A Novel Calcium-Sensing Receptor Antagonist Transiently Stimulates Parathyroid Hormone Secretion in Vivo
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内分泌学杂志 2005年第4期
Departments of Osteoporosis and Frailty (B.J.A., R,S., Z.M., J.M., J.S., J.H.M.F.), Metabolism and Pharmacokinetics (A.F., V.V.), and Discovery Chemistry (W.Y., J.K.D.), Pharmaceutical Research Institute, Bristol-Myers Squibb Company, Hopewell, New Jersey 08534
Address all correspondence and requests for reprints to: Jean H. M. Feyen, Ph.D., Metabolic and Cardiovascular Drug Discovery, Bristol-Myers Squibb Co., 311 Pennington-Rocky Hill Road, Pennington, New Jersey 08534.
Abstract
Circulating calcium (Ca2+) is a primary regulator of bone homeostasis through its action on PTH secretion. Extracellular Ca2+ modulates PTH secretion through a cell surface G protein-coupled receptor, the calcium-sensing receptor (CaR). The expression of the CaR suggests a critical role in cellular regulation by calcium in various organs, including parathyroid gland, bone, and kidney. Despite an obvious pharmacological utility for CaR antagonists in the treatment of disease, only a limited number of such classes of compounds exist. We have identified a novel class of small molecules with specific activity at the CaR. This class of compounds is represented by compound 1. It possesses potent antagonist activity at the human CaR with IC50 values of 64 nM and 230 nM in inhibiting intracellular Ca2+ flux and inositol phosphate generation in vitro, respectively. When administered to male rats in vivo, compound 1 robustly increased serum PTH levels. The stimulation of PTH secretion was rapid and transient when administered either iv or orally. The pharmacokinetic profile of compound 1 after oral administration revealed that maximal plasma levels of compound were reached within 1 h and the half-life of the compound to be approximately 2 h in rats. These data describe a representative compound of a novel chemical class than previously described allosteric modulators that offer a new avenue for the development of improved treatments of osteoporosis.
Introduction
THE SKELETAL ARCHITECTURE provides the scaffolding for the organization of organ systems and, as such, is key to normal physiological function of these systems. The maintenance of this scaffolding throughout lifetime is accomplished through the regulation of bone mineral density. Bone mineral density, in turn, is maintained through a delicate balance between bone formation and bone resorption. One key regulatory factor of both bone formation and bone resorption is circulating plasma Ca2+ concentrations. Calcium exerts its effects on bone metabolism both at the level of the parathyroid gland and at bone osteoclasts (1, 2) and osteoblasts (3, 4). In the case of the parathyroid gland, low circulating levels of Ca2+ stimulate the secretion of PTH that stimulates osteoclast resorption of bone, with a concomitant rise in circulating Ca2+ (5). At the osteoclast, Ca2+ released from metabolized bone matrix acts to inhibit osteoclast function (5).
Calcium exerts its effects on the parathyroid gland through binding to a cell surface receptor, the calcium-sensing receptor (CaR) (6, 7, 8, 9). The CaR is a G protein-coupled receptor that is preferentially coupled to Gq (6, 10). Indeed, in response to increasing extracellular Ca2+, a rapid and transient increase in intracellular inositol triphosphate levels can be detected in parathyroid cells in vitro (6). The coupling of CaR to PTH secretion is exquisitely sensitive, as suggested by the fact that the range of normal serum Ca2+ concentrations falls within the steeply cooperative region of the Ca2+ dose-response curve. The dose-response relationship of CaR is abnormally steep, with a Hill coefficient of approximately 2–3 (11).
Interestingly, the physiological consequence of PTH release is strongly determined by the temporal pattern of release of the hormone. That is, long-term elevation of serum PTH levels leads to the classically described stimulation of osteoclastic bone resorption, resulting in increased serum Ca2+ levels (12). In contrast, transient elevation of PTH secretion leads to activation of osteoblasts, resulting in bone formation (12, 13). Thus, this tight control of both bone formation and bone resorption make the CaR an attractive target for the development of therapeutic strategies for the treatment of osteoporosis or other bone-related diseases. Recently, Nemeth et al. (14) have reported the characterization of selective small-molecule allosteric modulators of the CaR. Both positive allosteric modulators (referred to as calcimimetics) and negative allosteric modulators (referred to as calcilytics) were reported. The calcilytic compound, NPS-2143, demonstrated potent (IC50 50 nM) inhibition of extracellular Ca2+ (Ca2+)o-induced influx of intracellular Ca2+ (Ca2+)i by recombinant CaR expressed in HEK-293 cells (15). In addition, this compound dose-dependently stimulated PTH release from bovine parathyroid cells with similar potency. NPS-2143 also stimulated PTH release in rats when administered either iv or orally (15, 16). However, administration of NPS-2143 to ovariectomized rats led to a sustained release of PTH, with increased bone turnover, but did not increase bone mineral density (16). This may be due to the pharmacokinetic profile of this compound, because it is well known that sustained elevation of PTH levels leads to the catabolic effects of the hormone. Therefore, we set out to identify novel chemical entities capable of selective antagonism/allosteric modulation of the human CaR that would have improved pharmacokinetic profiles.
Materials and Methods
Cell fluorescence assay
Calcilytic activity of test compounds was measured in HEK293 cells stably expressing the human recombinant CaR (HEK-CaR) by determining the ability of these compounds to block increases in (Ca2+)i after addition of Ca2+ to the culture medium. HEK-CaR cells were maintained in T-150 flasks in cell growth medium [DMEM, high glucose (Life Technologies, Inc. no. 11960–044), 10% FBS, 146 μg/ml glutamate, 0.4 mg/ml G418 (geneticin)] in 5% CO2-95% air at 37 C to 90% confluency. Cells were plated at 4,000 cells/well in 96-well poly D-lysine-coated black view plates (Falcon, VWR no. 62406–036) and incubated for 16–24 h in a humidified tissue culture incubator at 37 C and gassed with 5% CO2-95% air. An increase in (Ca2+)i concentration was measured using Fluo3 AM (Molecular Probes, no. F-1242) as an indicator dye after the addition of 0.8 mM CaCl2 to the culture wells. Fluorescence was monitored using a fluorescence imaging plate reader (FLIPR) (Molecular Devices; Palo Alto, CA).
At the beginning of each experiment, the cell medium was aspirated, and the cells were loaded with Fluo3 [50 μg dissolved in 25 μl DMSO and 25 μl 20% (vol/vol) pluronic acid] in 10 ml buffer A (10 mM HEPES, pH 7.5, containing Hanks’ salts without Ca2+ and Mg2+; 0.1% BSA; 0.05% D-glucose; 0.4 mM CaCl2) for 1 h at 37 C. After this incubation, the loading buffer was removed, and 120 μl/well buffer A was added. The compounds were prepared in buffer A and added to respective wells as 30-μl aliquots at 6x the final drug concentration. The fluorescence signals were then read by FLIPR (Molecular Devices, Sunnyvale, CA) to obtain the baseline fluorescence. Subsequently, 30 μl of 2.4-mM CaCl2 (0.8 mM final concentration) was added to each well. The fluorescence signal was read at 1-sec intervals for 60 sec and at 3-sec intervals for the next 60 sec. To evaluate the calcilytic activity of the compounds, dose-response curves for the compounds (half-log concentrations in triplicate) were generated based upon the ability of the compounds to inhibit the (Ca2+)o-induced increase in (Ca2+)i-related fluorescence.
Inositol phosphate (IP) determination
To study the CaR activity in response to CaR antagonists, the total IP accumulation in HEK-CaR cells was measured in the presence of 3 mM Ca2+. After labeling the cells with [3H]myo-inositol for 18–24 h, IPs were extracted from cells and isolated by anion-exchange chromatography. This was accomplished by plating HEK-CaR cells at 20,000 cells/well in 96-well poly-D-lysine-coated tissue culture plates in growth medium for 24 h. After this incubation, the growth medium was removed, and 100 μl labeling medium [Life Technologies, Inc. Medium 199 with 2% FBS and 5 μCi/ml [3H]myo-inositol, Amersham (Piscataway, NJ) no. TRK883] was added to each well and the cells incubated for 18–24 h at 37 C. The labeling medium was then removed from the wells, and the cells were washed once with 100 μl PBS without Ca2+ and Mg2+. To each well, 80 μl Hepes-buffered saline medium (126 mM NaCl, 4 mM KCl, 1 mM MgSO4, 0.7 mM K2HPO4/KH2PO4, 20 mM HEPES, 0.1 mM CaCl2, 20 mM LiCl, and 0.2% BSA) was added and incubated for 20 min at 37 C. Compounds were added to triplicate wells, at varying concentration, as 10-μl aliquots. Cells were incubated for another 20 min at 37 C, followed by the addition of 10 μl of 30-mM CaCl2 (diluted in HBS medium) to each well (to achieve a final concentration of 3 mM) and the cells incubated for 2 h at 37 C. After this incubation, the reaction medium was aspirated, and 40 μl/well ice-cold formic acid (10 mM, pH 3) was added to each well and the plate incubated at room temperature for 30 min. The medium was neutralized by addition of 160 μl of 5-mM NH4OH(pH 9) per well. An anion exchange resin plate was prepared by loading 200 μl/well of formate anion exchange resin (Bio-Rad, Hercules, CA: AG1-x8) suspended in deionized water (1:1) into a 96-well filter plate (Millipore, Billerica, MA; MultiScreen MAHVN45). Column chromatography and elution were performed using a vacuum manifold (Millipore). The neutralized medium (200 μl/well) was added to the resin-packed plate and passed through the resin column through the vacuum manifold (0.5 psi). Column plates were washed twice with 200 μl/well deionized water to remove free inositol. IPs in the samples were eluted with 100 μl/well of 1-M ammonium formate/formic acid (pH 5) into a 96-well plate. About 30 μl eluate was mixed with 200 μl MicroScint PS (Packard, Boston, MA) in Packard OptiPlates (96-well-white); the plates were sealed with TopSeal (Packard no. 6005185) and mixed at room temperature overnight and counted for 1 min/well in a TopCount NXT (Packard).
CaR scintillation proximity ligand binding assay (SPA)
To each well of a 384-well OptiPlate (Packard), 35 μl of 2x SPA binding buffer (50 mM Tris-HCl, pH 7.6, containing 5 mM KCl, 3 mM CHAPS, 1 mM PMSF, 5 mM MgCl2, 50% NaCl, and 0.1% BSA), 30 μl of 0.5-mg/ml cell membranes (15 μg), 2 μl of 2.6-μM [3H]compound 2 (50 nM final except where indicated), 3 μl of 35x compound 1 or unlabeled compound 2, 0.5 mg YSi-WGA SPA beads in 35 μl of 1x SPA binding buffer were added. The total reaction mixture vol of 105 μl was incubated at room temperature for 3 h. The plate was counted in a TopCount for 1 min/well to determine the radioactive ligand bound to the receptor.
In vivo pharmacology of CaR antagonists
All procedures involving animals were approved by the Hopewell Animal Care and Use Committee. The efficacy (maximal effect) of calcilytic compounds in vivo was studied using 200-g male Sprague Dawley rats (Charles River, Boston, MA). Compound activity was assessed after iv administration through an indwelling jugular catheter in anesthetized rats or after oral administration (gavage) in conscious, freely moving rats. Animals were allowed to equilibrate to the institutional vivarium for 48 h before use in these studies. Rats were anesthetized with ketamine/xylazine (40 mg/kg:5 mg/kg, im), and a SILASTIC (Dow Corning Corp., Midland, MI) catheter was implanted via the right jugular vein then secured to the pectoral muscle and vein with 4–0 silk sutures. The cannula was then exteriorized from the nape of the neck. The catheter was maintained patent by filling it with 150 IU/ml sterile, heparinized saline.
In the case of iv studies, after recovery for 18–24 h, the animals were anesthetized with 40 mg/kg ketamine im. The heparinized saline was removed from the catheter, and the catheter was flushed with 1 ml sterile saline without heparin. Thirty minutes after flushing of the catheter, a baseline blood sample was withdrawn and the volume replaced with sterile saline. Immediately after baseline sampling, the animals were administered varying concentrations of compound in EtOH:Tween 80-H2O (10:20:70) as vehicle. The compounds were administered at varying doses in a total vol of 0.2 ml directly into the catheter that was subsequently flushed with 0.3 ml saline. Serial blood samples (0.4 ml) were then drawn at 2, 5, and 10 min after administration of the compound. Blood samples were allowed to clot for 2–3 h at 4 C and then serum collected by centrifugation for measurement of PTH and analysis of serum concentration of compound.
For studies in which the compound was administered orally, animals were allowed to recover from surgery in shoebox caging within our vivarium procedure room for 24 h. Catheter extensions, consisting of PE50 tubing containing a 21-g blunt connector, were used to maintain the connection between the extension and the indwelling catheter. The extension was run out of the cage through the wire cage top and attached to a 1-ml tuberculin syringe. After connection of the extensions, the catheter was flushed with 0.5 ml sterile saline and the animals allowed to equilibrate for 30–45 min. A baseline blood sample was taken (0-time) and the compounds or vehicle (45% 4,5-hydroxypropyl-?-cyclodextrin) administered by gavage (3 ml). Serial blood samples (0.3 ml) were withdrawn from the indwelling catheters at 30, 60, 120, 180, and 240 min after administration from the conscious, freely moving animals. After withdrawal of each blood sample, an equal volume of sterile saline was used to replace that taken from the animal. Both treatment groups were performed in the same study.
The pharmacokinetic profile of compound 1 was investigated in similar fashion as the pharmacology studies. Male rats were catheterized via the right jugular vein and then administered 54 μmol/kg compound 1 prepared in 45% 2-hydroxypropyl-?-cyclodextrin by oral gavage. Serial blood samples were collected at 0, 0.25, 0.5, 0.75, 1, 2, 4, 6, and 8 h after dosing via the right jugular vein. Serum samples were obtained by centrifugation and were stored at –20 C until analysis. Before analysis, the samples were thawed and then extracted by the protein precipitation method, using acetonitrile. The supernatant was then analyzed for compound 1 by liquid chromatography in conjunction with tandem mass spectrometry (LC/MS/MS).
Rat PTH ELISA
PTH levels in serum samples were measured using a commercially available heterologous ELISA kit (Immutopics Inc., San Clemente, CA) according to the manufacturer’s instructions. The intraassay and interassay variations for this assay are 2.4% and 6.0%, respectively.
Statistics
Fluorescence data generated by FLIPR was processed by ActivityBase (ID Business Solutions, Persippany, NJDBS), and the IC50 values were determined from inhibition curves using the FLIPR associated software. Statistical analyses of in vitro and in vivo data were completed using ANOVA in conjunction with Duncan’s multiple range test using SigmaStat software (Systat Software, Richmond, CA). Before each analysis, data were tested for equal variance and normality. In some cases, data were found not to have equal variance. In these cases, statistical analyses were performed on log-transformed data. Differences between treatments were considered significant if P < 0.05. Dose-response curves were fitted and IC50 and EC50 values estimated using a four-parameter logistic equation as previously described (17).
Results
To assess the biological activity of potential compounds, we developed an in vitro fluorescence-based bioassay using the mobilization of (Ca2+)i as an end-point. Figure 1 illustrates the specificity of this assay, in which the ability of exogenous Ca2+ to stimulate the mobilization of (Ca2+)i was observed in both the HEK-CaR cells and the parental HEK-293 cells loaded with the Ca2+-sensitive dye Fluo 3-AM. Figure 1 displays an overlay of Ca2+ response curves in these cell lines. (Ca2+)i response was determined with 2 mM CaCl2. The addition of Ca2+-free medium to Fluo 3-loaded HEK-CaR cells did not induce an increase in fluorescence. However, addition of 2 mM Ca2+ to Fluo 3-loaded HEK-CaR cells induced a rapid increase in intracellular fluorescence that remained steady after reaching a maximal response within 20–30 sec. In the parental cell line devoid of CaR (HEK-293 cells) the effect of Ca2+ was qualitatively and quantitatively different. Addition of Ca2+ to these cells led to a much slower increase in fluorescence, as is evident in Fig. 1. Furthermore, the maximum signal attained was 25% (at 120 sec) of that observed with HEK-CaR cells. Thus, these data suggest that the rapid (Ca2+)o-induced rise in fluorescence in HEK-CaR cells is a receptor-mediated (Ca2+)i response that is specific for CaR.
FIG. 1. External calcium stimulates intracellular Ca2+ flux in HEK-CaR cells. HEK-CaR cells or the parental HEK cells were exposed to 2 mM Ca2+ after loading with the fluorescent dye Fluo 3-AM as described in Materials and Methods. Fluorescence was detected over a 2-min period after addition of extracellular Ca2+. Data are expressed as fluorescence units after normalization to baseline levels. Exogenous calcium induced an immediate and sustained increase in fluorescence in HEK-CaR cells (black circles). Exogenous Ca2+ induced only a slight increase in fluorescence in the parental HEK cells (white diamonds) that was much slower in onset. No increase in fluorescence was detected in the presence of medium alone (white triangles).
The intracellular Ca2+-mobilization assay was used to screen a large compound collection for inhibitors of (Ca2+)o-induced fluorescence. Through this process, we identified several compounds with calcilytic activity, albeit of weak potency. Through modulation of the chemical structure of one of these compounds, we were able to synthesize a number of higher potency calcilytic compounds. One such compound, compound 1, is represented in Fig. 2. The structure of compound 1 is depicted alongside that of the NPS calcilytic, NPS-2143, as well as a close analog of NPS-2143, compound 2. As is evident, compound 1 is structurally different from either NPS-2143 or compound 2. There is only a slight difference in structure between NPS-2143 and compound 2, where a 4-methoxyphenyl replaces the naphthyl group in the NPS compound. Unpublished data from our laboratory have shown compound 2 to have similar efficacy to that of NPS-2143 (data not shown).
FIG. 2. Comparison of the structures of compound 1 and compound 2 with the known calcilytic, NPS-2143. A large compound collection was screened for the ability of compounds to inhibit the calcium-induced increase in internal calcium concentration as determined by fluorescence (see Materials and Methods for details). One compound identified from structure-activity relationship studies of analogs of compounds identified in the screen was compound 1. This compound has a chemical structure distinct from the known calcilytic NPS-2143. Compound 2 represents a closely related analog of NPS-2143 that was used as a representative of the NPS chemical class of compounds as comparator. The shaded area highlights the 4-methoxyphenyl of compound 2 that is a replacement for the naphthyl group in NPS-2143.
To study the activity of compound 1, HEK-CaR cells were challenged with Ca2+ in the presence or absence of varying concentrations of the compound (Fig. 3). In these studies, the EC50 for (Ca2+)o-induced fluorescence was used (0.8 mM). Compound 1 dose-dependently inhibited the intracellular (Ca2+)o-induced fluorescence generated by the agonist calcium as seen in the overlay of FLIPR Ca2+ curves in Fig. 3A. To obtain the IC50 of the compound, the percent maximum signal was plotted against the log concentration of the compound. Compound 2 was used for comparison between the NPS compound series and the novel compound 1 (Fig. 3B). The IC50 for compound 1 was 64 nM in inhibiting (Ca2+)o-induced (Ca2+)i in HEK-CaR cells, whereas the NPS-2143 analog, compound 2, inhibited Ca2+-induced fluorescence with an IC50 of 4 nM. Thus, compound 1 is a potent inhibitor of the intracellular Ca2+ mobilization induced by exogenous Ca2+ in HEK-CaR cells, albeit approximately 15-fold less potent than a representative of the NPS calcilytic chemotype.
FIG. 3. Compound 1 dose-dependently inhibits extracellular Ca2+-induced fluorescence in HEK-CaR cells. A, Fluorescence profiles of HEK-CaR cells coincubated with 1.2 mM Ca2+ and increasing concentrations of compound 1. Fluorescence curves of cells cultured in the presence of differing concentrations of compound 1 are illustrated with different line properties (see legend within the figure for details). Increasing concentrations of compound 1 led to dose-dependent inhibition in Ca2+-induced fluorescence. B, Dose-response relationship for the inhibition in maximal Ca2+-induced fluorescence in the presence of either compound 2 (triangles) or compound 1 (circles). Compound 2 (EC50 = 4 nM) was approximately 15-fold more potent than compound 1 (EC50 = 64 nM).
CaR is a member of the G protein-coupled receptor superfamily and couples to phospholipase C. Activation of the CaR results in the phospholipase C-induced hydrolysis of phosphatidylinositol, raising the level of free IPs. To further characterize compound 1, we studied the effect of the compound on (Ca2+)o-induced total IP accumulation in HEK-CaR cells. Figure 4 illustrates that compound 1 dose-dependently decreased the IP accumulation induced by 3 mM Ca2+ in HEK-CaR cells. The potency for this effect was 229 nM compared with an IC50 of 99 nM for compound 2. Efficacy of compound 1 was equivalent to that of compound 2.
FIG. 4. Compound 1 and compound 2 inhibit extracellular Ca2+-induced IP generation in HEK-CaR cells. HEK-CaR cells prelabeled with [3H]myo-inositol were incubated in the presence of 3 mM Ca2+ and varying concentrations of either compound 1 (circles) or compound 2 (triangles). Accumulation of IP, generated over a 2-h incubation period, was measured according to the description in Materials and Methods. Data are presented as inhibition of maximal Ca2+-induced IP generated. Compound 2 inhibited Ca2+-induced IP accumulation, with an IC50 of 99 nM. Compound 1 was slightly less potent, with an IC50 of 229 nM.
The natural ligand calcium binds to CaR with low affinity (dissociation constant 1–2 mM), precluding its use as a ligand in a radioligand binding assay. We have developed a SPA as a binding assay for CaR using 3H-compound 2 as a radioligand. As shown in Fig. 1, compound 2 is of the same chemical class as NPS-2143, which has been demonstrated to be a negative allosteric modulator of CaR (15, 18). Therefore, these compounds do not compete for binding with Ca2+ to CaR. However, not all CaR allosteric modulators need bind at the same allosteric site to inhibit CaR functional activity. In Fig. 5, data from the SPA binding assay are shown in which we studied the ability of compound 1 to compete for binding of 3H-compound 2 to the human CaR. These studies were performed to determine whether these compounds share a common binding site(s) on CaR. In this assay, compound 1 did not compete for binding of 3H-compound 2 at concentrations as high as 100 μM. In contrast, unlabeled compound 2 competed for binding of 3H-compound 2 to the HEK-CaR cell membranes, with an IC50 of 2 μM (Fig. 5). This suggests that compound 1 does not bind at the same site as 3H-compound 2. Nevertheless, compound 1 still is capable of modulating the calcium-induced rise in (Ca2+)i and the Ca2+-induced IP accumulation in HEK-CaR cells with equal efficacy as compound 2 (see Figs. 3 and 4).
FIG. 5. Compound 1 does not compete with compound 2 for binding to HEK-CaR membranes. A SPA was used to study the ability of compound 1 to compete for binding of compound 2 to the CaR in HEK-CaR membranes. HEK-CaR membranes were incubated with 50 nM [3H]compound 2 in the presence of increasing concentrations of either compound 1 (black symbols) or unlabeled compound 2 (white symbols). Unlabeled compound 2 competed for binding of [3H]compound 2, with an IC50 of approximately 2 μM. Compound 1 did not compete for binding of compound 2 to HEK-CaR membranes.
To study the efficacy of compound 1 in vivo, intact male rats were catheterized, via the right jugular vein, for serial blood sampling studies. Figure 6 illustrates the effects of compound 1 on serum PTH levels in male rats administered the compound iv. Compound 1 rapidly stimulated PTH release, within 2 min (Fig. 6A, triangles). The timing of this effect was transient, because PTH levels rapidly declined toward baseline thereafter. Maximal efficacy of compound 1 was equivalent to that of the analog of NPS-2143. In addition, the temporal character of the effects of these compounds was also similar. The dose-response relationship for compound 1 is illustrated in Fig. 6B. The effect of compound 1 on maximal PTH levels was dose-dependent, with increasing doses leading to greater secretory profiles (Fig. 6A). This was reflected in greater area under the curve (data not shown). The maximal PTH release for each dose was used for determining potency of the compound. As is shown in Fig. 6B, compound 1 was a potent stimulator of PTH secretion, with an EC50 of 0.4 μmol/kg. Thus, compound 1 is capable of transiently stimulating PTH release in vivo.
FIG. 6. Compound 1 transiently stimulates serum PTH secretion after iv dosing in the rat in vivo. A, Male Sprague Dawley rats were catheterized, via the right jugular vein, 24 h before treatment with compounds. Rats were anesthetized with ketamine/xylazine im 30 min before withdrawal of a baseline (zero-time) blood sample. After collection of this sample, animals were treated with either vehicle (circles) or increasing concentrations of compound 1 (0.06–18 μmol/kg) iv. Compound 2 (7 μmol/kg, square symbols) was given to a separate group of animals as a positive control. Data are expressed as percent of baseline levels. B, The potency of compound 1 to stimulate PTH secretion is illustrated. Maximal PTH levels attained in response to treatment of rats with differing doses of compound 1 (black symbols) are plotted as percent of baseline value. Compound 1 stimulated PTH secretion, with an apparent ED50 of 0.4 μmol/kg. The maximal PTH level attained after administration of vehicle is represented by the white bar. *, P < 0.05; n = 5–6 animals per group.
Pharmacokinetic studies were performed in rats to better understand the characteristics of this compound after oral administration. Figure 7A illustrates the plasma concentrations of compound 1 after a single oral dose of 30 mg/kg (54 μmol/kg). Maximal plasma levels of approximately 2 μM were achieved within 1 h of administration of compound 1. The compound was rapidly cleared, with an oral half-life of approximately 2 h. Eight hours after dosing, levels of the compound in plasma were approximately 1.5% of maximum and were undetectable 24 h after administration. In a separate study, rats were administered 50 μmol/kg compound 1 or vehicle and blood samples collected over a 4-h period from the conscious, freely moving animals. Figure 7B illustrates the effect of this single oral dose of compound 1 on maximal PTH levels attained during the 4-h sampling period. Upon dosing, serum PTH levels rapidly increased, to reach maximal levels within 30–60 min. Serum PTH levels declined thereafter, reaching a plateau from 60–180 min after administration. PTH levels declined to that of vehicle-treated animals within 4 h after dosing. Overall, compound 1 increased serum PTH levels approximately 3-fold compared with vehicle-treated animals. These data demonstrate compound 1 to be an orally available negative allosteric modulator of the CaR that transiently stimulates PTH release in vivo.
FIG. 7. Serum levels of compound 1 and PTH after oral dosing in rats. A, Serum concentration vs. time profile of compound 1 in rats after oral administration of 54 μmol/kg compound 1. Data are expressed as means ± SEM; n = 3. B, Serum PTH levels from male rats administered 50 μmol/kg compound 1 (black symbols) or vehicle (45% 2-hydroxypropyl-?-cyclodextrin, white symbols) po. Serial blood samples were collected at 0, 0.5, 1, 1.5, 2, and 4 h after dosing. Sera were then assayed for PTH levels described in Materials and Methods. Oral dosing of compound 1 to male rats induced a rapid increase in serum PTH levels that was maximal within 30 min of dosing. Levels then slowly decreased thereafter, achieving vehicle levels within 2 h. Data are expressed as percent of baseline PTH levels ± SEM. *, P < 0.05; n = 8 animals per group.
Discussion
Recent data from the development of pharmacological agents modulating the CaR have provided a suitable proof-of-concept for the use of calcilytics as a potential therapeutic strategy for the treatment of osteoporosis. Given the tight association between serum Ca2+ concentrations and PTH release, it is not surprising that many have focused on this mechanism to induce bone anabolism. Despite the report of selective allosteric modulators of CaR that can stimulate PTH release, room for improvement in this class of compounds remains. This was especially evident when a representative of this class (NPS-2143) was studied in vivo. Data from these experiments demonstrated a prolonged half-life, both when administered po and iv (15, 16, 18). In their description of NPS-2143, Nemeth et al. found that plasma PTH levels were elevated more than 1 h after termination of infusion. Additionally, this compound had a plasma half-life of approximately 8 h when administered orally (16). The prolonged half-life with a resultant elevated concentration of serum PTH may have been a contributing factor to the lack of efficacy seen on bone mineral density when given alone to osteopenic rats (16). Therefore, we sought to synthesize antagonists to the CaR with the goal to identify a series of compounds with potent antagonizing and/or allosteric modulatory activity toward the CaR and possessing an improved pharmacokinetic profile in vivo.
Screening of a large compound collection revealed a number of chemotypes that were able to stimulate Ca2+ flux in HEK-293 cells. A representative compound derived from one of these chemotypes (compound 1) was capable of inhibiting the (Ca2+)o-induced rise in (Ca2+)i fluorescence in this assay, with an apparent IC50 of 64 nM. Similarly, compound 1 inhibited (Ca2+)o-induced accumulation of IP, with an IC50 of 230 nM.
To investigate the specificity of the effects of compound 1, we studied the receptor dependency of the action of the compound in cell lines not expressing the CaR. The data from these experiments demonstrate that in the absence of CaR, compound 1 has no effect on calcium flux. These data suggest that the effects of compound 1 observed in the cell lines reflect CaR-dependent activity. Subsequent study of the activity profile of this compound vs. other G protein-coupled receptors demonstrated a clear selectivity of compound 1 for the CaR. Although significant interaction was detected vs. the histamine H2 and serotonin receptors (data not shown), compound 1 demonstrated at least 50-fold selectivity for CaR.
In our experiments, we studied whether compound 1 was capable of acting through the same or an overlapping site with NPS-2143 by observing the ability of compound 1 to compete for binding of compound 2 (a closely related analog of NPS-2143, see Fig. 1) to CaR. Our data reveal that compound 1 was incapable of competing for binding with compound 2, suggesting that these two compounds do not share a common binding site on CaR or that the overlap in binding sites is not sufficient for compound 1 to displace compound 2. It is likely that NPS-2143 and compound 2 do act at the CaR through a similar site because these two molecules are so closely related; and thus, compound 2 affords a suitable surrogate for NPS-2143 and comparator for compound 1 activity. Unfortunately, the nature of the binding studies provides no insight into the site(s) of interaction of compound 1 with the CaR. In their description of the properties of NPS-2143, Nemeth et al. (15, 18, 19, 20) suggest that the mechanism of action of NPS-2143 and related calcilytic compounds is similar to type II calcimimetic compounds (e.g. NPS-568). These authors have suggested that these structurally related compounds act through an allosteric mechanism to alter the sensitivity (affinity) of the CaR to (Ca2+)o (20). Data to support this hypothesis can be found in studies employing a truncated form of the CaR that is devoid of the extracellular domain of the receptor. In these experiments, cells expressing this mutant receptor responded to the calcimimetic NPS-568 with an increase in IP3 production (21). Alanine substitutions in the second transmembrane domain of this truncated receptor blocked the ability of the receptor to signal in the presence of NPS-568 (21). These data suggest that, although the extracellular portion of the CaR is the primary point of binding for Ca2+ (22), the second transmembrane region of the receptor is intimately involved in activating signaling of the ligand bound receptor. Studies of naturally occurring mutations in CaR support this hypothesis (11, 23, 24, 25). In both rats and humans, mutations in the second transmembrane domain of CaR lead to a disruption in the ability of the receptor to signal. Interestingly, mutations of this region of the receptor do not appreciably affect binding of the receptor. These data suggest that this region of the receptor is involved in generating the conformational changes of the CaR that lead to association with signaling proteins. This idea of transferring the signal of ligand binding from the extracellular domain to sites in the transmembrane and cytoplasmic regions of receptors has been hypothesized for many G protein-coupled receptors, including CaR (26). However, other portions of the receptor have been hypothesized to also play a role, including the Ala116-Pro136 region of the receptor (26). Alanine scanning mutagenesis of this segment of the second extracellular loop of the receptor increased the potency for Ca2+ and elevated the basal signaling activity of the receptor (26, 27). It has been hypothesized that these effects are due to stabilization of the active conformation of CaR by these mutations. Because compound 1 did not compete for binding with compound 2, these latter data suggest that this region of the extracellular loops of the receptor may be a potential site for action of compound 1. However, the possibility still exists that compound 1 could significantly compete directly for binding to CaR by Ca2+, because we were unable to study this option directly.
Regardless of the sites of action, administration of compound 1 to male rats produced a rapid and robust increase of serum PTH levels. Efficacy of compound 1 was equivalent to that of compound 2. The effects of compound 1 were transient, with PTH levels decreasing toward baseline within 10 min. This is in contrast to NPS-2143, which has been reported to maintain high plasma levels for more than 8 h after oral administration (16). The ability of compound 1 to increase serum PTH levels was dose-dependent, with an ED50 of approximately 0.4 μmol/kg. The pharmacokinetic analysis of compound 1 revealed that upon oral dosing, the compound swiftly attained peak plasma levels, after which the compound was rapidly cleared, such that the T1/2 for compound 1 was approximately 2 h. This pattern of exposure is significantly better than that reported for NPS-2143, which was reported to have a half-life greater than 8 h upon po dosing (16). This is an important feature of any calcilytic, because the anabolic action of calcium requires a transient activation of the receptor (12, 13). In the description of the activity of NPS-2143, it was noted that significant effects of this compound on bone mineral density in osteopenic rats was demonstrated only in the presence of the antiresorptive effects of estrogen. Lone administration of NPS-2143 did not result in improvement in bone mineral density. These observations could be due to the long half-life of NPS-2143, which would lead to a more resorptive pattern of CaR modulation. The pharmacokinetic profile of absorption of compound 1 after oral administration was verified by effects on serum PTH levels. Serum PTH reached maximal levels within 1 h of administration. Together, these data show compound 1 to have equal efficacy with, but a more advantageous pharmacokinetic profile for stimulating PTH secretion than, NPS-2143.
In conclusion, we have identified and characterized a novel compound capable of antagonizing the human and rat CaR. The data demonstrate the compound to be equally efficacious as, but slightly less potent than, a close analog of the best characterized calcilytic, NPS-2143. These data offer a new avenue for the development of novel anabolic agents for the treatment of osteoporosis.
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Address all correspondence and requests for reprints to: Jean H. M. Feyen, Ph.D., Metabolic and Cardiovascular Drug Discovery, Bristol-Myers Squibb Co., 311 Pennington-Rocky Hill Road, Pennington, New Jersey 08534.
Abstract
Circulating calcium (Ca2+) is a primary regulator of bone homeostasis through its action on PTH secretion. Extracellular Ca2+ modulates PTH secretion through a cell surface G protein-coupled receptor, the calcium-sensing receptor (CaR). The expression of the CaR suggests a critical role in cellular regulation by calcium in various organs, including parathyroid gland, bone, and kidney. Despite an obvious pharmacological utility for CaR antagonists in the treatment of disease, only a limited number of such classes of compounds exist. We have identified a novel class of small molecules with specific activity at the CaR. This class of compounds is represented by compound 1. It possesses potent antagonist activity at the human CaR with IC50 values of 64 nM and 230 nM in inhibiting intracellular Ca2+ flux and inositol phosphate generation in vitro, respectively. When administered to male rats in vivo, compound 1 robustly increased serum PTH levels. The stimulation of PTH secretion was rapid and transient when administered either iv or orally. The pharmacokinetic profile of compound 1 after oral administration revealed that maximal plasma levels of compound were reached within 1 h and the half-life of the compound to be approximately 2 h in rats. These data describe a representative compound of a novel chemical class than previously described allosteric modulators that offer a new avenue for the development of improved treatments of osteoporosis.
Introduction
THE SKELETAL ARCHITECTURE provides the scaffolding for the organization of organ systems and, as such, is key to normal physiological function of these systems. The maintenance of this scaffolding throughout lifetime is accomplished through the regulation of bone mineral density. Bone mineral density, in turn, is maintained through a delicate balance between bone formation and bone resorption. One key regulatory factor of both bone formation and bone resorption is circulating plasma Ca2+ concentrations. Calcium exerts its effects on bone metabolism both at the level of the parathyroid gland and at bone osteoclasts (1, 2) and osteoblasts (3, 4). In the case of the parathyroid gland, low circulating levels of Ca2+ stimulate the secretion of PTH that stimulates osteoclast resorption of bone, with a concomitant rise in circulating Ca2+ (5). At the osteoclast, Ca2+ released from metabolized bone matrix acts to inhibit osteoclast function (5).
Calcium exerts its effects on the parathyroid gland through binding to a cell surface receptor, the calcium-sensing receptor (CaR) (6, 7, 8, 9). The CaR is a G protein-coupled receptor that is preferentially coupled to Gq (6, 10). Indeed, in response to increasing extracellular Ca2+, a rapid and transient increase in intracellular inositol triphosphate levels can be detected in parathyroid cells in vitro (6). The coupling of CaR to PTH secretion is exquisitely sensitive, as suggested by the fact that the range of normal serum Ca2+ concentrations falls within the steeply cooperative region of the Ca2+ dose-response curve. The dose-response relationship of CaR is abnormally steep, with a Hill coefficient of approximately 2–3 (11).
Interestingly, the physiological consequence of PTH release is strongly determined by the temporal pattern of release of the hormone. That is, long-term elevation of serum PTH levels leads to the classically described stimulation of osteoclastic bone resorption, resulting in increased serum Ca2+ levels (12). In contrast, transient elevation of PTH secretion leads to activation of osteoblasts, resulting in bone formation (12, 13). Thus, this tight control of both bone formation and bone resorption make the CaR an attractive target for the development of therapeutic strategies for the treatment of osteoporosis or other bone-related diseases. Recently, Nemeth et al. (14) have reported the characterization of selective small-molecule allosteric modulators of the CaR. Both positive allosteric modulators (referred to as calcimimetics) and negative allosteric modulators (referred to as calcilytics) were reported. The calcilytic compound, NPS-2143, demonstrated potent (IC50 50 nM) inhibition of extracellular Ca2+ (Ca2+)o-induced influx of intracellular Ca2+ (Ca2+)i by recombinant CaR expressed in HEK-293 cells (15). In addition, this compound dose-dependently stimulated PTH release from bovine parathyroid cells with similar potency. NPS-2143 also stimulated PTH release in rats when administered either iv or orally (15, 16). However, administration of NPS-2143 to ovariectomized rats led to a sustained release of PTH, with increased bone turnover, but did not increase bone mineral density (16). This may be due to the pharmacokinetic profile of this compound, because it is well known that sustained elevation of PTH levels leads to the catabolic effects of the hormone. Therefore, we set out to identify novel chemical entities capable of selective antagonism/allosteric modulation of the human CaR that would have improved pharmacokinetic profiles.
Materials and Methods
Cell fluorescence assay
Calcilytic activity of test compounds was measured in HEK293 cells stably expressing the human recombinant CaR (HEK-CaR) by determining the ability of these compounds to block increases in (Ca2+)i after addition of Ca2+ to the culture medium. HEK-CaR cells were maintained in T-150 flasks in cell growth medium [DMEM, high glucose (Life Technologies, Inc. no. 11960–044), 10% FBS, 146 μg/ml glutamate, 0.4 mg/ml G418 (geneticin)] in 5% CO2-95% air at 37 C to 90% confluency. Cells were plated at 4,000 cells/well in 96-well poly D-lysine-coated black view plates (Falcon, VWR no. 62406–036) and incubated for 16–24 h in a humidified tissue culture incubator at 37 C and gassed with 5% CO2-95% air. An increase in (Ca2+)i concentration was measured using Fluo3 AM (Molecular Probes, no. F-1242) as an indicator dye after the addition of 0.8 mM CaCl2 to the culture wells. Fluorescence was monitored using a fluorescence imaging plate reader (FLIPR) (Molecular Devices; Palo Alto, CA).
At the beginning of each experiment, the cell medium was aspirated, and the cells were loaded with Fluo3 [50 μg dissolved in 25 μl DMSO and 25 μl 20% (vol/vol) pluronic acid] in 10 ml buffer A (10 mM HEPES, pH 7.5, containing Hanks’ salts without Ca2+ and Mg2+; 0.1% BSA; 0.05% D-glucose; 0.4 mM CaCl2) for 1 h at 37 C. After this incubation, the loading buffer was removed, and 120 μl/well buffer A was added. The compounds were prepared in buffer A and added to respective wells as 30-μl aliquots at 6x the final drug concentration. The fluorescence signals were then read by FLIPR (Molecular Devices, Sunnyvale, CA) to obtain the baseline fluorescence. Subsequently, 30 μl of 2.4-mM CaCl2 (0.8 mM final concentration) was added to each well. The fluorescence signal was read at 1-sec intervals for 60 sec and at 3-sec intervals for the next 60 sec. To evaluate the calcilytic activity of the compounds, dose-response curves for the compounds (half-log concentrations in triplicate) were generated based upon the ability of the compounds to inhibit the (Ca2+)o-induced increase in (Ca2+)i-related fluorescence.
Inositol phosphate (IP) determination
To study the CaR activity in response to CaR antagonists, the total IP accumulation in HEK-CaR cells was measured in the presence of 3 mM Ca2+. After labeling the cells with [3H]myo-inositol for 18–24 h, IPs were extracted from cells and isolated by anion-exchange chromatography. This was accomplished by plating HEK-CaR cells at 20,000 cells/well in 96-well poly-D-lysine-coated tissue culture plates in growth medium for 24 h. After this incubation, the growth medium was removed, and 100 μl labeling medium [Life Technologies, Inc. Medium 199 with 2% FBS and 5 μCi/ml [3H]myo-inositol, Amersham (Piscataway, NJ) no. TRK883] was added to each well and the cells incubated for 18–24 h at 37 C. The labeling medium was then removed from the wells, and the cells were washed once with 100 μl PBS without Ca2+ and Mg2+. To each well, 80 μl Hepes-buffered saline medium (126 mM NaCl, 4 mM KCl, 1 mM MgSO4, 0.7 mM K2HPO4/KH2PO4, 20 mM HEPES, 0.1 mM CaCl2, 20 mM LiCl, and 0.2% BSA) was added and incubated for 20 min at 37 C. Compounds were added to triplicate wells, at varying concentration, as 10-μl aliquots. Cells were incubated for another 20 min at 37 C, followed by the addition of 10 μl of 30-mM CaCl2 (diluted in HBS medium) to each well (to achieve a final concentration of 3 mM) and the cells incubated for 2 h at 37 C. After this incubation, the reaction medium was aspirated, and 40 μl/well ice-cold formic acid (10 mM, pH 3) was added to each well and the plate incubated at room temperature for 30 min. The medium was neutralized by addition of 160 μl of 5-mM NH4OH(pH 9) per well. An anion exchange resin plate was prepared by loading 200 μl/well of formate anion exchange resin (Bio-Rad, Hercules, CA: AG1-x8) suspended in deionized water (1:1) into a 96-well filter plate (Millipore, Billerica, MA; MultiScreen MAHVN45). Column chromatography and elution were performed using a vacuum manifold (Millipore). The neutralized medium (200 μl/well) was added to the resin-packed plate and passed through the resin column through the vacuum manifold (0.5 psi). Column plates were washed twice with 200 μl/well deionized water to remove free inositol. IPs in the samples were eluted with 100 μl/well of 1-M ammonium formate/formic acid (pH 5) into a 96-well plate. About 30 μl eluate was mixed with 200 μl MicroScint PS (Packard, Boston, MA) in Packard OptiPlates (96-well-white); the plates were sealed with TopSeal (Packard no. 6005185) and mixed at room temperature overnight and counted for 1 min/well in a TopCount NXT (Packard).
CaR scintillation proximity ligand binding assay (SPA)
To each well of a 384-well OptiPlate (Packard), 35 μl of 2x SPA binding buffer (50 mM Tris-HCl, pH 7.6, containing 5 mM KCl, 3 mM CHAPS, 1 mM PMSF, 5 mM MgCl2, 50% NaCl, and 0.1% BSA), 30 μl of 0.5-mg/ml cell membranes (15 μg), 2 μl of 2.6-μM [3H]compound 2 (50 nM final except where indicated), 3 μl of 35x compound 1 or unlabeled compound 2, 0.5 mg YSi-WGA SPA beads in 35 μl of 1x SPA binding buffer were added. The total reaction mixture vol of 105 μl was incubated at room temperature for 3 h. The plate was counted in a TopCount for 1 min/well to determine the radioactive ligand bound to the receptor.
In vivo pharmacology of CaR antagonists
All procedures involving animals were approved by the Hopewell Animal Care and Use Committee. The efficacy (maximal effect) of calcilytic compounds in vivo was studied using 200-g male Sprague Dawley rats (Charles River, Boston, MA). Compound activity was assessed after iv administration through an indwelling jugular catheter in anesthetized rats or after oral administration (gavage) in conscious, freely moving rats. Animals were allowed to equilibrate to the institutional vivarium for 48 h before use in these studies. Rats were anesthetized with ketamine/xylazine (40 mg/kg:5 mg/kg, im), and a SILASTIC (Dow Corning Corp., Midland, MI) catheter was implanted via the right jugular vein then secured to the pectoral muscle and vein with 4–0 silk sutures. The cannula was then exteriorized from the nape of the neck. The catheter was maintained patent by filling it with 150 IU/ml sterile, heparinized saline.
In the case of iv studies, after recovery for 18–24 h, the animals were anesthetized with 40 mg/kg ketamine im. The heparinized saline was removed from the catheter, and the catheter was flushed with 1 ml sterile saline without heparin. Thirty minutes after flushing of the catheter, a baseline blood sample was withdrawn and the volume replaced with sterile saline. Immediately after baseline sampling, the animals were administered varying concentrations of compound in EtOH:Tween 80-H2O (10:20:70) as vehicle. The compounds were administered at varying doses in a total vol of 0.2 ml directly into the catheter that was subsequently flushed with 0.3 ml saline. Serial blood samples (0.4 ml) were then drawn at 2, 5, and 10 min after administration of the compound. Blood samples were allowed to clot for 2–3 h at 4 C and then serum collected by centrifugation for measurement of PTH and analysis of serum concentration of compound.
For studies in which the compound was administered orally, animals were allowed to recover from surgery in shoebox caging within our vivarium procedure room for 24 h. Catheter extensions, consisting of PE50 tubing containing a 21-g blunt connector, were used to maintain the connection between the extension and the indwelling catheter. The extension was run out of the cage through the wire cage top and attached to a 1-ml tuberculin syringe. After connection of the extensions, the catheter was flushed with 0.5 ml sterile saline and the animals allowed to equilibrate for 30–45 min. A baseline blood sample was taken (0-time) and the compounds or vehicle (45% 4,5-hydroxypropyl-?-cyclodextrin) administered by gavage (3 ml). Serial blood samples (0.3 ml) were withdrawn from the indwelling catheters at 30, 60, 120, 180, and 240 min after administration from the conscious, freely moving animals. After withdrawal of each blood sample, an equal volume of sterile saline was used to replace that taken from the animal. Both treatment groups were performed in the same study.
The pharmacokinetic profile of compound 1 was investigated in similar fashion as the pharmacology studies. Male rats were catheterized via the right jugular vein and then administered 54 μmol/kg compound 1 prepared in 45% 2-hydroxypropyl-?-cyclodextrin by oral gavage. Serial blood samples were collected at 0, 0.25, 0.5, 0.75, 1, 2, 4, 6, and 8 h after dosing via the right jugular vein. Serum samples were obtained by centrifugation and were stored at –20 C until analysis. Before analysis, the samples were thawed and then extracted by the protein precipitation method, using acetonitrile. The supernatant was then analyzed for compound 1 by liquid chromatography in conjunction with tandem mass spectrometry (LC/MS/MS).
Rat PTH ELISA
PTH levels in serum samples were measured using a commercially available heterologous ELISA kit (Immutopics Inc., San Clemente, CA) according to the manufacturer’s instructions. The intraassay and interassay variations for this assay are 2.4% and 6.0%, respectively.
Statistics
Fluorescence data generated by FLIPR was processed by ActivityBase (ID Business Solutions, Persippany, NJDBS), and the IC50 values were determined from inhibition curves using the FLIPR associated software. Statistical analyses of in vitro and in vivo data were completed using ANOVA in conjunction with Duncan’s multiple range test using SigmaStat software (Systat Software, Richmond, CA). Before each analysis, data were tested for equal variance and normality. In some cases, data were found not to have equal variance. In these cases, statistical analyses were performed on log-transformed data. Differences between treatments were considered significant if P < 0.05. Dose-response curves were fitted and IC50 and EC50 values estimated using a four-parameter logistic equation as previously described (17).
Results
To assess the biological activity of potential compounds, we developed an in vitro fluorescence-based bioassay using the mobilization of (Ca2+)i as an end-point. Figure 1 illustrates the specificity of this assay, in which the ability of exogenous Ca2+ to stimulate the mobilization of (Ca2+)i was observed in both the HEK-CaR cells and the parental HEK-293 cells loaded with the Ca2+-sensitive dye Fluo 3-AM. Figure 1 displays an overlay of Ca2+ response curves in these cell lines. (Ca2+)i response was determined with 2 mM CaCl2. The addition of Ca2+-free medium to Fluo 3-loaded HEK-CaR cells did not induce an increase in fluorescence. However, addition of 2 mM Ca2+ to Fluo 3-loaded HEK-CaR cells induced a rapid increase in intracellular fluorescence that remained steady after reaching a maximal response within 20–30 sec. In the parental cell line devoid of CaR (HEK-293 cells) the effect of Ca2+ was qualitatively and quantitatively different. Addition of Ca2+ to these cells led to a much slower increase in fluorescence, as is evident in Fig. 1. Furthermore, the maximum signal attained was 25% (at 120 sec) of that observed with HEK-CaR cells. Thus, these data suggest that the rapid (Ca2+)o-induced rise in fluorescence in HEK-CaR cells is a receptor-mediated (Ca2+)i response that is specific for CaR.
FIG. 1. External calcium stimulates intracellular Ca2+ flux in HEK-CaR cells. HEK-CaR cells or the parental HEK cells were exposed to 2 mM Ca2+ after loading with the fluorescent dye Fluo 3-AM as described in Materials and Methods. Fluorescence was detected over a 2-min period after addition of extracellular Ca2+. Data are expressed as fluorescence units after normalization to baseline levels. Exogenous calcium induced an immediate and sustained increase in fluorescence in HEK-CaR cells (black circles). Exogenous Ca2+ induced only a slight increase in fluorescence in the parental HEK cells (white diamonds) that was much slower in onset. No increase in fluorescence was detected in the presence of medium alone (white triangles).
The intracellular Ca2+-mobilization assay was used to screen a large compound collection for inhibitors of (Ca2+)o-induced fluorescence. Through this process, we identified several compounds with calcilytic activity, albeit of weak potency. Through modulation of the chemical structure of one of these compounds, we were able to synthesize a number of higher potency calcilytic compounds. One such compound, compound 1, is represented in Fig. 2. The structure of compound 1 is depicted alongside that of the NPS calcilytic, NPS-2143, as well as a close analog of NPS-2143, compound 2. As is evident, compound 1 is structurally different from either NPS-2143 or compound 2. There is only a slight difference in structure between NPS-2143 and compound 2, where a 4-methoxyphenyl replaces the naphthyl group in the NPS compound. Unpublished data from our laboratory have shown compound 2 to have similar efficacy to that of NPS-2143 (data not shown).
FIG. 2. Comparison of the structures of compound 1 and compound 2 with the known calcilytic, NPS-2143. A large compound collection was screened for the ability of compounds to inhibit the calcium-induced increase in internal calcium concentration as determined by fluorescence (see Materials and Methods for details). One compound identified from structure-activity relationship studies of analogs of compounds identified in the screen was compound 1. This compound has a chemical structure distinct from the known calcilytic NPS-2143. Compound 2 represents a closely related analog of NPS-2143 that was used as a representative of the NPS chemical class of compounds as comparator. The shaded area highlights the 4-methoxyphenyl of compound 2 that is a replacement for the naphthyl group in NPS-2143.
To study the activity of compound 1, HEK-CaR cells were challenged with Ca2+ in the presence or absence of varying concentrations of the compound (Fig. 3). In these studies, the EC50 for (Ca2+)o-induced fluorescence was used (0.8 mM). Compound 1 dose-dependently inhibited the intracellular (Ca2+)o-induced fluorescence generated by the agonist calcium as seen in the overlay of FLIPR Ca2+ curves in Fig. 3A. To obtain the IC50 of the compound, the percent maximum signal was plotted against the log concentration of the compound. Compound 2 was used for comparison between the NPS compound series and the novel compound 1 (Fig. 3B). The IC50 for compound 1 was 64 nM in inhibiting (Ca2+)o-induced (Ca2+)i in HEK-CaR cells, whereas the NPS-2143 analog, compound 2, inhibited Ca2+-induced fluorescence with an IC50 of 4 nM. Thus, compound 1 is a potent inhibitor of the intracellular Ca2+ mobilization induced by exogenous Ca2+ in HEK-CaR cells, albeit approximately 15-fold less potent than a representative of the NPS calcilytic chemotype.
FIG. 3. Compound 1 dose-dependently inhibits extracellular Ca2+-induced fluorescence in HEK-CaR cells. A, Fluorescence profiles of HEK-CaR cells coincubated with 1.2 mM Ca2+ and increasing concentrations of compound 1. Fluorescence curves of cells cultured in the presence of differing concentrations of compound 1 are illustrated with different line properties (see legend within the figure for details). Increasing concentrations of compound 1 led to dose-dependent inhibition in Ca2+-induced fluorescence. B, Dose-response relationship for the inhibition in maximal Ca2+-induced fluorescence in the presence of either compound 2 (triangles) or compound 1 (circles). Compound 2 (EC50 = 4 nM) was approximately 15-fold more potent than compound 1 (EC50 = 64 nM).
CaR is a member of the G protein-coupled receptor superfamily and couples to phospholipase C. Activation of the CaR results in the phospholipase C-induced hydrolysis of phosphatidylinositol, raising the level of free IPs. To further characterize compound 1, we studied the effect of the compound on (Ca2+)o-induced total IP accumulation in HEK-CaR cells. Figure 4 illustrates that compound 1 dose-dependently decreased the IP accumulation induced by 3 mM Ca2+ in HEK-CaR cells. The potency for this effect was 229 nM compared with an IC50 of 99 nM for compound 2. Efficacy of compound 1 was equivalent to that of compound 2.
FIG. 4. Compound 1 and compound 2 inhibit extracellular Ca2+-induced IP generation in HEK-CaR cells. HEK-CaR cells prelabeled with [3H]myo-inositol were incubated in the presence of 3 mM Ca2+ and varying concentrations of either compound 1 (circles) or compound 2 (triangles). Accumulation of IP, generated over a 2-h incubation period, was measured according to the description in Materials and Methods. Data are presented as inhibition of maximal Ca2+-induced IP generated. Compound 2 inhibited Ca2+-induced IP accumulation, with an IC50 of 99 nM. Compound 1 was slightly less potent, with an IC50 of 229 nM.
The natural ligand calcium binds to CaR with low affinity (dissociation constant 1–2 mM), precluding its use as a ligand in a radioligand binding assay. We have developed a SPA as a binding assay for CaR using 3H-compound 2 as a radioligand. As shown in Fig. 1, compound 2 is of the same chemical class as NPS-2143, which has been demonstrated to be a negative allosteric modulator of CaR (15, 18). Therefore, these compounds do not compete for binding with Ca2+ to CaR. However, not all CaR allosteric modulators need bind at the same allosteric site to inhibit CaR functional activity. In Fig. 5, data from the SPA binding assay are shown in which we studied the ability of compound 1 to compete for binding of 3H-compound 2 to the human CaR. These studies were performed to determine whether these compounds share a common binding site(s) on CaR. In this assay, compound 1 did not compete for binding of 3H-compound 2 at concentrations as high as 100 μM. In contrast, unlabeled compound 2 competed for binding of 3H-compound 2 to the HEK-CaR cell membranes, with an IC50 of 2 μM (Fig. 5). This suggests that compound 1 does not bind at the same site as 3H-compound 2. Nevertheless, compound 1 still is capable of modulating the calcium-induced rise in (Ca2+)i and the Ca2+-induced IP accumulation in HEK-CaR cells with equal efficacy as compound 2 (see Figs. 3 and 4).
FIG. 5. Compound 1 does not compete with compound 2 for binding to HEK-CaR membranes. A SPA was used to study the ability of compound 1 to compete for binding of compound 2 to the CaR in HEK-CaR membranes. HEK-CaR membranes were incubated with 50 nM [3H]compound 2 in the presence of increasing concentrations of either compound 1 (black symbols) or unlabeled compound 2 (white symbols). Unlabeled compound 2 competed for binding of [3H]compound 2, with an IC50 of approximately 2 μM. Compound 1 did not compete for binding of compound 2 to HEK-CaR membranes.
To study the efficacy of compound 1 in vivo, intact male rats were catheterized, via the right jugular vein, for serial blood sampling studies. Figure 6 illustrates the effects of compound 1 on serum PTH levels in male rats administered the compound iv. Compound 1 rapidly stimulated PTH release, within 2 min (Fig. 6A, triangles). The timing of this effect was transient, because PTH levels rapidly declined toward baseline thereafter. Maximal efficacy of compound 1 was equivalent to that of the analog of NPS-2143. In addition, the temporal character of the effects of these compounds was also similar. The dose-response relationship for compound 1 is illustrated in Fig. 6B. The effect of compound 1 on maximal PTH levels was dose-dependent, with increasing doses leading to greater secretory profiles (Fig. 6A). This was reflected in greater area under the curve (data not shown). The maximal PTH release for each dose was used for determining potency of the compound. As is shown in Fig. 6B, compound 1 was a potent stimulator of PTH secretion, with an EC50 of 0.4 μmol/kg. Thus, compound 1 is capable of transiently stimulating PTH release in vivo.
FIG. 6. Compound 1 transiently stimulates serum PTH secretion after iv dosing in the rat in vivo. A, Male Sprague Dawley rats were catheterized, via the right jugular vein, 24 h before treatment with compounds. Rats were anesthetized with ketamine/xylazine im 30 min before withdrawal of a baseline (zero-time) blood sample. After collection of this sample, animals were treated with either vehicle (circles) or increasing concentrations of compound 1 (0.06–18 μmol/kg) iv. Compound 2 (7 μmol/kg, square symbols) was given to a separate group of animals as a positive control. Data are expressed as percent of baseline levels. B, The potency of compound 1 to stimulate PTH secretion is illustrated. Maximal PTH levels attained in response to treatment of rats with differing doses of compound 1 (black symbols) are plotted as percent of baseline value. Compound 1 stimulated PTH secretion, with an apparent ED50 of 0.4 μmol/kg. The maximal PTH level attained after administration of vehicle is represented by the white bar. *, P < 0.05; n = 5–6 animals per group.
Pharmacokinetic studies were performed in rats to better understand the characteristics of this compound after oral administration. Figure 7A illustrates the plasma concentrations of compound 1 after a single oral dose of 30 mg/kg (54 μmol/kg). Maximal plasma levels of approximately 2 μM were achieved within 1 h of administration of compound 1. The compound was rapidly cleared, with an oral half-life of approximately 2 h. Eight hours after dosing, levels of the compound in plasma were approximately 1.5% of maximum and were undetectable 24 h after administration. In a separate study, rats were administered 50 μmol/kg compound 1 or vehicle and blood samples collected over a 4-h period from the conscious, freely moving animals. Figure 7B illustrates the effect of this single oral dose of compound 1 on maximal PTH levels attained during the 4-h sampling period. Upon dosing, serum PTH levels rapidly increased, to reach maximal levels within 30–60 min. Serum PTH levels declined thereafter, reaching a plateau from 60–180 min after administration. PTH levels declined to that of vehicle-treated animals within 4 h after dosing. Overall, compound 1 increased serum PTH levels approximately 3-fold compared with vehicle-treated animals. These data demonstrate compound 1 to be an orally available negative allosteric modulator of the CaR that transiently stimulates PTH release in vivo.
FIG. 7. Serum levels of compound 1 and PTH after oral dosing in rats. A, Serum concentration vs. time profile of compound 1 in rats after oral administration of 54 μmol/kg compound 1. Data are expressed as means ± SEM; n = 3. B, Serum PTH levels from male rats administered 50 μmol/kg compound 1 (black symbols) or vehicle (45% 2-hydroxypropyl-?-cyclodextrin, white symbols) po. Serial blood samples were collected at 0, 0.5, 1, 1.5, 2, and 4 h after dosing. Sera were then assayed for PTH levels described in Materials and Methods. Oral dosing of compound 1 to male rats induced a rapid increase in serum PTH levels that was maximal within 30 min of dosing. Levels then slowly decreased thereafter, achieving vehicle levels within 2 h. Data are expressed as percent of baseline PTH levels ± SEM. *, P < 0.05; n = 8 animals per group.
Discussion
Recent data from the development of pharmacological agents modulating the CaR have provided a suitable proof-of-concept for the use of calcilytics as a potential therapeutic strategy for the treatment of osteoporosis. Given the tight association between serum Ca2+ concentrations and PTH release, it is not surprising that many have focused on this mechanism to induce bone anabolism. Despite the report of selective allosteric modulators of CaR that can stimulate PTH release, room for improvement in this class of compounds remains. This was especially evident when a representative of this class (NPS-2143) was studied in vivo. Data from these experiments demonstrated a prolonged half-life, both when administered po and iv (15, 16, 18). In their description of NPS-2143, Nemeth et al. found that plasma PTH levels were elevated more than 1 h after termination of infusion. Additionally, this compound had a plasma half-life of approximately 8 h when administered orally (16). The prolonged half-life with a resultant elevated concentration of serum PTH may have been a contributing factor to the lack of efficacy seen on bone mineral density when given alone to osteopenic rats (16). Therefore, we sought to synthesize antagonists to the CaR with the goal to identify a series of compounds with potent antagonizing and/or allosteric modulatory activity toward the CaR and possessing an improved pharmacokinetic profile in vivo.
Screening of a large compound collection revealed a number of chemotypes that were able to stimulate Ca2+ flux in HEK-293 cells. A representative compound derived from one of these chemotypes (compound 1) was capable of inhibiting the (Ca2+)o-induced rise in (Ca2+)i fluorescence in this assay, with an apparent IC50 of 64 nM. Similarly, compound 1 inhibited (Ca2+)o-induced accumulation of IP, with an IC50 of 230 nM.
To investigate the specificity of the effects of compound 1, we studied the receptor dependency of the action of the compound in cell lines not expressing the CaR. The data from these experiments demonstrate that in the absence of CaR, compound 1 has no effect on calcium flux. These data suggest that the effects of compound 1 observed in the cell lines reflect CaR-dependent activity. Subsequent study of the activity profile of this compound vs. other G protein-coupled receptors demonstrated a clear selectivity of compound 1 for the CaR. Although significant interaction was detected vs. the histamine H2 and serotonin receptors (data not shown), compound 1 demonstrated at least 50-fold selectivity for CaR.
In our experiments, we studied whether compound 1 was capable of acting through the same or an overlapping site with NPS-2143 by observing the ability of compound 1 to compete for binding of compound 2 (a closely related analog of NPS-2143, see Fig. 1) to CaR. Our data reveal that compound 1 was incapable of competing for binding with compound 2, suggesting that these two compounds do not share a common binding site on CaR or that the overlap in binding sites is not sufficient for compound 1 to displace compound 2. It is likely that NPS-2143 and compound 2 do act at the CaR through a similar site because these two molecules are so closely related; and thus, compound 2 affords a suitable surrogate for NPS-2143 and comparator for compound 1 activity. Unfortunately, the nature of the binding studies provides no insight into the site(s) of interaction of compound 1 with the CaR. In their description of the properties of NPS-2143, Nemeth et al. (15, 18, 19, 20) suggest that the mechanism of action of NPS-2143 and related calcilytic compounds is similar to type II calcimimetic compounds (e.g. NPS-568). These authors have suggested that these structurally related compounds act through an allosteric mechanism to alter the sensitivity (affinity) of the CaR to (Ca2+)o (20). Data to support this hypothesis can be found in studies employing a truncated form of the CaR that is devoid of the extracellular domain of the receptor. In these experiments, cells expressing this mutant receptor responded to the calcimimetic NPS-568 with an increase in IP3 production (21). Alanine substitutions in the second transmembrane domain of this truncated receptor blocked the ability of the receptor to signal in the presence of NPS-568 (21). These data suggest that, although the extracellular portion of the CaR is the primary point of binding for Ca2+ (22), the second transmembrane region of the receptor is intimately involved in activating signaling of the ligand bound receptor. Studies of naturally occurring mutations in CaR support this hypothesis (11, 23, 24, 25). In both rats and humans, mutations in the second transmembrane domain of CaR lead to a disruption in the ability of the receptor to signal. Interestingly, mutations of this region of the receptor do not appreciably affect binding of the receptor. These data suggest that this region of the receptor is involved in generating the conformational changes of the CaR that lead to association with signaling proteins. This idea of transferring the signal of ligand binding from the extracellular domain to sites in the transmembrane and cytoplasmic regions of receptors has been hypothesized for many G protein-coupled receptors, including CaR (26). However, other portions of the receptor have been hypothesized to also play a role, including the Ala116-Pro136 region of the receptor (26). Alanine scanning mutagenesis of this segment of the second extracellular loop of the receptor increased the potency for Ca2+ and elevated the basal signaling activity of the receptor (26, 27). It has been hypothesized that these effects are due to stabilization of the active conformation of CaR by these mutations. Because compound 1 did not compete for binding with compound 2, these latter data suggest that this region of the extracellular loops of the receptor may be a potential site for action of compound 1. However, the possibility still exists that compound 1 could significantly compete directly for binding to CaR by Ca2+, because we were unable to study this option directly.
Regardless of the sites of action, administration of compound 1 to male rats produced a rapid and robust increase of serum PTH levels. Efficacy of compound 1 was equivalent to that of compound 2. The effects of compound 1 were transient, with PTH levels decreasing toward baseline within 10 min. This is in contrast to NPS-2143, which has been reported to maintain high plasma levels for more than 8 h after oral administration (16). The ability of compound 1 to increase serum PTH levels was dose-dependent, with an ED50 of approximately 0.4 μmol/kg. The pharmacokinetic analysis of compound 1 revealed that upon oral dosing, the compound swiftly attained peak plasma levels, after which the compound was rapidly cleared, such that the T1/2 for compound 1 was approximately 2 h. This pattern of exposure is significantly better than that reported for NPS-2143, which was reported to have a half-life greater than 8 h upon po dosing (16). This is an important feature of any calcilytic, because the anabolic action of calcium requires a transient activation of the receptor (12, 13). In the description of the activity of NPS-2143, it was noted that significant effects of this compound on bone mineral density in osteopenic rats was demonstrated only in the presence of the antiresorptive effects of estrogen. Lone administration of NPS-2143 did not result in improvement in bone mineral density. These observations could be due to the long half-life of NPS-2143, which would lead to a more resorptive pattern of CaR modulation. The pharmacokinetic profile of absorption of compound 1 after oral administration was verified by effects on serum PTH levels. Serum PTH reached maximal levels within 1 h of administration. Together, these data show compound 1 to have equal efficacy with, but a more advantageous pharmacokinetic profile for stimulating PTH secretion than, NPS-2143.
In conclusion, we have identified and characterized a novel compound capable of antagonizing the human and rat CaR. The data demonstrate the compound to be equally efficacious as, but slightly less potent than, a close analog of the best characterized calcilytic, NPS-2143. These data offer a new avenue for the development of novel anabolic agents for the treatment of osteoporosis.
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