51 (69) 96 (62) 215 (34) 402 (31) 327 (78)
NA ND 2.0 (1.0, 2.0 (0.8, 2.8 (1.0, 2.3 (1.3, 2.0 (1.3, 3.0 (1.0, 2.8 (1.3,
NA ND ND ND 1.4 (22) 1.7 (22) 2.1 (33) 2.4 (21) 3.7 (26)
776 (33) 529 (22) 732 (51) 743 (29) 779 (33) 878 (40) 1073 (37) 1507 (35) 1003 (35)
42 (33) 29 (25) 42 (33) 44 (40) 54 (53) 69 (36) 131 (57) 191 (43) 113 (31)
8.0 8.0 2.5 8.0 2.3 1.0 1.4 2.0 1.5
NA 4.2 (−62.6, 71.0) 2.5 (−62.3, 67.3) 0.6 (−70.5, 71.6) 81.2 (32.2, 130) 97.7 (32.8, 163) 262 (196, 328) 423 (357, 488) 301 (236, 365)
3.0) 3.5) 4.0) 4.0) 3.5) 4.0) 3.0)
(0.3, 23.9) (1.8, 12.0) (0.5, 12.0) (1.8, 12.0) (0.5, 12.0) (0.5, 3.5) (0.8, 2.5) (0.5, 2.5) (0.7, 3.0)
NA = not applicable, ND = not determined, AUC(0–t) = area under the concentration-time curve from time zero (pre-dose) to time of last quantifiable concentration, Cmax = maximum plasma concentration, tmax = time of maximum observed concentration, t1/2 = terminal phase half-life, PTH Cmax = maximum PTH concentration post-dose, AUC (0–24 h) = area under the concentration-time curve from time zero to 24 h post-dose determined by the trapezoidal method. a Geometric mean (CV%) reported for AUC and Cmax. b Median (range) reported for tmax. c Maximum percent change defined as 100 ⁎ ((PTH Cmax − baseline)/baseline)); baseline defined as pre-dose concentration. Reported as point estimate (95% confidence interval). Parameter point estimate is the difference of adjusted LSmeans between active and placebo regimens.
and bone strength, demonstrating a preclinical proof of concept for this new class of compound. Overall, the bone-forming effect of SB-423557 in the rat was of a somewhat lesser magnitude compared with subcutaneously admin-
Fig. 7. Mean (±SEM) SB-423562 concentrations in blood (A) and parathyroid hormone (PTH) concentration in plasma (B) following oral administration of SB-423557 to healthy human subjects. Doses b 50 mg are not shown for clarity.
istered PTH(1–34). Although the OVX rat model is well established to study the effect of PTH on bone, concentrations of PTH several-fold higher are required compared with doses of PTH that are used in clinical practice [7,25,26]. It may not be possible to achieve this level of PTH release from the parathyroid gland in the rat via CaR antagonism, presumably due to either a physiologic limit in the amount of endogenous PTH available for immediate release from the parathyroid or due to feedback mechanisms. While caution should be applied when directly comparing PTH concentrations due to differences in the immunoassays for PTH(1–34) and PTH(1–84), on a molar basis, peak concentrations of PTH in the rat were ∼4-fold lower following SB-423557 compared to s.c. PTH(1–34). Accepting this difference, the lower PTH exposure following SB-423557 likely contributed to the somewhat lower bone-forming effects of this compound compared to s.c. PTH(1–34). Repeated antagonism of the CaR could theoretically lead to parathyroid gland hyperplasia. However, whether chronic stimulation of the parathyroid gland by, for example, dietary calcium deficiency increases parathyroid cell proliferation is controversial. Increased proliferation has been seen in weanling [27], but not in older rats fed low calcium diets [28]. Similarly, secondary or tertiary hyperparathyroidism is seen in patients with hypophosphatemic osteomalacia or Xlinked dominant hypophosphatamic rickets, respectively, on longterm oral phosphate therapy [29,30]. However, these may or may not
Fig. 8. Mean (±SEM) serum calcium concentrations in healthy human subjects following oral administration with either 400 mg of SB-423557 or placebo.
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involve CaR. Therefore, we examined the effect of SB-423557 on the parathyroid gland in the rat model. This study showed that daily administration of SB-423557 to OVX rats for 12 weeks did not increase parathyroid cell proliferation, confirming previous results with the longer acting compound NPS2143 [16]. While longer duration studies will be required to confirm the effect of long-term treatment, the current data indicate that repeated antagonism of parathyroid CaR by a calcilytic for up to 12 weeks does not lead to parathyroid hyperplasia. The duration of PTH exposure is an important determinant of the relative anabolic versus catabolic effects on bone. In the rat, a transient increase in plasma PTH concentrations over l–2 h has been shown to be optimal for bone formation [31,32]. In humans, continuous elevation of PTH, as seen in moderate-to-severe hyperparathyroidism, is associated with decreased BMD [33]. In contrast, following s.c. administration of 20 μg PTH(1–34), a tmax of approximately 30 min and a duration of elevated PTH of up to 3 h have been reported, which led to improved BMD and decreased fracture risk in patients with osteoporosis [34]. The PTH response following oral administration of a range of doses of SB423557 to humans correlates with that seen following exogenous s.c. PTH therapy, with a tmax ranging from 1 to 2 h and a duration of elevated PTH from 0.5 to 6.5 h for doses of 50 to 500 mg. The duration of PTH elevation appears directly proportional to the systemic exposure of SB423562. Thus i.v. administration of SB-423562 resulted in a SB-423562 and PTH exposure that was shorter in duration whereas oral administration of SB-423557 led to a more extended exposure. In the study with i.v. administered SB-423562, no significant increases in serum ionized calcium was noted. It is important to note that calcemic response is a function of the duration and magnitude of the PTH exposure. Thus, a lack of serum Ca2+ elevation in the i.v. SB423562 study is likely due to shorter duration of PTH exposure in this study. Such transient ∼3- to 5-fold increase in endogenous PTH of a duration lasting b 30 min is unlikely to produce Ca2+ changes from either skeleton or kidney. In contrast, higher doses of orally administered SB-423557 resulted in comparable maximal PTH concentrations but with a duration lasting several hours, which were associated with modest increases in serum Ca2+. Although we did not measure renal function in these studies, no change in urinary calcium excretion was noted. Overall, single administration of SB-423562 and SB-423557 in humans elicited PTH release in a profile consistent with bone-forming activity. In addition, administration of these compounds to humans demonstrated excellent tolerability in this short-term study. However, repeat dose studies will be required to evaluate longer term safety and impact on renal function and calcium homeostasis in humans. In summary, we have designed SB-423562, a CaR antagonist, and SB-423557, an orally active ethyl ester precursor of SB-423562. These compounds elicit a transient PTH profile in animal models and humans resulting in a bone-forming effect in the OVX rat model of bone loss. Further assessment of CaR antagonists is ongoing in longterm studies to evaluate safety and efficacy parameters, including PK– PD relationships, bone biomarker response and BMD in patients with osteoporosis. Acknowledgments We thank Dr. Scott Miller and Mary Beth Bowman of the Department of Radiobiology at the University of Utah for analysis of proliferating cells in the parathyroid glands. We thank Drs. Larry Suva and Dominique Ethgen for their contributions. We also acknowledge the editorial assistance provided by Lucy Richardson during the preparation of this manuscript. References [1] Tarantino U, Cannata G, Lecce D, Celi M, Cerocchi I, Iundusi R. Incidence of fragility fractures. Aging Clin Exp Res 2007;19:7–11.
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