Antagonizing the parathyroid calcium receptor stimulates parathyroid hormone secretion and bone formation in osteopenic rats

Parathyroid hormone (PTH) is an effective bone anabolic agent, but it must be administered parenterally. An orally active anabolic agent would provide a valuable alternative for treating osteoporosis. NPS 2143 is a novel, selective antagonist (a “calcilytic”) of the parathyroid cell Ca²⁺ receptor. Daily oral administration of NPS 2143 to osteopenic ovariectomized (OVX) rats caused a sustained increase in plasma PTH levels, provoking a dramatic increase in bone turnover but no net change in bone mineral density. Concurrent oral administration of NPS 2143 and subcutaneous infusion of 17β-estradiol also resulted in increased bone turnover. However, the antiresorptive action of estrogen decreased the extent of bone resorption stimulated by the elevated PTH levels, leading to an increase in bone mass compared with OVX controls or to either treatment alone. Despite the sustained stimulation to the parathyroid gland, parathyroid cells did not undergo hyperplasia. These data demonstrate that an increase in endogenous PTH secretion, induced by antagonism of the parathyroid cell Ca²⁺ receptor with a small molecule, leads to a dramatic increase in bone turnover, and they suggest a novel approach to the treatment of osteoporosis.

Osteoporosis is a serious and prevalent disease associated with significant mortality and morbidity, particularly after hip fracture (1). Osteoporotic fractures are a significant health problem, estimated to cost $13.8 billion in the United States in 1995 (2). Currently available therapies such as estrogen, raloxifene, calcitonin, and bisphosphonates prevent further bone loss by inhibiting bone resorption. These can lead to a significant reduction in fracture risk. However, these antiresorptive therapies reduce the risk of fracture by only 40–50%, at best. The ability to enhance bone formation and to increase bone mass to levels approaching the young adult mean would represent a significant advance in the treatment of osteoporosis. Parathyroid hormone (PTH) is the most widely studied bone anabolic agent (3, 4). Intermittent treatment of both rats (3, 5–7) and osteoporotic men and women (4, 8–13) with PTH increases bone mass and strength markedly, without significant adverse effects. Unfortunately, PTH must be administered parenterally; to date this has been restricted to subcutaneous injection in humans. This is far from ideal for a chronic therapy.

The control of PTH secretion by extracellular Ca²⁺ is mediated by a Ca²⁺ receptor expressed on the surface of parathyroid cells (14). The cloning and characterization of the bovine form of this G protein–coupled receptor was first described by Brown and colleagues (15). Subsequently, the human homologue was isolated (16). This receptor is negatively coupled to PTH secretion, such that increasing the concentration of extracellular ligand (Ca²⁺) leads to inhibition of PTH secretion. Numerous natural activating and inactivating mutations in the human Ca²⁺ receptor have been identified and linked to several disorders of calcium metabolism (17). Recently, small molecule activators of the Ca²⁺ receptor (calcimimetics) have been identified that inhibit PTH secretion in vitro (18, 19) and in patients with primary or secondary hyperparathyroidism (20, 21).

It has been proposed that an antagonist of the Ca²⁺ receptor (a calcilytic) would mimic a state of hypocalcemia and stimulate PTH secretion. The resulting transient increases in circulating PTH levels, if repeated on a daily basis, might produce an anabolic effect in bone (14). We showed previously that the induction of hypocalcemia by infusion of the calcium chelator EGTA raised circulating PTH to levels that were sufficient to stimulate bone formation in osteopenic rats (22). If this could be achieved with an orally active small molecule antagonist of the Ca²⁺ receptor, it might represent a novel anabolic therapy for osteoporosis. The identification and pharmacological characterization of such a molecule, NPS 2143, has recently been described (23). Briefly, screening of compound libraries in human embryonic kidney (HEK 293) cells that stably express the human parathyroid Ca²⁺ receptor identified a compound that was a weak (IC₅₀ = 11 μM) inhibitor of increases in cytoplasmic Ca²⁺ levels induced by extracellular Ca²⁺. Structural modification of this compound yielded NPS 2143, a more potent (IC₅₀ = 43 nM) and selective compound (Figure 1). In particular, NPS 2143 did not inhibit cytoplasmic Ca²⁺ responses to glutamate in HEK 293 cells engineered to express the structurally related metabotropic glutamate receptors, or to γ-amino butyric acid (GABA) in similar cells expressing similarly related GABA_B receptors (24). Moreover, NPS 2143 stimulated PTH secretion from bovine parathyroid cells in vitro (EC₅₀ = 39 nM) and increased plasma PTH levels when administered to normal rats by intravenous infusion. Here we describe the effect of daily oral administration of NPS 2143 on bone in aged ovariectomized (OVX) rats with established osteopenia, and demonstrate for the first time that antagonism of the parathyroid cell Ca²⁺ receptor in vivo by an orally active small molecule leads to an increase in bone turnover.

Figure 1

Structure of NPS 2143.

Study 1: Small but sustained elevation of PTH levels increases bone turnover with no net gain or loss of bone

Animals that had undergone ovariectomy 3 months previously had significantly lower BMD at all three skeletal sites measured. The reductions were 15% at the lumbar spine and proximal tibia, and 24% at the distal femur. BMD in the proximal tibia was unaffected by daily treatment with NPS 2143, but was returned to preovariectomy levels after 8 weeks of treatment with PTH (Figure 2). Similar changes were seen in the distal femur and lumbar vertebrae (not shown). Plasma PTH levels were measured after dosing in the last week of the experiment. PTH levels were elevated (> 100 pg/mL) within 30 minutes after dosing with NPS 2143; levels remained high for at least 4 hours (Figure 3). PTH levels in animals given the 5 μg/kg subcutaneous dose of PTH were similar to those dosed with NPS 2143, but had returned to baseline by 2 hours after the injection (Figure 3). The differences in the duration of the PTH response can be explained by sustained systemic exposure to NPS 2143, which in a separate experiment was found to be elevated for up to 8 hours after dosing (Figure 4). NPS 2143 levels were undetectable (< 10 ng/mL) at 24 hours.

Figure 2

Effect of daily administration of NPS 2143 (100 μmol/kg, per os) or rat PTH(1–34) (5 μg/kg, subcutaneously) on bone mineral density (BMD) in the proximal tibia of osteopenic OVX rats. Values shown as mean ± SE; n = 10–14 per group. ^ASignificantly different from sham-vehicle; P < 0.05. ^BSignificantly different from OVX-vehicle; P < 0.05. ^CSignificantly different from OVX–NPS 2143; P < 0.05.

Figure 3

Plasma levels of PTH after the administration of NPS 2143 (100 μmol/kg, per os) or rat PTH(1–34) (5 μg/kg, subcutaneously) to OVX rats. Both endogenous and exogenous PTH levels are of the 1-34 peptide. Values are mean ± SE; n = 3–4 per timepoint.

Figure 4

Plasma levels of NPS 2143 after its oral administration (100 μmol/kg) to rats. Values are mean ± SE; n = 3.

The different plasma PTH profiles obtained under these two dosing conditions induced markedly different effects on bone turnover. Dynamic histomorphometric analysis of cancellous bone in the proximal tibial metaphysis showed that bone formation (% O.Pm and % L.Pm) was further elevated above the OVX control level by both PTH and NPS 2143 (Figure 5). MAR was unaffected by any treatment (data not shown). However, bone resorption, as measured by percent E.Pm, was significantly higher in the NPS 2143 group than in the PTH or OVX control groups. This is exemplified further by the dramatic increase in bone turnover (BFR/B.Ar) in rats treated with NPS 2143 compared with the other two groups (Figure 5). Thus, the modest but prolonged elevation of PTH achieved by administration of NPS 2143 resulted in a dramatic increase in both bone formation and resorption, with no net bone gain or loss. PTH injections also increased both resorption and formation, but formation exceeded resorption, resulting in increased bone mass (Figure 2).

Figure 5

Histomorphometric analysis of cancellous bone in the proximal tibial metaphysis from osteopenic OVX rats treated daily for 8 weeks with NPS 2143 (100 μmol/kg, per os) or rat PTH(1–34) (5 μg/kg, subcutaneously). See Methods for abbreviations. Values are mean ± SE; n = 10–14 per group. ^ASignificantly different from sham-vehicle; P < 0.05. ^BSignificantly different from OVX-vehicle; P < 0.05. ^CSignificantly different from OVX–NPS 2143; P < 0.05.

Study 2: Small but sustained elevation of PTH levels in the presence of an antiresorptive agent increases bone turnover with net bone gain

A second study was performed in animals in which NPS 2143 was administered daily for 5 weeks in the presence or absence of estrogen. Figure 6 shows representative sections of proximal tibiae from sham-operated and OVX controls treated with vehicle, an OVX rat treated with 17β-estradiol alone, and an OVX rat treated with 17β-estradiol plus NPS 2143. It is obvious visually that coadministration of NPS 2143 and 17β-estradiol increased cancellous bone area over and above that achieved with 17β-estradiol alone. These changes were quantified histomorphometrically; the results are shown in Table 1. Tb.Ar was 72% lower in OVX animals than in sham-operated animals, a loss that was not significantly restored by treatment with 17β-estradiol. Treatment with NPS 2143 alone also had no effect on OVX-induced osteopenia. However, NPS 2143 given in combination with 17β-estradiol resulted in a significant, twofold increase in percent Tb.Ar over the OVX group, an increase that was due primarily to an increase in Tb.Th (Table 1). The apparent small increase in Tb.N in rats given 17β-estradiol probably resulted from prevention of further cancellous bone loss during the treatment period. Treatment with 17β-estradiol alone completely reversed the OVX-induced increases in indices of bone formation and resorption (Figure 7). As was observed in Study 1, treatment with NPS 2143 alone also increased most of these indices above levels in OVX controls. However, in rats treated with NPS 2143 plus 17β-estradiol, tissue-referent BFR was significantly elevated compared with all other groups (Figure 7). Elevation of bone formation rate/tissue area shows that bone mass was increased in the area measured, reflecting new bone formation on bone surfaces that were not being remodeled, a classic feature of PTH action. This appears to be a result of a decrease in resorption (presumably due to concurrent estrogen treatment) relative to the NPS 2143–treated animals in the face of maintained elevation of bone formation (Figure 7).

Figure 6

Sections of proximal tibia stained with von Kossa stain from OVX rats with established osteopenia, showing the anabolic effects on cancellous bone of daily oral administration for 5 weeks of NPS 2143 (100 μmol/kg) when given in combination with continuous subcutaneous infusion of 17β-estradiol. Shown are a sham-operated rat given vehicle (a), an OVX rat given vehicle (b), an OVX rat given 17β-estradiol alone (c), and an OVX rat treated with NPS 2143 and 17β-estradiol (d).

Figure 7

Histomorphometric analysis of cancellous bone in the proximal tibial metaphysis from osteopenic OVX rats treated daily for 5 weeks with 17β-estradiol (continuous subcutaneous infusion), NPS 2143 (100 μmol/kg, per os.), or NPS 2143 and 17β-estradiol. See Methods for abbreviations. Values are mean ± SE, n = 9–10 per group. ^ASignificantly different from sham-vehicle; P < 0.05. ^BSignificantly different from OVX-vehicle; P < 0.05. ^CSignificantly different from OVX-estradiol; P < 0.05. ^DSignificantly different from OVX–NPS 2143; P < 0.05.

Table 1

Effect of administration of 17β-estradiol and NPS 2143 on cancellous bone in the proximal tibial metaphysis of OVX rats with established osteopenia

These changes in histomorphometric indices of bone formation and resorption and in cancellous bone content were reflected by changes in BMD in the distal femur. BMD was 0.3725 ± 0.0095 g/cm² in the sham-operated rats at baseline, and changed little (final value 0.3744 ± 0.0068 g/cm²) during the 5-week treatment period. BMD was substantially reduced in the OVX rats at baseline (0.3134 ± 0.0032 g/cm²), and was not significantly different between groups (Figure 8). BMD did not change significantly (0.4 ± 0.8% increase) in vehicle-treated OVX rats, and decreased slightly (2.0 ± 1.7% decrease) in rats given NPS 2143 alone. However, BMD increased 2.9 ± 1.5% in rats given 17β-estradiol alone, and 7.2 ± 1.4% in rats given NPS 2143 and 17β-estradiol. The magnitude of the increase in BMD in rats given the combination of 17β-estradiol and NPS 2143 was significantly greater (P < 0.02) than in rats given estradiol alone.

Figure 8

Effects of daily treatment of osteopenic OVX rats for 5 weeks with 17β-estradiol (continuous subcutaneous infusion), NPS 2143 (100 μmol/kg, per os), or NPS 2143 and 17β-estradiol on BMD in the distal femur. Values are mean ± SE; n = 9–10 per group. ^BSignificantly different from OVX-vehicle; P < 0.05. ^CSignificantly different from OVX–NPS 2143; P < 0.05.

Parathyroid cell proliferation

Approximately 6,000 cells from three separate sections from both parathyroid glands were evaluated for BrdU uptake in each rat. The rate of cell proliferation in the parathyroid glands of the animals in this study was low. On average, only about 30 of the 6,000 cells (0.5%) had divided during the last 4 days of the study (Figure 9). In contrast, in the ileum, almost 100% of surface epithelial cells and about 30% of cells within the central compartment of the villus had taken up BrdU (not shown). The rate of cell division was not significantly affected by ovariectomy or by treatment with 17β-estradiol, NPS 2143, or both.

Figure 9

Absence of changes in cell proliferation in the parathyroid gland of osteopenic OVX rats treated daily for 5 weeks with 17β-estradiol (continuous subcutaneous infusion), NPS 2143 (100 μmol/kg, per os), or NPS 2143 and 17β-estradiol. Values are mean ± SE; n = 4–5 per group; P = 0.26.

NPS 2143 has no direct effects on osteoblasts and osteoclasts in vitro

Osteoblast activity. Whereas PTH caused a 2.6-fold increase in cyclic AMP levels in TF274 osteoblastic cells, NPS 2143 (0.1–10.0 μmol/L) had no effect on either basal or PTH-induced cyclic AMP levels (Figure 10a). Essentially identical results were seen in primary human osteoblasts (not shown). PTH treatment of TF274 cells increased alkaline phosphatase activity 7.6-fold; the addition of NPS 2143 (0.01–1.0 μmol/L) did not affect either basal or PTH-stimulated activity (Figure 10b). NPS 2143 (10 μmol/L) tended to reduce the alkaline phosphatase response to PTH in TF274 cells, although this is more likely to be a result of cellular toxicity at this very high concentration.

Figure 10

NPS 2143 does not affect basal or PTH-stimulated cyclic AMP formation or alkaline phosphatase activity in TF274 osteoblastic cells. Values are mean ± SE of experiments performed in triplicate.

Osteoclast activity. NPS 2143 had no effect on bone resorption by isolated human osteoclasts plated on bovine cortical bone particles at concentrations up to 3 μmol/L. In contrast, an inhibitor of cathepsin K, SB-240314 (28), inhibited this production with an IC₅₀ of 0.9 μmol/L. This assay is limited by its sensitivity to DMSO, so concentrations of NPS 2143 above 3 μmol/L could not be tested. In the fetal rat long bone assay, NPS 2143 inhibited resorption significantly, with an IC₅₀ of 11.3 ± 3.0 μmol/L.

We have demonstrated that an orally active calcilytic compound can be designed that blocks the parathyroid Ca²⁺ receptor and thereby raises plasma PTH to levels that are sufficient to stimulate bone turnover. The pharmacokinetic characteristics of this molecule are such that, after oral administration, a prolonged elevation of PTH (> 4 hours) is obtained. In contrast, when PTH was elevated for up to 2 hours after PTH injection, bone turnover was elevated but the balance was positive, resulting in net bone gain. When PTH was elevated to similar levels for longer than 4 hours in NPS 2143–treated animals, bone turnover was further elevated. However, both resorption and formation remained in balance, leading to no net loss or gain in bone mass.

It is well known that chronic elevation of PTH, such as that seen in hyperparathyroidism, leads to bone loss and abnormal bone histology (33, 34). Dobnig and Turner (35) showed that subcutaneous infusion of high doses of PTH (40 μg/kg and 80 μg/kg) over periods of 2 or more hours each day for 7 days led to rapid body weight loss, hypercalcemia, and histological abnormalities in the skeleton similar to those seen in patients with hyperparathyroidism. In our study, the much smaller increases in PTH, although sustained, did not lead to these adverse effects. It has been shown previously that substantial cancellous bone loss occurs in rats with mild secondary hyperparathyroidism (two- to threefold increases in PTH levels) resulting from chronic renal insufficiency (34). Although an anabolic effect of PTH was not obtained with the more sustained exposure in the current study, the fact that bone loss was not exaggerated in rats given NPS 2143 suggests that the duration rather than the magnitude of the PTH response was insufficient to cause a worsening of the osteopenia. Indeed, plasma levels of PTH were normal, and NPS 2143 was undetectable in the circulation by 24 hours after the oral dose. This suggests that a shorter-acting compound that induces a more transient increase in PTH levels might induce a net anabolic effect on bone.

A further point of interest with regard to the amount of PTH secreted in response to NPS 2143 is the small magnitude of increase in PTH levels required to have profound effects on bone turnover. We showed previously that doses of rat PTH(1–34) as low as 1 μg/kg (which increase PTH levels by approximately threefold) increase bone formation in OVX rats (22). In contrast, most published studies on effects of PTH in rats have used a dose of 80 μg/kg. When injected subcutaneously, this dose leads to a circulating level of approximately 10,000 pg/mL (35), compared with the 100–200 pg/mL in our studies. This clearly demonstrates that very small increases in the plasma levels of PTH are effective in stimulating bone metabolism. This is also illustrated by the much lower doses used in the clinical studies performed recently in which PTH doses of about 0.4–0.8 μg/kg body weight increased bone mass substantially (8–13). The 0.4 μg/kg dose increased plasma levels of PTH(1–34) to approximately 90 pmol/L at 30 minutes after dosing (36). This represents an approximately threefold increase over the baseline circulating level of endogenous PTH in these patients. Thus it appears that the repeated transient elevation of circulating PTH levels by a short-acting Ca²⁺ receptor antagonist are quite sufficient to cause an anabolic effect in bone.

One potential concern with repeated stimulation of the parathyroid gland is that it may induce parathyroid cell hyperplasia. It is well recognized that the parathyroid glands are larger in rats fed a low-calcium diet (37). Naveh-Many et al. (38) showed markedly increased parathyroid cell proliferation in weanling rats fed a low-calcium diet for 3 weeks. In contrast, Wernerson et al. (39) showed in older calcium-deficient rats that this glandular enlargement resulted exclusively from hypertrophy of the parathyroid cells, and not from hyperplasia. The results of our studies, showing that a hyperplastic response does not occur even with the extended duration of stimulation to parathyroid cells induced by treatment with NPS 2143, are consistent with those of Wernerson et al.

Numerous studies have been performed in the OVX rat to determine whether concurrent antiresorptive treatment affects the anabolic effect of PTH. Studies have shown that the bone formation effect of PTH in OVX rats is not ablated by estrogen, calcitonin, or the bisphosphonate risedronate (40–43). However, one study did show an inhibition of bone turnover and a dramatic decrease in bone formation rate when the bisphosphonate tiludronate was used in conjunction with PTH in elderly ewes (44). The authors of that study suggested that bone turnover is required in order for PTH to have an anabolic effect. Our study showed that new bone formation occurred in the absence of previous resorption, which would appear to contradict those findings. It is possible that the effects in rats and sheep may be different. However, it is clear that in postmenopausal women, the anabolic effect of PTH is demonstrable in the presence of estrogen (10, 11, 13). Our studies also suggest that even a long-acting calcilytic could have therapeutic benefits if administered concurrently with estrogen.

There have been reports of Ca²⁺ receptors on both osteoclasts and osteoblasts. Receptors that are both similar to (45, 46) and distinct from (47) the one known receptor have been reported on osteoblasts in vitro. Ca²⁺ receptors have also been characterized pharmacologically on osteoclasts (48), but there is no evidence to suggest that this receptor is structurally similar to that on the parathyroid cell, and it is known to be distinct pharmacologically (49). Moreover, although previous studies have shown that NPS 2143 does not act on structurally similar metabotropic glutamate and GABA_B receptors (23), it may act on other receptors. Particularly important in this regard are PTH receptors in bone. Any activity of a Ca²⁺ receptor antagonist on PTH receptors at this site could easily abolish the desired effect on bone and instead cause something quite undesirable. Indeed, actions of NPS 2143 on any of a number of different receptors expressed in bone could render this class of compounds therapeutically useless. For this reason, we examined the effects of NPS 2143 in a number of assays that monitor functional activity of bone cells in vitro, both in the presence and absence of PTH. Of the five different assays used, only the fetal rat long bone assay detected an effect of NPS 2143. The inhibitory effect on resorption in this assay was not seen using isolated human osteoclasts, and even in the rat assay, inhibitory effects of NPS 2143 were seen only at concentrations (IC₅₀ = 11 μM) far in excess of those required to block the parathyroid Ca²⁺ receptor (IC₅₀ = 0.043 μM). Taken together, the data suggest that NPS 2143, when used at concentrations that maximally inhibit Ca²⁺ receptor activity, does not affect bone cell activity. This suggests that the increased bone resorption seen in rats treated with NPS 2143 results solely from the prolonged exposure to elevated PTH levels.

It has not escaped our attention that the failure of a potent Ca²⁺ receptor antagonist to affect bone resorption in vitro probably has implications for the controversy regarding the presence (or absence) of the Ca²⁺ receptor in skeletal tissues (46–50). However, our data do not directly address this issue and therefore should not be used to support either argument. The pharmacological properties of receptors are known to differ depending on the cellular environment in which they are expressed (51). The Ca²⁺ receptor is no exception, and displays a very different pharmacology in C cells than in parathyroid cells, at least in response to Ca²⁺ receptor agonist (calcimimetic) compounds (52, 53).

In conclusion, we have provided proof-of-concept for the view that stimulation of endogenous PTH secretion using an antagonist of the parathyroid cell Ca²⁺ receptor results in increased bone formation and resorption. In the presence of an antiresorptive agent, NPS 2143 caused an increase in bone mass. These findings provide the basis for the development of calcilytics, a novel class of anabolic agents for the treatment of established osteoporosis.

The authors thank Sandra Hoffman, Peter Liang, Janice Vasco-Moser, Beata Lechowska, and Uma Prabhakar for excellent technical assistance, and Michael W. Lark, Larry J. Suva, and John C. Lee for editorial input.

Parts of this work were presented at the Second Joint Meeting of the American Society for Bone and Mineral Research and the International Bone and Mineral Society, San Francisco, California, USA, December 1–6, 1998, and have appeared in abstract form (1998. Bone. 23[Suppl.]:S163).

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