Calcilytic Compounds: Potent and Selective Ca Receptor Antagonists That Stimulate Secretion of Parathyroid Hormone

Despite the discovery of many ions and molecules that activate the Ca receptor, there are no known ligands that block this receptor. Reported here are the pharmacodynamic properties of a small molecule, NPS 2143, which acts as an antagonist at the Ca receptor. This compound blocked (IC50 of 43 nM) increases in cytoplasmic Ca concentrations [Ca ]i elicited by activating the Ca receptor in HEK 293 cells expressing the human Ca receptor. NPS 2143, even when tested at much higher concentrations (3 M), did not affect the activity of a number of other G protein-coupled receptors, including those most structurally homologous to the Ca receptor. NPS 2143 stimulated parathyroid hormone (PTH) secretion from bovine parathyroid cells (EC50 of 41 nM) over a range of extracellular Ca concentrations and reversed the effects of the calcimimetic compound NPS R-467 on [Ca ]i and on secretion of PTH. When infused intravenously in normal rats, NPS 2143 caused a rapid and large increase in plasma levels of PTH. Ca receptor antagonists are termed calcilytics and NPS 2143 is the first substance (either atomic or molecular) shown to possess such activity. The pharmacodynamic properties of NPS 2143 together with the recently demonstrated effects of this compound on bone formation support the view that orally active calcilytic compounds might provide a novel anabolic therapy for osteoporosis. The cell surface Ca receptor is the primary molecular entity regulating the secretion of parathyroid hormone (PTH). Activation of this receptor by increased levels of extracellular Ca inhibits PTH secretion, whereas its presumed inactivation by lowered extracellular Ca leads to an increase in PTH secretion. PTH regulates systemic Ca homeostasis by acting on target cells in both the kidney and the skeleton to increase plasma levels of Ca (Brown and MacLeod, 2001). In bone, PTH increases bone turnover but the resulting overall effect on bone mineral density is highly dependent on the temporal changes in the circulating levels of PTH. Thus, sustained elevations in plasma PTH levels, such as occur in hyperparathyroidism, result in increased bone resorption and a net decrease in bone mass at most skeletal sites (Antonsen and Sherrard, 1995; Silverberg et al., 1995). In contrast, temporary increases in plasma levels of PTH or its 1-34 fragment, achieved by the daily (or near daily) injection of either peptide, stimulate new bone formation in animal models of osteopenia (Dempster et al., 1993; Kimmel et al., 1993; Ejersted et al., 1995; Li and Wronski, 1995; Fox et al., 1997) and in clinical studies of osteoporotic patients (Reeve, 1996; Hodsman et al., 1997; Lindsay et al., 1997; Lane et al., 1998; Fujita et al., 1999, Rittmaster et al., 2000). The results of these studies using peptides administered intermittently demonstrate that PTH is a potent anabolic agent that increases bone mineral density and bone strength to a greater extent than that achieved by antiresorptive therapies (Mosekilde et al., 1994; Hodsman et al., 1997; Lindsay et al., 1997; Lane et al., 1998; Fujita et al., 1999; Rittmaster et al., 2000). The profound stimulatory effect of these peptides on bone formation has generated interest in the use of PTH or its fragments as a novel anabolic therapy for osteoporosis. However, the therapeutic use of these peptides is compromised by the need for systemic administration of a costly biological agent. An alternative approach that might overcome these drawbacks, and yet achieve similar anabolic effects on bone, is based on the use of small, orally active compounds that regulate plasma levels of endogenous PTH (Nemeth, 1996; Fox et al., 1997). This hypothesis holds that blocking Ca receptor activity with small molecules will stimulate PTH secretion. With the appropriate pharmacokinetic profile, such compounds would be expected to cause a marked but transient increase in circulating PTH levels, sufficient to 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, CA, December 1–6, 1998, and appeared in an abstract [(1998) Bone 23 (Suppl):S156]. ABBREVIATIONS: PTH, parathyroid hormone; [Ca ]i, cytoplasmic Ca 2 concentration; mGluR, metabotropic glutamate receptor; GABABR, -aminobutyric acid type B receptor; HEK, human embryonic kidney. 0022-3565/01/2991-323–331$3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 299, No. 1 Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics 4006/928002 JPET 299:323–331, 2001 Printed in U.S.A. 323 at A PE T Jornals on A ril 2, 2017 jpet.asjournals.org D ow nladed from stimulate new bone formation. While this hypothesis is in line with conventional thinking, it remains untested because no ligand has been found that blocks activation of the Ca receptor. In contrast, a wide variety of inorganic or organic polycations and certain phenylalkylamines have been shown to act as agonists or allosteric activators of this receptor (Nemeth and Fox, 1999). Like G protein-coupled receptor agonists in general, those that activate the Ca receptor exhibit marked tissue selectivity (Lavigne et al., 1998; Fox et al., 1999) and are not ideal ligands to study Ca receptor function, particularly in those tissues that are not involved in systemic Ca homeostasis and that often express much lower levels of the Ca receptor than classic “calcemic tissues” such as the parathyroid glands and kidney. In contrast, G protein-coupled receptor antagonists typically do not show profound tissue selectivity. Because of this, receptor antagonists are more valuable tools to study receptor function in a variety of different tissues. Such compounds, if capable of stimulating secretion of PTH, might also provide structures for novel drugs capable of transiently increasing levels of plasma PTH and stimulating new bone formation. The need for such an anabolic therapy is underscored by the serious health problem posed by osteoporosis, the incidence of which is increasing as the general population ages. Already there are nearly 6 million women and about 2 million men with osteoporosis in the United States and a far greater number of individuals with osteopenia or low bone mineral density. While currently available antiresorptive therapies, such as estrogen or bisphosphonates prevent further bone loss, they cause relatively small increases in new bone formation. The ability to stimulate new bone formation and thereby increase bone mass to levels approaching those in young adults, would constitute a significant advance in the treatment of osteoporosis. This report describes the salient pharmacodynamic properties of NPS 2143, a small molecule (Fig. 1) that blocks the parathyroid gland Ca receptor and stimulates PTH secretion in vitro and in vivo. This compound, which is one member of a family of structurally similar compounds, is the first substance, either ionic or molecular, shown to possess inhibitory activity at the Ca receptor. Materials and Methods Assays for Assessing Potency and Selectivity of Compounds on Ca Receptor. HEK 293 cells engineered to express the human parathyroid Ca receptor have been described in detail previously (Nemeth et al., 1998). This clonal cell line, referred to as HEK 293 4.0-7 cells, has been used in a high-throughput screening format to detect agonists and allosteric activators (calcimimetics) of the Ca receptor (Nemeth et al., 1998). Changes in the concentration of cytoplasmic Ca ([Ca ]i) provide a quantitative and functional assessment of Ca receptor activity in these cells and the results using this assay parallel those obtained using a homologous expression system of bovine parathyroid cells. On-line continuous measurements of fluorescence in fluo-3or fura-2-loaded HEK 293 4.0-7 cells were obtained using a custom-built spectrofluorimeter (Racke and Nemeth, 1993) or a fluorescence imaging plate reader instrument (Molecular Devices, Sunnyvale, CA). Test compounds were incubated with cells for 1 min before increasing the concentration of extracellular Ca from 1.0 to 1.75 mM. Compounds were tested individually at a concentration of 100 g/ml (20–80 M) and those causing more than a 40% inhibition of the control response were considered to be biologically active. To determine the potencies (IC50) of compounds with biological activity, concentration-response curves were obtained and then, as an initial assessment of selectivity, the effects of compounds on [Ca ]i evoked by other G protein-coupled receptors were examined at a concentration several times their IC50. Wild-type HEK 293 cells (and HEK 293 4.0-7 cells) express receptors for thrombin, bradykinin, and ATP, which couple to the mobilization of intracellular Ca . These responses can be studied to quickly assess any nonselective action of compounds on G protein-coupled receptors. Additional assays for selectivity included HEK 293 cells engineered to express receptors most homologous in sequence and topology to the Ca receptor. These included native or chimeric receptors for various metabotropic glutamate (mGluRs) and -aminobutyric acid type B receptors (GABABRs). Chimeric receptors were created using partial sequences of metabotropic glutamate receptors and Ca receptors, engineered to couple to activation of phospholipase C and release of intracellular Ca in HEK 293 cells as described in the legend to Fig. 4 and in Table 1. Compounds lacking pan-activity were then subjected to structural modifications and their potencies and selectivities monitored using these HEK 293 4.0-7 cell assays in an iterative process. Screening of compound libraries using measurements of [Ca ]i in HEK 293 4.0-7 cells resulted in the identification of a number of structurally diverse compounds. Many of these compounds, however, failed to meet subsequent criteria and were eliminated. Several of Fig. 1. Chemical structure of NPS 2143: N-[(R)-2-hydroxy-3-(2-cyano-3chlorophenoxy)propyl]-1,1-dimethyl-2-(2-naphthyl)ethylamine. The compound was used as the monohydrochloride salt. Shown is the R-enantiomer which, depending on the assay, is 10to 100-fold more potent than the corresponding S-enantiomer. TABLE 1 Cytoplasmic Ca responses elicited by several G protein-coupled receptors that are homologous to the Ca receptor are unaffected by NPS 2143 See Materials and Methods for description of cell lines used. Using the plate reader instrument, each cell line was treated with test compounds at the indicated concentrations prior to addition of the appropriate receptor agonist. The relative fluorescence units (RFU) shown are the means of 4 to 16 wells from two different plates and are not corrected for basal fluorescence. RFU % Control Response mGluR1 20 M Glutamate (stable) 1125 100 10 M NPS 2390 341 30 300 nM NPS 2143 1171 104 3 M NPS 2143 1166 104 20 M Glutamate (transient) 1886 100 300 nM NPS 2143 2262 120 10 M DHPG (transient) 1887 100 10 M NPS 2390 128 7 300 nM NPS 2143 1924 102 3 M NPS 2143 1977 105 mGluR5d (transient) 10 M DHPG 882 100 10 M NPS 2390 139 16 300 nM NPS 2143 848 96 3 M NPS 2143 923 105 GABABR1 GABABR2 (stable) 3 M Baclofen 2142 100 10 M SCH 50911 0 0 300 nM NPS 2143 2144 100 3 M NPS 2143 1884 88 10 M GABA 2334 100 10 M SCH 50911 26 1 300 nM NPS 2143 2225 95 3 M NPS 2143 2106 90 324 Nemeth et al. at A PE T Jornals on A ril 2, 2017 jpet.asjournals.org D ow nladed from these original compounds did possess the requisite potency and selectivity; structural modifications to one of these (IC50 of 11 M) led to a number of compounds that were profiled in detail. The pharmacodynamic properties of one of these, NPS 2143 (Fig. 1), is described in this report. Assays of Ca Receptor Activity Using Bovine Parathyroid Cells. The effect of NPS 2143 on Ca receptor-dependent regulation of PTH secretion and cyclic AMP formation was assessed using primary cultures of dissociated bovine parathyroid cells. Following overnight culture, the cells were removed from the flasks by decanting and washed with buffer containing 126 mM NaCl, 4 mM KCl, 1 mM MgSO4, 0.5 mM CaCl2, 0.7 mM K2HPO4/KH2PO4, 20 mM NaHEPES, pH 7.45, 1 mg/ml glucose, and 0.1% bovine serum albumin (ICN Biomedicals, Cleveland, OH). Portions (0.2 ml) of this cellular suspension were added to 1275-mm polystyrene tubes with or without NPS 2143 and/or varying concentrations of CaCl2. Incubations (in triplicate) at 37°C for 20 or 30 min were terminated by placing the tubes on ice. Cells were pelleted by centrifugation (500g for 10 min at 4°C) and 0.1 ml of supernatant was immediately assayed for PTH content. A portion of the cellular suspension was left on ice during the incubation period and then processed in parallel with the other tubes. The amount of PTH in the supernatant from the tubes maintained on ice was defined as “basal release” and was subtracted from all other samples. PTH levels were quantified using a rat PTH(1-34) immunoradiometric assay kit, which also detects bovine PTH (Immutopics, San Clemente, CA). For each experiment, results were expressed as picograms of PTH released/10 cells and then normalized to PTH released in 0.5 mM Ca . Cell numbers were determined by counting nuclei in a hemocytometer after lysing the cells and staining the nuclei with cresyl violet. For measurements of cyclic AMP formation, parathyroid cells were incubated for 15 min at 37°C in 96-well plates (120,000 cells/ well) in buffer containing 0.5 or 2 mM CaCl2 in the presence or absence of isoproterenol (1 M) and/or NPS 2143 (300 nM) before cells were lysed. All wells additionally contained 0.5 mM isobutylmethylxanthine. Levels of total cyclic AMP (cells plus medium) were determined using the BIOTRAK cyclic AMP scintillation proximity assay kit (Amersham Pharmacia Biotech, Piscataway, NJ). Luminescence was measured on a Microbeta 1450 Tri-Lux instrument (Wallac, Gaithersburg, MD). Plasma Levels of PTH in Rats. Normal male Sprague-Dawley rats (250–275 g) with unrestricted access to commercial rodent chow (Teklad 8640; Harlan Teklad, Madison, WI) and tap water were used. The animals were anesthetized by intraperitoneal injection of ketamine/xylazine (90:7 mg/kg) and chronic indwelling catheters were implanted in the inferior vena cava (for compound infusion) and in the abdominal aorta (for blood sampling) accessed by the femoral vein and artery, respectively. Following catheterization, the rats were housed individually and allowed to recover for at least 3 days before study. The protocol was approved by the Institutional Animal Care and Use Committee of NPS Pharmaceuticals, Inc. (Salt Lake