The discovery of the Calcium Sensing Receptor led to the development of calcimimetics, which are agents that facilitate the function of the CaSR. In this essay, calcium homeostasis and the structure of the CaSR will be briefly touched to explain the complex interaction between the calcimimetic agent, cinacalcet, and the CaSR in its role in calcium homeostasis. Cinacalcet binds to the TMD of the CaSR, which is distinct from the other highly conserved binding sites, and enhances the affinity of the CaSR for calcium. Cinacalcet's therapeutic value in treating systemic calcium impaired diseases will also be evaluated in this essay, primarily focusing in hyperparathyroidism. The Food and Drug Administration has approved of the use of cinacalcet in treating secondary hyperparathyroidism; however research has shown that it may also have a significant role in treating primary and tertiary hyperparathyroidism. Furthermore, the CaSR has been identified on the pancreatic islets, juxtaglomerular cells, and endothelial cells with no apparent function in systemic calcium regulation. It has been postulated that these CaSR function as mediators that facilitate insulin secretion, manage renin release, and control vascular regulation. This has brought light to the wide range of roles cinacalcet may have beyond regulating calcium homeostasis. However, clinical trials must be conducted regarding these roles beyond calcium homeostasis to truly understand the full impact cinacalcet may have.
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Calcimimetics are inorganic and organic polycations that mimic or potentiate the effects of extracellular Ca2+. These molecules activate the CaRS and inhibit PTH secretion with a leftward shift in the set point for calcium-regulated PTH secretion (Fox et al., 1999). There are two types of calcimimetics- type I and type II. Type I calcimimetics, which act as classic agnoists, are inorganic and organic polycations such as Mg2+ and polyarginine. Type II calcimimetics, which act as allosteric modulators, increase sensitivity of the receptor to extracellular Ca2+. This essay will specifically focus on the type II calcimimetic cinacalcet, as shown in Figure 1, and its role in calcium homeostasis and other processes.
Calcium Homeostasis and the Structure of the Calcium Sensing Receptor
Systemic calcium is controlled and tightly regulated by several organs in the body- the parathyroid and thyroid glands, bone, kidney, and intestine. The calcium sensing receptor (CaSR) is a crucial component in our understanding of calcium homeostasis. CaSR is a G protein-coupled receptor (GPCR) expressed on the cell surface in order to sense the levels of extracellular Ca2+ and activate signaling pathways. In parathyroid cells, the receptor controls the secretion of parathyroid hormone (PTH), which is a hormone that increases extracellular Ca2+. When there is high extracellular Ca2+levels, the CaSR initiates signaling that inhibits PTH secretion. On the other hand, when there is low extracellular Ca2+ levels, the CaSR initiates signaling that increases PTH secretion. As a result, calcium resorption from the bone (Dempster et al., 1993) and calcium reabsorption from the kidney filtrate (Peacock et al., 1969) is stimulated and the level of extracellular Ca2+ is raised.
Not only does CaSR control the secretion of PTH, it also controls the release of calcitonin from the parafolicular C-cells located in the thyroid gland (Garrett et al., 1995). Calcitonin is a hormone that has a reverse role to PTH, which is to decrease extracellular Ca2+. When there is high levels of extracellular Ca2+, the CaSR stimulates the secretion of calcitonin, which then stimulates bone formation and decreases renal calcium readsorption to decrease extracellular Ca2+. Calcium homeostasis is primarily maintained by the inhibition of the release of PTH and the stimulation of the release of calcitonin by the CaSR (Harrington and Fotsch., 2007).
The Structure of and Signaling by the Calcium-Sensing Receptor
The CaSR is a 1078 amino acid glycoprotein with an extracellular N-terminal nutrient-binding Venus Flytrap (VFT) domain; a Cys-rich domain that couples nutrient binding to receptor activation; a 7-transmembrane domain (TMD); and an intracellular C-terminal signaling domain (ICD) that induces intracellular signaling (Brown et al., 2001). It is a promiscuous receptor that provides binding sites for multiple charged molecules in the VFT domain. The binding site for Ca2+ is located in the extra cellular domain (EDC) of the CaSR, which is also known as the orthosteric site (Conigrave et al., 2000). There is multiple Ca2+ binding sites- some of which are truly selective for Ca2+ ions and some of which are activated by multivalent cations, such as Sr2+, Gd3+, polycations, and cation peptides (Handlogten et al., 2000). However, there are also binding sites present on the CaSR that have specificity and selectivity which are distinct from the Ca2+ binding site. The binding sites are found on the TMD, and are present for allosteric modulators.
The Interaction Between Cinacalcet and CaSR in Calcium Homeostasis
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A general model for the binding of allosteric antagonists (calcimimetics and calcilytics) of the CaSR was proposed by Miedlich et al. in 2004 (Miedlich et al., 2004). The calcimimetic and calcilytic were docked in silico with the homology model of the CaSR, and it was speculated that a salt bridge formed between the protonated basic amine of the ligand and Glu-837 located in the TMD VII as a primary interaction with the receptor, as shown in Figure 2. This was further supported by the results obtained from the site-direct mutagenesis of the CaSR (Hu et al., 2002), which showed that both calcimimetics and calcilytics were less active on the Glu to Ala-837 mutant of the CaSR. It was also postulated that both calcimimetics and calcilytics form hydrophobic and Ï€-stacking interactions with Phe-668 and Phe-684 since they are less active on the Ala-668 and Ala-684 CaSR mutants. However, it was suggested that Arg-680 of the CaSR did not significantly interact with calcimimetics whereas it interacted with the hydroxyl group of calcilytics because the Arg to Ala-680 CaSR mutant do not reduce the activity of calcimimetics but reduce the activity of calcilytics. Data obtained from these experiments were used to design a general template for type II calcimimetics such as cinacalcet HCl.
Cinacalcet, a positive allosteric modulator, bind to target sites on the CaSR that are distinct from the highly conserved orthosteric agonist binding sites (Chattopadhyay et al., 1998). Cinacalcet display receptor subtype selectivity by binding in the TMD of the CaSR. Their high specificity for the CaSR is a result of their unique ability to selectively regulate responses specifically in certain tissues in which the endogenous agonist exerts its physiological effects (Christopoulos., 2002).Once bound to the binding sites in the TMD, cinacalcet enhances the affinity of the CaSR for calcium (Miedlich et al., 2004; Petrel et al., 2004). In the presence, but not in the absence of Ca2+o, cinacalcet shifts the effect Ca2+o has on the formation of PTH, Ca2+I, and inositol phoasphate to the left (Nemeth et al., 1998).
Clinical Trials with Cinacalcet
The development of calcimimetics has brought new light to the treatment of extracellular calcium homeostasis disorders. The drug has been used in various experimental studies and clinical trials, which has so far led to the approval by the Food and Drug Administration (FDA) for use in secondary hyperparathyroidism (HPT). Primary HPT is generally caused by an autonomous adenoma of the parathyroid glands or parathyroid carcinoma in rare cases. The excessive secretion of PTH results in an increase of serum ionized calcium. Secondary HPT is typically a response to chronic kidney disease (CKD). The reduced serum calcium levels induces a response in which there is an increase in PTH. Tertiary HPT is a result of untreated secondary HPT or kidney transplantation. Prolonged and persistent stimulus to PTH secretion leads to high PTH levels and hypercalcaemia. The drug cinacalcet has undergone and is currently undergoing clinical trials to become a viable option of treatment for irregular calcium homeostasis.
Cinacalcet in Primary HPT
Primary HPT is characterized as a disease with asymptomatic, milder hypercalcaemia, and high levels of serum PTH. The prevalence is approximately 1-4 per 1000 and the female to male ratio is 3:1 (Melton, 1991). In most cases parathyroidectomy (PTX) is the recommended treatment for primary HPT. However, patients who do not meet the eligibility parameter, as illustrated in Table 1, face an unmet medical need for a non-surgical treatment option.
A potential medical alternative to PTX is the calcimimetic drug, cinacalcet. Its ability to increase the sensitivity of the CaSR to serum calcium and to decrease PTH and calcium levels makes cinacalcet a possible candidate for treatment. Shobak et al. studied the effects of cinacalcet in a randomized, double-blind, dose-finding study of 21 days duration with 22 primary HPT patients (Shoback et al., 2003). There was a significant decrease in serum calcium and an increase in serum phosphorus level through the 15 days of dosing in different groups. The mean reduction was 16% (P=0.004) in serum calcium level, and 8.9 -0.7 mg/dl in (meanSD) serum calcium. There was a larger reduction in serum calcium level when the cinacalcet doses were 40mg and 50mg opposed to 30mg. The second part of the experiment, which studied the safety of cinacalcet, used doses that were approximately one half of the 50-, 75-, and 100-mg once-daily doses of cinacalcet administered twice-daily. A 7 day follow-up period occurred after 15 days of twice daily dosing, reaching the overall indication that cinacalcet formed normalization of serum calcium in primary HPT without adverse effects.
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This was followed with a study that assessed the long-term efficiency and safety of cinacalcet in reducing serum calcium and PTH levels in patients with primary HPT. In this randomized, double blind, placebo-controlled study of 52 weeks duration (n=78), twice daily doses of 30-50mg/d of cinacalcet was administered. Results obtained indicated that after 12 months the serum calcium in 73% of cinacalcet-treated patients was normalized, whereas only 5% of the placebo group's serum calcium normalized (P<0.001). The PTH level dropped by 7.6% which was significantly lower than the placebo group that increased by 7.7% (P<0.01). Bone mineral density did not change in either groups, however an increase was seen in the biochemical markers of bone turnover in the presence of cinacalcet (Peacock et al., 2005). Hence, these studies show that cinacalcet is an effective and relatively safe treatment for primary HPT.
Cinacalcet in Secondary HPT
In most cases, secondary HPT develops early in CKD when the serum calcium and phosphate levels are within normal limits. As the disease progresses, calcitriol production by the kidney decreases, and the serum PTH and phosphate levels increases (Martinez et al., 1997). Several treatment options have been proposed and are used in daily practice, such as dialysis, phosphate binders, calcitriol and other active vitamin D metabolites (Koshimies et al., 1996). The National Kidney Foundation's Kidney Disease Outcomes Quality Initiative (NKF-K/DOQI) set guidelines for the treatment of secondary HPT. The recommended target levels of serum PTH, calcium, phosphate, and calciumÃ-phosphate product is expected to be the key contributor in reducing mortality, hospitalization, fracture, and parathyroidectomy in secondary HPT patients (National Kidney Foundation., 2003).
Block et al. studied the safety and efficiency of cinacalcet in a randomized, double-blind, placebo-controlled study (n=741, duration=26 weeks) with patients on maintenance dialysis and secondary HPT (PTH>300 pg m/L). Treatment with cinacalcet was significantly more effective in lowering serum PTH than placebo (-43% vs. +9% respectively, P<0.001), as well as serum calcium (-6.8% vs. +0.4%, P<0.001), including serum phosphate (-8.4% vs. +0.2&, P<0.001), and calciumÃ-phosphate product (-14.6% vs. +0.5%, P<0.001) (Block et al., 2004). The one adverse effect found more commonly with cinacalcet treated patients than placebo patients was nausea and vomiting, however they were mild and limited to single episodes. Similar results were obtained by Lindberg et al. in another phase 3, multicenter, randomized, placebo-controlled, double-blind study in dialysis patients with secondary PTH300 pg m/L despite traditional therapy (Lindberg et al., 2005).
Furthermore, an experiment was conducted to analyze the effect of cinacalcet on the four parameters of the recommended NKF-K/DOQI target levels. In a pooled analysis of data from three placebo-controlled, double-blind, randomized study, patients on dialysis and HPT secondary to CKD received traditional therapy plus cinacalcet or placebo (n=1136, duration=26 weeks). Data from this experiment showed that significantly more patients achieved the recommended NKF-K/DOQI target level for all parameters with cinacalcet (P<0.001 for all) than to standard therapy which consisted of phosphate binders and vitamin D sterols, as shown in Figure 3 (Moe et al., 2005). Thus, the collection of consistent data indicating achievement of NKF-K/DOQI targets with cinacalcet lead to the approval of AMG073 (cinacalcet) for use in secondary HPT by the FDA.
Cinacalcet in Tertiary HPT
Often metabolic imbalances persist after kidney transplantation. Statistics show that one year after transplantation 50% of the patients have incomplete resolution of HPT (Lobo et al., 1995); over 2.5 years after transplantation only 23% of patients have normal level of PTH and intact renal transplant function (Torres et al., 1998); and 3 months after transplantation more than 50% of renal transplant recipients have hypercalcaemia (Reinhardt et al., 1998).
Serra et al. investigated the effect of cinacalcet in tertiary HPT in 11 kidney allograft recipients in a prospective open label study for up to 10 weeks. The patients were administered different doses of cinacalcet to achieve and maintain serum calcium levels within the target range of 8.4-10.4 mg d/L (2.1-2.6 mmol/L). Data obtained indicated a fast and sustained normalization of serum calcium from 2.730.05mmol/L to 2.440.05mmol/L and 2.420.04mmol/L (9.76 and 9.68 mg d/L) after 2 and 10 weeks, respectively, in all patients treated with cinacalcet. There was an increase in serum phosphate, and there was no change in the calciumphosphate product. Serum PTH significantly decreased at weeks 2 and 10 by 16.1% and 21.8% respectively. Throughout the study, kidney function was stable, there was no rejection of allograft, and cyclosporine levels remained unchanged. 30 mg of cinacalcet daily successfully treated the majority of the patients (Serra et al., 2005).
In addition, Serra et al. confirmed these short-term results by recently publishing an extension focusing on the sustainability over six months (Serra et al., 2007). Similar results were obtained by Kruse et al. in 2005, Leca et al. in 2006, and Kruse et al. in 2007 on the effects of cinacalcet in patients with normal kidney allograft function and hypercalcaemia (Kruse et al. 2005, Leca et al. 2006, Kruse et al. 2007). Therefore, in the case of kidney allograft recipients with tertiary HPT and hypercalcaemia, cinacalcet is a potential treatment to correct abnormal calcium homeostasis. However, ongoing clinical trials in these patients are necessary to determine the appropriate treatment duration, long-term efficiency, and safety regarding cinacalcet.
The Effect of Cinacalcet Beyond Calcium Homeostasis
The discovery of CaSR and the development of calcimimetics have brought light to calcium homeostasis and new therapeutic opportunities for HPT. However, the CaSR is also involved in other cellular processes, as shown in Figure 4, and the application of calcimimetics may not be exclusively for calcium regulation only. It has been suggested that the calcimimetic, cinacalcet, could also play a role in insulin secretion, rennin release, and vascular regulation.
The CaSR expressed on the pancreatic islets of Langerhans is highly unlikely to be involved in calcium homeostasis. The function of the CaSR in the pancreatic islet, where insulin secretion provides the basis of energy regulation, is not fully understood. However, it is commonly suggested that it has an important function of mediating cell-to-cell communication to facilitate insulin secretory responses (Jones et al., 2007). Studies suggest that neighboring cells expressing the CaSR are recruited by the extrusion of calcium from stimulated cells, which allow amplification and integration of a tissue-wide response, as shown in Figure 5 (Hofer et al., 2004). It has also been suggested that the CaSR plays a role in cell adhesion and proliferation in the pancreatic islet. A study on human epidermal keratinocytes suggests that the inactivation of the CaSR suppresses the assembly of the ECAD-catenin-phosphotidylinositol3-kinase complex (P13K), which is a complex of specialized cell adhesion molecules that form the architecture of the islet (Tue et al., 2008). Hence, the CaSR is thought to influence several functions that regulate calcium activity between Î²-cells, which in turn has an effect on insulin secretion.
Since the CaSR is proposed to have a role in insulin secretion, calcimimetics such as cinacalcet could possibly play a part in activating the CaSR to enhance insulin secretion from human islets. Nemeth and Fox studied the effect of calcimimetics on the activation of CaSR, and stated that it does not directly activate the receptor but acts to sensitize it to Ca2+ (Nemeth and Fox, 1999). The presence of Ca2+ is crucial for the calcimimetic to induce an increase in secretory response. The magnitude of insulin response is dependent more heavily on the concentration of calcium than of the calcimimetic. Research by Straub et al. in 2000 and Gray et al. in 2006 is consistent with the observation that calcimimetics could further enhance the maximal insulin secretion in pancreatic islets. Gray et al. further discovered that calcimimetic activation of the CaSR in human and rodent Î²-cell increased insulin secretion without the need for an associated increase in nutrient stimulation (Gray et al. 2006). These studies indicate that calcimimetic therapies, such as cinacalcet, may have a wider purpose beyond calcium regulation, and more research is necessary to fully explore the therapeutic potential of cinacalcet in diseases impairing insulin secretion.
The CaSR is also found on the juxtaglomerular (JG) cell in the glomerulus, which releases renin. Renin is the rate-limiting enzymatic step in the production of angiotensin, and integrates cardiovascular and renal function in the control of blood pressure, salt, and volume homeostasis (Persson, 2003). Two main intracellular second messenger systems, the cyclic nucleotide, cyclic adenosine monophosphate (cAMP) and intracellular calcium, regulate renin synthesis and secretion (Schweda and Kurtz, 2004). When the CaSR is stimulated, it decreases in cAMP formation and inhibits renin release.
An experiment was conducted by Ortiz-Capisano et al. in 2009 to investigate whether CaSR activation by cinacalcet decreases cAMP and renin release, in part, by stimulating a calcium calmodulin-activated phosphodiesterase 1 (PDE 1). Stimulation of the JG cell CaSR with cinacalcet resulted in a decrease in cAMP from a basal of 1.130.14 to 0.690.08 pM/mg protein (P<0.001) and a decrease in renin release from a basal of 0.890.16 to 0.380.08 Î¼g ANG I/mlÎ‡h-1Î‡mg protein-1 (P<0.001), as shown in Figure 6 (Ortiz-Capisano et al., 2009). This data suggests that cinacalcet could have beneficial effects on renal disease progression by interfering with the renin-angiotensin system, but more studies must be conducted to investigate the potential benefits of cinacalcet in high renin models.
The CaSR has also been found in endothelial cells and possibly in vascular smooth muscle cells and adventitial cells in experimental animal models. The CaSR is speculated to have the function of regulating the myogenic tone in rat subcutaneous arteries when activated in ex vivo experiments. Treatment with cinacalcet led to a concentration dependent vasodilation of isolated pre-contracted aortae (Ohanian et al., 2005). Furthermore, the effects of cinacalcet on selected clinical outcomes was performed on a retrospective pooled analysis of safety data from four randomized, placebo-controlled, double-blind clinical trials with patients with ESRD and uncontrolled secondary HPT (daily cinacalcet dose = 30-180 mg, n= 1184, intact PTH300 pg m/L). As shown in Figure 7, there was a significant reduction of 39% in cardiovascular hospitalization with cinacalcet (P=0.005 vs. placebo) (Cunningham et al., 2005). However, there were no significant reductions in all-cause hospitalizations and all-cause mortality (+3% and -19% respectively, P=n.s. for both).
Currently, there are two ongoing major trials investigating the effects of cinacalcet on cardiovascular outcomes. The Evaluation Of Cincalcet HCL Therapy to Lower Cardio Vascular Events (EVOVLE) is a randomized, double- blind, placebo-controlled trial underway in 422 locations globally. It aims to study the effects of cinacalcet on cardiovascular events and death in 3883 patients with CKD and secondary HPT receiving maintenance hemodialysis. The other trial is the ADVANCE trial that is a randomized open-label clinical trial that aims to evaluate the effect of cinacalcet and low-dose vitamin D on the progression of coronary artery calcification scores. 330 patients with CKD receiving hemodialysis with base-line coronary artery calcification score of at least 30 are involved in this trial (Al-Aly, 2010). Although these two trials are underway at the moment, more clinical trials must be conducted in order to determine the beneficial pleitropic or harmful off-target effects of cinacalcet on vascular conditions. With further research, there may be a potential translational application of cinacalcet to vascular health in the far future.
In conclusion, the calcimimetic agent cinacalcet has a prominent role in the control of systemic calcium. It's unique ability to shift the effect of Ca2+o on the formation of PTH, Ca2+I, and inositol phoasphate to the left in order to increase the sensitivity of the CaSR to serum calcium and to decrease PTH and calcium levels makes cinacalcet a possible therapeutic treatment for various diseases linked to calcium homeostasis disorder. The FDA has approved its role in the treatment of secondary HPT, however it may also be a viable option of treatment for primary and tertiary HPT. Research by Shoback et al., Peacock et al., Serra et al. and such suggests that cinacalcet may have a significant non-surgical therapeutic role in disease related to calcium homeostasis other than secondary HPT.
Not only does cinacalcet have the potential of treating systemic calcium impaired diseases, it may also have a role in treating insulin secretion, renin release, and vascular regulation. The CaSR has been found on the pancreatic islets, the JG cells in the glomerulus, and endothelial cells with no apparent function in systemic calcium regulation. It has been postulated that the CaSR expressed on the pancreatic islets facilitate insulin secretion, the CaSR expressed on the JG cells manage renin release, and the CaSR expressed on endothelial cells control vascular regulation. Studies by Gray et al., Oritz-Capisano et al., Ohanian et al., and such suggest that cinacalcet may have a therapeutic role beyond regulating calcium homeostasis. Nevertheless, more clinical trials must be performed to reach solid conclusions on the full effect cinacalcet has on these areas. The FDA should not only primarily focus on the use of cinacalcet in systemic calcium regulation but its use in other various areas.