Calcimimetics are positive allosteric modulators to the calcium-sensing receptor (CaSR). Activation of the CaSR inhibits the secretion of parathyroid hormone (PTH), stimulates the secretion of calcitonin, and decreases serum calcium (Ca2+). Cinacalcet, a second-generation calcimimetic, is used therapeutically to control PTH in patients with chronic kidney disease who are on dialysis with secondary hyperparathyroidism. A calcimimetic that displays increased separation of PTH versus Ca2+ lowering in patients would potentially allow the use of calcimimetics to treat patients in earlier stages of renal disease because hypocalcemia can develop in this population. Toward this end, we developed a third-generation calcimimetic, determined the molecular pharmacological properties of it using an operation model of allosteric modulation/agonism, and measured the compound effects on PTH, serum ionized Ca2+, and calcitonin levels in 5/6 nephrectomized rats. We found the new molecule effectively reduced PTH levels without promoting calcitonin secretion or hypocalcemia. Furthermore, our third-generation molecule was less efficacious at promoting calcitonin secretion from human thyroid carcinoma cells compared with 3-(2-chlorophenyl)-N-((1R)-1-(3-methoxyphenyl)ethyl)-1-propanamine (R-568), a first-generation calcimimetic. These data provide evidence that calcimimetics with increased potency can be used to lower PTH without production of significant hypocalcemia because the threshold for inhibition of PTH secretion is much lower than the threshold for calcitonin secretion.
The calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor originally identified from bovine parathyroid cells and isolated by an expression cloning strategy in Xenopus laevis oocytes (Brown et al., 1993). The highest level of expression of the CaSR is found in parathyroid tissue where it plays a crucial role in calcium and phosphate homeostasis. The receptor is also expressed in other tissues including the thyroid, kidney, brain, stomach, and gastrointestinal tract (Brown, 2007). CaSR activation by millimolar concentrations of calcium in the parathyroid gland inhibits parathyroid hormone (PTH) secretion, thereby reducing bone resorption (Brown, 1991). In addition, CaSR activation in C cells of the thyroid stimulates secretion of calcitonin, an inhibitor of bone resorption (Fox et al., 1999a). Stimulation of the receptor by millimolar concentrations of Ca2+ can activate both Gq and Gi signaling pathways, leading to inositol phosphate (IP) production, intracellular Ca2+ flux, and ERK phosphorylation (Chen et al., 1989; Hawkins et al., 1989; Kifor et al., 1997). In the kidney, the CaSR and the PTH receptor regulate calcium reabsorption (Chang and Shoback, 2004). Activation of the receptor in the stomach is associated with increased acid secretion, whereas activation of the CaSR in the gastrointestinal tract has been shown to inhibit fluid secretion from colonic crypt cells (Dufner et al., 2004; Geibel et al., 2006).
Disorders of Ca2+ and phosphate homeostasis develop in end-stage renal disease (ESRD) because of reduced serum calcitriol and elevated serum PTH levels. Calcimimetics are positive allosteric modulators that bind in the transmembrane domain on the CaSR. They increase the potency and/or affinity of Ca2+ to the receptor (Hammerland et al., 1998; Petrel et al., 2004). Modulation of the CaSR with calcimimetics effectively reduced serum PTH in rodent models of ESRD (Fox et al., 1999b). Cinacalcet is a second-generation calcimimetic indicated for the treatment of secondary hyperparathyroidism (SHPT) in patients with chronic kidney disease (CKD) on dialysis (Goodman et al., 2002; Block et al., 2004). In addition, cinacalcet is indicated for the treatment of hypocalcemia in patients with parathyroid carcinoma and in some countries for the treatment of primary hyperparathyroidism. Furthermore, three reports demonstrate the effectiveness of cinacalcet in treating familial hypocalciuric hypercalcemia (Timmers et al., 2006; Festen-Spanjer et al., 2008; Alon and Vandervoorde, 2010).
Treatment of SHPT in CKD stages 3 and 4 with calcimimetics has not been approved by the Food and Drug Administration and requires further evaluation. Patients with stages 3 and 4 CKD also have elevated PTH but at lower levels than patients with ESRD. In a randomized, double-blind, placebo-controlled study to assess efficacy and safety of cinacalcet in nondialysis patients, cinacalcet effectively decreased plasma intact PTH. However, hypocalcemia (generally asymptomatic) was observed (Chonchol et al., 2009). Hence, it is desirable to develop a molecule that is able to suppress PTH secretion without causing hypocalcemia for use is this group of patients. Toward this end, we developed (1R)-1-(6-methoxy-4′-(trifluoromethyl)-3-biphenylyl)-N-((1R)-1-phenylethyl)ethanamine (calcimimetic B) that we compared with 3-(2-chlorophenyl)-N-((1R)-1-(3-methoxyphenyl)ethyl)-1-propanamine (R-568), a first-generation calcimimetic (Harrington et al., 2010). We determined the molecular pharmacological parameters of each calcimimetic and measured the compound effects on PTH, serum-ionized calcium (Ca2+), and calcitonin levels in 5/6 nephrectomized (Nx) rats. Measurements were conducted up to 4 h postdose to explore the differential effects on Ca2+ lowering of these two calcimimetics. We found that calcimimetic B was more potent and had less Ca2+-lowering effect relative to PTH lowering in 5/6 Nx rats compared with R-568.
Materials and Methods
Chemicals, Plasmids, and Reagents.
R-568 and calcimimetic B were prepared at Amgen, Inc. (Thousand Oaks, CA). HEK293, Chinese hamster ovary, and TT cells were obtained from the American Type Culture Collection (Manassas, VA). HEK293 cells stably expressing the human CaSR were obtained from NPS Pharmaceuticals (Bedminster, NJ). Yttrium silicate SPA beads and tritiated inositol (50–80 Ci/mmol) were obtained from GE Healthcare (Chalfont St. Giles, Buckinghamshire, UK). pcDNA3.1-aequorin and pcDNA3.1-human CaSR (hCaSR) were prepared by standard cloning techniques. Plasmids were prepared with a QIAGEN plasmid mega DNA preparation kit (QIAGEN, Valencia, CA).
Male Sprague-Dawley rats (249–424 g) were purchased from Harlan (Indianapolis, IN). Rats were pair-housed under a 12-h/12-h light/dark cycle and given ad libitum access to standard rat chow (1.2% calcium, 0.9% phosphorus) and water. Experiments were performed under protocols approved by Amgen's Internal Animal Care and Use Committee.
Six million Chinese hamster ovary cells were seeded in a 150-cm2 dish in DMEM/F12/10% fetal calf serum without antibiotics. One hour after plating, the cells were transfected with 10 μg of hCaSR plasmid and 10 μg of aequorin plasmid with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The cells were incubated at 37°C with 5% CO2 for 6 h, and the medium was replaced. One day after transfection, the cells were detached by brief treatment with trypsin/EDTA, washed, and resuspended in 10 ml of HBSS containing 0.5 mM CaCl2 and 0.1% BSA. Coelenterazine F was added to a final concentration of 1 μg/ml. The cells were gently agitated every 15 min for a total of 2 h. The cells were then diluted 3-fold in the same buffer lacking coelenterazine F. R-568 was prepared as a 100× stock in DMSO. Just before the assay, the R-568 was diluted in 100 μl of calcium-free HBSS with 0.1% BSA and placed in a 96-well white plate (Corning Life Sciences, Lowell, MA). One hundred microliters of cells was then added to the plate on a MicroLumat luminometer (Berthold Technologies USA, Oak Ridge, TN), and the luminescence was recorded. The lowest concentration of calcium tested was 0.25 mM.
Fluorescent Imaging Plate Reader Assay.
Ca2+ flux was measured using a fluorescent imaging plate reader (FLIPR; Molecular Devices, Sunnyvale, CA). Twenty thousand HEK cells stably expressing the hCaSR were placed in a 384-well, poly-d-lysine-coated plate with a clear bottom and black sides in 50 μl of cell media (DMEM/10% fetal bovine serum). The next day, the media were removed and the cells were incubated in 50 μl of dye buffer (BD Biosciences Discovery Labware, Bedford, MA) and allowed to incubate for 1.5 h at room temperature. Compounds were dissolved in 100% DMSO at 100× concentration. The compounds were then diluted 100-fold in HEPES-buffered saline containing 1.0 mM CaCl2. Twenty five microliters of compound was then added to the cells, and Ca2+ flux was monitored for 90 s on FLIPR. Maximum observed fluorescence values were used in the calculation of LogEC50.
Inositol Phosphate Accumulation Assay.
HEK293 cells were dispensed into a poly-d-lysine tissue culture-treated, 96-well plate at a density of 25,000 cells per well. The next day, the cells (∼80–90% confluent) were transfected with 100 ng of hCaSR plasmid per well using Lipofectamine 2000 according to the manufacturer's instructions. Six hours after transfection the medium was replaced with inositol-free DMEM/10% dialyzed fetal calf serum supplemented with 1μCi/ml tritiated inositol. After incubation overnight, the cells were washed once in calcium-free HBSS and then treated with the 100 μl of calcium free HBSS/0.01% BSA containing various concentrations of CaCl2, R-568 (prepared as above in DMSO), and 10 mM LiCl and incubated at 37°C for 1 h. The media were aspirated and the cells were lysed with ice-cold 20 mM formic acid. After incubation at 4°C for 5 h, the lysate was added to yttrium silicate SPA beads, allowed to settle overnight, and read on a Beckman TopCount scintillation counter (Beckman Coulter, Fullerton, CA).
ERK Phosphorylation Assay.
HEK293 cells stably expressing the hCaSR were dispersed in a 96-well, tissue culture-treated plate at a density of 45,000 cells per well for 36 h. The cells were then starved overnight in serum-free medium and then washed with phosphate-free buffer without Ca2+. ERK phosphorylation was induced by compound addition for 5 min, and the media were aspirated. The cells were lysed by quick-freezing in liquid nitrogen. Fifty microliters of Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA) was added to each well and denatured by heating to 100°C for 5 min, and then 10 μl of the cell suspension was loaded on a 4 to 12% gradient SDS-polyacrylamide gel electrophoresis gel. The gel was transferred to a polyvinylidene difluoride membrane and probed with primary phosphor-ERK antibody (Cell Signaling Technology, Danvers, MA) at a 1:1000 dilution at 4°C overnight. The membrane was then probed with a goat anti-rabbit IgG secondary antibody (Thermo Fisher Scientific, Waltham, MA) at a dilution of 1:5000 for 1 h at room temperature. The membrane signal was detected using SuperSignal Substrate (Thermo Fisher Scientific).
Subtotal Nx (5/6) male Sprague-Dawley rats were used to evaluate calcimimetic effects on mediators of serum Ca2+, including PTH and calcitonin secretion. All studies and procedures were performed in accordance with policies and procedures approved by the Internal Animal Care and Use Committee of Amgen Inc.
Surgical Procedures for 5/6 Nx Rats.
A two-step procedure was performed starting with ligation of renal artery (left kidney; uppermost bifurcation) in anesthetized rats [intramuscular cocktail: two parts ketamine (100 mg/ml), one part xylazine (100 mg/ml), 0.3 parts acepromazine (10 mg/ml)]. After a 1-week recovery period after ligation of the left kidney, the right kidney was removed in anesthetized animals. This procedure reduces the original functional renal mass by five-sixths and induces CKD accompanied by SHPT. After the surgical procedures, animals recovered for 6 weeks before the initiation of studies.
Six weeks after 5/6 Nx rats (n = 7–8/group) were fasted overnight (14–16 h) and then treated with single oral doses of the research calcimimetics, R-568 (1, 10, 30, or 100 mg/kg) or calcimimetic B (0.03, 0.1, or 0.3 mg/kg). Vehicle-treated control rats (n = 8) received 2% hydroxypropyl methylcellulose, 1% pluronic F68, and 10% Captisol in water in equivolume amounts (5 ml/kg) as the calcimimetic-treated groups.
Blood was collected from the retro-orbital sinus from anesthetized rats (2% isofluorane in O2). Whole blood ionized Ca2+ and serum PTH and calcitonin were determined at 0, 0.25, 0.5, 1, 2, and 4 h postdose. Immediately after the animals recovered from the last blood sample collection they were given free access to food. For ionized Ca2+, a Ciba-Corning 634 ISE Ca2+/pH Analyzer (Ciba-Corning Diagnostics Corp., Medfield, MA) was used. Serum PTH and calcitonin levels were determined using commercial ELISA rat PTH[1-34] and rat calcitonin assay kits (Immutopics, San Clemente, CA).
In Situ Hybridization.
A standard in situ hybridization protocol was performed to examine the expression of the rat CaSR in normal rat parathyroid and thyroid tissue derived from archived blocks of immersion-fixed, paraffin-embedded material (Wilcox, 1993). In brief, a 282-base pair 33P-labeled RNA antisense probe corresponding to nucleotides 2154 to 2435 of the rat CaSR gene (GenBank accession number U10354) was used as a probe. Hybridization was conducted at 60°C in a hybridization solution containing 1 × 106 cpm of 33P-labeled antisense riboprobe, followed by RNase digestion, and a series of high-stringency washes at 55°C for 30 min. The slides were coated with Kodak NTB emulsion (Eastman Kodak, Rochester, NY), exposed for 3 weeks in the dark at 4°C, developed, and then counterstained with hematoxylin and eosin.
Calcitonin Secretion Assay.
TT cells were seeded in 96-well plates at a density of 30,000 cells per well and grown for 48 h until they reached 80% confluence, washed in phosphate-free buffer without Ca2+ and serum, and Ca+2-starved for 2 h. Cells were subsequently treated with 1 mM CaCl2 or 1 mM CaCl2 plus different concentrations of calcimimetic (stock prepared at 100× in DMSO) for 12 h. The medium from treated or untreated cells was centrifuged (1500g for 3 min), and the concentration of calcitonin in the culture medium was determined using a commercial ELISA method per the manufacturer's instructions (Immutopics) and normalized to cell protein content. Control medium, which was not exposed to the cells, had no detectable levels of calcitonin. Absorbance at 450 nM was measured using an Envision instrument (PerkinElmer Life and Analytical Sciences, Waltham, MA) and normalized to cell protein content. Values were plotted against a calcitonin standard curve, and each treatment dose was conducted in triplicate.
Individual dose-response curves were fitted to the variable slope Hill equation by nonlinear regression analysis using Prism 4.01 (GraphPad Software Inc., San Diego, CA). Fitting of data from the inositol phosphate accumulation assay in Fig. 4 was performed using the following form of an operational model of allosterism/agonism (see Fig. 3; also see Lu et al., 2009): where A and B denote the molar concentrations of the orthosteric ligand and allosteric ligand, respectively; E denotes response; Em denotes the maximum response; KB denotes the dissociation constant of allosteric ligand B; EC50A denotes the half-maximal response of orthosteric full agonist A; τB denotes the efficacy of ligand B; α denotes a cooperativity factor that governs the magnitude and extent of the allosteric effect on the binding affinity of each ligand; β is a measure of the allosteric effect of the modulator on the signaling efficacy of the orthosteric agonist; and n denotes a logistic slope factor. KB, EC50, τB, and αβ (determined as a composite parameter) were estimated as base 10 logarithms (Christopoulos, 1998). Experimental error is reported as S.D. unless otherwise noted.
We accessed the molecular pharmacological properties of R-568 and calcimimetic B using Ca2+ flux, IP accumulation, and ERK phosphorylation assays. Because calcimimetics are positive allosteric modulators to the CaSR, we performed allosteric titration experiments in 1 to 1.2 mM CaCl2, a concentration that is similar to the level of ionizable Ca2+ in human serum. Shown in Table 1 are the LogEC50 values for R-568 and calcimimetic B against the hCaSR. R-568 displayed an average LogEC50 of −7.2, −6.4, and −6.4 in the FLIPR, aequorin, and IP assays, respectively (Fig. 1, A–C). Calcimimetic B displayed an average LogEC50 of −8.1, − 7.8, and −7.7 in the FLIPR, aequorin, and IP assays, respectively. Similar potency values were obtained against the rat CaSR (data not shown). Calcimimetic B was significantly more potent than R-568 in all three assay formats. A typical dose-response curve of CaCl2 compared with R-568 and calcimimetic B in Ca2+-free buffer is shown in Fig. 1D. Both compounds displayed partial agonism in addition to their allosteric modulatory properties.
Shown in Fig. 2 are the effects of R-568 and calcimimetic B on ERK phosphorylation in the presence and absence of extracellular Ca2+. As observed in the IP accumulation assay, both R-568 and calcimimetic B promoted ERK phosphorylation in the absence of extracellular Ca2+, indicating the compounds are agonists as well as allosteric modulators to the CaSR. Potency values for R-568 and calcimimetic B in the absence of extracellular Ca2+ were LogEC50 = −5.4 ± 0.15 (n = 3) and −7.2 ± 0.46 (n = 3), respectively (Fig. 2, A and C). In the presence of 1 mM extracellular Ca2+, the potency of R-568 and calcimimetic B was increased approximately 10-fold to LogEC50 = −6.8 ± 0.14 (n = 3) and −8.2 ± 0.44 (n = 3), respectively (Fig. 2, B and D).
Activation and modulation of the CaSR by R-568 and calcimimetic B is a composite of 1) the affinity of the compound at the receptor denoted KB, 2) the intrinsic efficacy denoted τB, 3) the ability of the compound to modulate the affinity of Ca2+ to the CaSR denoted α (cooperativity), and 4) the ability of the compound to modulate the coupling of the Ca2+ occupied CaSR to downstream signaling pathways denoted β (efficacy modulation). These parameters were quantitated using an operational model of allosteric modulation and allosteric agonism (Leach et al., 2007; Aurelio et al., 2009; Lu et al., 2009).
Shown in Fig. 3A is a diagram depicting the parameters of the model along with three graphs showing a simulation that demonstrates the effect of the parameters (Fig. 3, B–D). Shown in black is the agonist (Ca2+) dose-response curve and shown in red is the agonist dose-response curve in the presence of a positive allosteric modulator. In the case of positive allosteric modulators such as calcimimetics (α > 1), affinity modulation has the effect of left-shifting the agonist dose-response curve (Fig. 3B). If the calcimimetic also possesses intrinsic efficacy, as is the case of R-568 and calcimimetic B, the agonist dose-response curve is again left-shifted with an increase in basal response caused by modulator efficacy (Fig. 3C). Allosteric modulators that possess efficacy modulation (β > 1) and intrinsic efficacy also left-shift the agonist dose-response curve in addition to increasing the basal response (Fig. 3D). It is noteworthy that affinity modulation (α) cannot be distinguished from efficacy modulation (β) when the orthosteric agonist (Ca2+ in this case) is a full agonist (Lu et al., 2009).
Using this model, we performed a series of Ca2+ dose-response curves in the presence of increasing doses of calcimimetic (Fig. 4). The hCaSR displayed a robust response to Ca2+ in the inositol phosphate accumulation assay. The effect of R-568 and calcimimetic B was characterized by an increase in basal responsiveness and an enhancement of the potency of extracellular Ca2+. This result is consistent with both compounds being positive allosteric modulators as well as partial agonists. There was no significant effect on maximum orthosteric agonist response, Emax, consistent with extracellular Ca2+ being a full agonist at the hCaSR. Global fitting of the entire family of curves to an operational model of allosterism/agonism yielded the estimates of orthosteric agonist potency (LogEC50), modulator affinity (LogKB), modulator efficacy (LogτB), and cooperativity (Logαβ) shown in Table 2. Because of the large number of parameters associated with the model, it was necessary to estimate the cooperativity (α) and efficacy modulation (β) as a composite parameter, αβ (Lu et al., 2009). We found that both R-568 and calcimimetic B are allosteric modulators as well as partial agonists because their τB values were greater than 0. Calcimimetic B showed a significant improvement in affinity compared with R-568 by 0.6 log units. In addition, calcimimetic B displayed greater efficacy than R-568 (τB = 1.2 versus 0.8; p = 0.001) as evidenced by the greater basal responsiveness observed in the absence of Ca2+ (Fig. 4). As a reference, a τ value of 1 indicates a partial agonist that achieves 50% of the maximum full-agonist response at saturating concentrations. A full agonist typically has τ values of 10 or more. Both compounds displayed positive cooperativity in the IP assay. However, the cooperativity observed for R-568 and calcimimetic B was not statistically different. Because the α and β values were obtained as one composite αβ value, we were unable to ascertain the relative contribution of affinity versus efficacy modulation. The use of eq. 1 requires that the value of τ for Ca2+ be large, indicating receptor reserve. This could be a borderline case for the CaSR. Hence, we also examined simulations using the expanded operational model of allosterism and agonism in which no assumptions about receptor reserve were made (Leach et al., 2007; Lu et al., 2009). The simulations revealed minor differences in the α and β values obtained compared with eq. 1 (data not shown). However, one is not able to fit the experimental data presented in Fig. 4 to the expanded operational model because there are too many parameters to be determined. In either case, both R-568 and calcimimetic B possessed composite cooperativity/efficacy values that were not statistically different.
To assess the activity of the calcimimetics in vivo, we used the 5/6 Nx rat as an animal model of SHPT (Henley et al., 2005). Rats were treated with single oral doses of calcimimetic B (0.03, 0.1, and 0.3 mg/kg) or R-568 (10, 30, and 100 mg/kg). Higher doses of R-568 versus calcimimetic B were selected based on pharmacokinetic data in rats: R-568 has a half-life of 2.3 h and an oral bioavailability of 4.6%, whereas calcimimetic B has a half-life of 9.3 h and an oral bioavailability of 60%. Serum Ca2+, PTH, and calcitonin were measured at 0, 0.25, 0.5, 1, 2, and 4 h postdose (Fig. 5). R-568 elicited a rapid significant decrease (0.25 h after drug administration) in serum PTH (Fig. 5A). In contrast, calcimimetic B effects on PTH lowering during the first 30 min after drug administration were less pronounced than R-568. However, by 1 h after treatment, PTH was lowered to approximately the same level by the highest dose (0.3 mg/kg) of calcimimetic B (PTH = 37 ± 17 pg/ml; n = 8) compared with R-568 (PTH = 35 ± 19; n = 8 and 37 ± 10; n = 7 pg/ml for the 30 and 100 mg/kg doses, respectively). By 2 h after treatment a lower dose of calcimimetic B (0.1 mg/kg) lowered PTH (102 ± 37 pg/ml; n = 8) to comparable levels achieved by R-568 (PTH = 114 ± 85 and 72 ± 44 pm/ml for the 30 and 100 mg/kg doses, respectively). Serum PTH measured 4 h after drug administration was lowest in the calcimimetic B (0.3 mg/kg) group (PTH = 14 ± 6 pg/ml) and the R-568 (100 mg/kg) group (PTH = 44 ± 13), followed by the calcimimetic B (0.1 mg/kg; PTH = 107 ± 47) and the R-568 (30 mg/kg; PTH 204 ± 84) groups. Effects of R-568 on Ca2+ lowering were greater and more immediate than the effects of calcimimetic B (Fig. 5B). In addition, doses of calcimimetic B that produced similar PTH lowering compared with R-568 at a given time after drug administration had lesser effects on blood Ca2+ levels (Fig. 5A). R-568 elicited a rapid and dose-dependent increase in calcitonin secretion that correlated with decreases in Ca2+ (Fig. 5C). In contrast, calcimimetic B had no effect on calcitonin at any dose, including the highest dose (0.3 mg/kg) that profoundly suppressed PTH. Shown in Fig. 5D is an situ hybridization experiment demonstrating high expression of the CaSR in parathyroid relative to the surrounding thyroid tissue.
The differential effects of R-568 and calcimimetic B on calcitonin secretion were further explored using the thyroid carcinoma cell line (TT) as an established in vitro model of calcitonin secretion (Leong et al., 1981). Western blot analysis revealed TT cells express the CaSR with the major band migrating at ∼160 kDa (Fig. 6A). This contrasts with an earlier report that found TT cells devoid of CaSR expression (Garrett et al., 1995). An allosteric titration experiment was performed in which cells were treated for 12 h with increasing concentrations of calcimimetics and a fixed concentration of Ca+2 (1 mM). The cells showed a dose-dependent secretion of calcitonin in response to increasing concentrations of R-568. In contrast, cells treated with calcimimetic B secreted much lower levels of calcitonin (Fig. 6B).
Calcimimetics are positive allosteric modulators that increase the potency of Ca2+ to the CaSR, resulting in a left shift of the calcium dose-response curve. However, the overall signaling efficacy of the CaSR is not affected by either R-568 or calcimimetic B. This result is consistent with Ca2+ acting as a full agonist. In allosteric titration experiments involving the IP, Ca2+ flux, and ERK phosphorylation assays, we found that calcimimetic B was significantly more potent than R-568. We showed that R-568 and calcimimetic B promoted IP accumulation and ERK phosphorylation in the absence of Ca2+, indicating that both compounds have inherent CaSR agonist activity in addition to being positive allosteric modulators. We used an operational model of allosteric modulation/agonism to demonstrate that calcimimetic B displayed higher affinity and greater intrinsic efficacy for the CaSR compared with R-568. However, we did not observe a statistically significant difference in cooperativity values between R-568 and calcimimetic B. The serum concentration of ionizable Ca2+ is approximately 1.4 mM in rodents. Serum Ca2+ cannot be lowered significantly without resulting in serious adverse effects on experimental animals. Hence, the effect of improved intrinsic efficacy of calcimimetic B (the activity present in the absence of Ca2+) was not possible to examine in vivo. The greater in vivo potency we observed with calcimimetic B in terms of its ability to suppress PTH secretion is likely a combination of factors, including higher potency, higher affinity and intrinsic efficacy, longer half-life, and higher oral bioavailability compared with R-568.
We found that calcimimetic B showed a greater separation of PTH versus ionized Ca2+ lowering compared with R-568. It is noteworthy that whereas R-568 was both an inhibitor of PTH secretion and stimulator of calcitonin secretion, calcimimetic B did not stimulate calcitonin secretion at doses sufficient to inhibit PTH secretion. This may explain, at least in part, why calcimimetic B has less effect on the reduction in ionized Ca2+ compared with R-568 because calcitonin administration in rodents lowers serum-ionized Ca2+ levels (Nishiki et al., 2003). The rate of onset of calcium lowering by calcimimetic B was less than that for R-568. It is plausible to hypothesize that this observation was caused by a lack of an effect of calcimimetic B on thyroid-mediated secretion of calcitonin. This notion is supported by the observation that in R-568-treated, thyroidectomized rats (i.e., no calcitonin-releasing target) with intact parathyroids the rate of onset of calcium lowering was slower than in normal rats with intact thyroid glands (Fox et al., 1999a). In addition, it has been shown that R-568 rapidly reduced Ca2+ levels in parathyroidectomized thyroid-intact rats that were given PTH infusions to restore normocalcemia after removal of the parathyroids (Fox et al., 1999a), supporting the notion that decreased PTH secretion was not solely responsible for calcimimetic-induced hypocalcemia. The ability of calcimimetic B to differentially affect PTH suppression compared with calcitonin secretion may in part be explained by its pharmacokinetic properties. A 30 mg/kg dose of R-568 administered to uremic rats produced R-568 exposure levels that were up to 100-fold higher than a 0.1 mg/kg dose of calcimimetic B (Supplemental Table 1). It has been shown in rodents that calcimimetics (R-568 and R-467) depress serum PTH at doses much lower than doses that increase plasma calcitonin (Nemeth and Fox, 1999). R-568 has been shown to be approximately 40 times more potent in reducing PTH levels than in increasing calcitonin levels (Fox et al., 1999a). Similar findings have been reported for cinacalcet HCl (Nemeth et al., 2004). In addition, R-568 in doses that suppressed PTH and minimally decreased serum calcium have been shown to increase serum calcitonin in dialysis patients (Antonsen et al., 1998).
It is plausible to hypothesize that the lower exposure levels of calcimimetic B translate into lower tissue levels that may be below the threshold for calcitonin secretion, yet sufficient for inhibition of PTH secretion. These data suggest that calcitonin secretion plays a role in the hypocalcemia observed in this CKD model. Calcitonin is a potent stimulator of bone formation in rodents, resulting in deceased serum Ca2+ levels (Baylink et al., 1969; Sørensen et al., 1970). Hence, calcimimetic B was able to achieve a desirable PTH suppression while minimizing hypocalcemia.
The functional selectivity of calcimimetic B to lower PTH while having minimal effect on calcitonin secretion may be a result of selective activation of CaSR pathways in the chief cells of the parathyroid compared with the C cells of the thyroid. The expression of the CaSR in parathyroid tissue is much higher than in thyroid tissue as demonstrated by the in situ hybridization experiment shown in Fig. 5D. Because compound efficacy is proportional to receptor density, this may in part explain the differential effects seen with calcimimetic B. Consistent with our in vivo data, we found that human thyroid carcinoma TT cells secrete calcitonin in response to R-568, whereas calcimimetic B showed reduced efficacy. It is noteworthy that no functional selectivity was observed for calcimimetic B over R-568 in our second-messenger and ERK phosphorylation assays. There are examples of a compound promoting activation of a receptor in one cell type but not another. For example, binding of nicotinic acid to the nicotinic acid receptor inhibits lipolysis in adipocytes and stimulates prostaglandin release from Langerhans cells in the skin. However, some synthetic nicotinic acid receptor agonists inhibit lypolysis in adipocytes but show no stimulation of prostaglandin secretion from Langerhans cells (Richman et al., 2007). This ligand bias was found to be a result of differential activation of β-arrestin pathways in Langerhans cells over G protein pathways. It remains to be determined whether calcimimetic B exhibits a signaling bias in chief cells over C cells via a similar mechanism. It is noteworthy that it has been reported that an autoantibody to the CaSR was found to selectively activate the Gq pathway while inhibiting ERK phosphorylation in CaSR expressing HEK293 cells (Makita et al., 2007). Whether or not these dual acting pathways exist in C cells remains to be determined. Of course, it is also plausible that a combination of the improved potency and pharmacokinetic parameters of calcimimetic B are responsible for the observed selective in vivo effects.
In conclusion, we have developed a calcimimetic that has the unique ability to increase the separation of PTH lowering versus Ca2+ lowering in 5/6 Nx rats compared with R-568, the first calcimimetic introduced into clinical trials. Our data support the hypothesis that calcimimetic B is able to accomplish this effect, in part, because of the lack of calcitonin secretion at doses sufficient to inhibit PTH secretion. We presented an allosteric model that demonstrates that calcimimetic B has higher affinity and greater intrinsic efficacy and improved pharmacokinetic properties compared with R-568. These properties taken together may explain how calcimimetic B attenuates PTH secretion at much lower exposure (concentration) levels than R-568. The amount of R-568 needed to maintain adequate PTH suppression seems to be sufficient enough to also target calcitonin secretion. The data we provide suggests that it is plausible that calcimimetics with specific properties could be developed for use in patients with renal disease where it is desirable to lower PTH without causing significant reduction in serum Ca2+ levels (Levin et al., 2007).
Participated in research design: Henley and Reagan.
Conducted experiments: Yang, Davis, Lu, Morony, Fan, Sun, Shatzen, Pretorius, and Reagan.
Contributed new reagents or analytic tools: Fan, Florio, St. Jean, and Fotsch.
Performed data analysis: Henley, Florio, Pretorius, Richards, and Reagan.
Wrote or contributed to the writing of the manuscript: Henley, Richards, and Reagan.
We thank Arthur Christopoulos (Monash University, Melbourne, Australia) for his contribution to the artwork in Fig. 3.
This research was wholly funded by Amgen, Inc.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- calcium-sensing receptor
- human CaSR
- chronic kidney disease
- end-stage renal disease
- fluorescent imaging plate reader
- inositol phosphate
- parathyroid hormone
- secondary hyperparathyroidism
- human embryonic kidney
- Dulbecco's modified Eagle's medium
- dimethyl sulfoxide
- Hanks' balanced salt solution
- bovine serum albumin
- enzyme-linked immunosorbent assay
- extracellular signal-regulated kinase.
- Received December 28, 2010.
- Accepted March 16, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics