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Takashi Shigematsu,1 Shinji Asada,2 Yuichi Endo,3 Takehisa Kawata,2,3 Masafumi Fukagawa,4 Tadao Akizawa5 1Department of Nephrology, Wakayama Medical University, Wakayama, Japan; 2Medical Affairs Department, Kyowa Kirin Co., Ltd., Tokyo, Japan; 3R&D Division, Kyowa Kirin Co., Ltd., Tokyo, Japan; 4Division of Nephrology, Endocrinology, and Metabolism, Department of Internal Medicine, Tokai University School of Medicine, Kanagawa, Japan; 5Division of Nephrology, Department of Medicine, Showa University School of Medicine, Tokyo, JapanCorrespondence: Takashi ShigematsuDepartment of Nephrology, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-8509, JapanTel +81-73-441-0638Fax +81-73-441-0639Email taki@wakayama-med.ac.jpPurpose: Evocalcet is a novel oral calcimimetic drug that has demonstrated similar efficacy to cinacalcet in regulating serum parathyroid hormone (PTH), calcium, and phosphate levels, with fewer upper gastrointestinal tract-related adverse drug reactions (ADRs) in patients with secondary hyperparathyroidism undergoing hemodialysis in Japan. We investigated the efficacy and safety of once-daily oral evocalcet under different dialysate calcium concentrations.Patients and Methods: A posthoc analysis by dialysate calcium concentration (2.5, 2.75, and 3.0 mEq/L) was performed using data from a previous Phase 3 study that included cinacalcet as an active control. Efficacy endpoints were the proportion of patients who achieved the target intact PTH levels of ≥ 60 and ≤ 240 pg/mL between Week 28 and Week 30; time-course changes in serum intact PTH; calcium and phosphorus levels, bone turnover markers, and fibroblast growth factor 23 (FGF23) over the 30-week study period. Safety endpoints were overall ADRs and hypocalcemia- and upper gastrointestinal tract-related ADRs.Results: A total of 634 patients were included in the analysis. Levels of intact PTH, calcium, phosphate, bone turnover markers, and FGF23 showed improvement in all sub-groups, irrespective of dialysate calcium concentration. The incidence of upper gastrointestinal tract-related ADRs was significantly lower in the evocalcet group than thecinacalcet group with dialysate calcium concentrations of 2.75 and 3.0 mEq/L (p< 0.05 for both concentrations).Conclusion: Evocalcet was effective and safe in regulating the levels of serum intact PTH, calcium, and phosphate in patients with secondary hyperparathyroidism undergoing hemodialysis, irrespective of dialysate calcium concentration.Keywords: hypocalcemia, intact parathyroid hormone, oral calcimimetic, posthoc analysis, upper gastrointestinal tract

To elucidate the effect of evocalcet, a new oral calcimimetic to bone of secondary hyperparathyroidism (SHPT) with chronic kidney disease (CKD), the rats were 5/6 nephrectomized and fed on a high-phosphate diet. The treated rats were then divided into vehicle groups and evocalcet administered groups. The rats in the vehicle groups exhibited increased levels of serum PTH and inorganic phosphate (Pi) levels, high bone turnover, and severe cortical porosity, mimicking SHPT (CKD-SHPT rats). The cortical bone of the CKD-SHPT rats showed broad demineralization around the osteocytes, suppression of Phex/small integrin-binding ligand N-linked glycoprotein-mediated mineralization in the periphery of the osteocytic lacunae, and increased levels of osteocytic cell death, all of which were considered as the first steps of cortical porosity. In contrast, evocalcet ameliorated the increased serum PTH levels, the enlarged osteocytic lacunae, and the cortical porosity of the CKD-SHPT rats. Osteocytes of CKD-SHPT rats strongly expressed PTH receptor and Pit1/Pit2, which sense extracellular Pi, indicating that PTH and Pi affected these osteocytes. Cell death of cultured osteocytes increased in a Pi concentration-dependent manner, and PTH administration rapidly elevated Pit1 expression and enhanced osteocytic death, indicating the possibility that the highly concentrated serum PTH and Pi cause severe perilacunar osteolysis and osteocytic cell death. It is likely therefore that evocalcet not only decreases serum PTH but also reduces the exacerbation combined with PTH and Pi to the demineralization of osteocytic lacunae and osteocytic cell death, thereby protecting cortical porosity in CKD-SHPT rats.

Calcimimetics allosterically activate the calcium receptor (CaR) and inhibit the secretion of parathyroid hormone (PTH). Cinacalcet hydrochloride (cinacalcet) has been approved as the first calcimimetic drug for the treatment of secondary hyperparathyroidism (SHPT) in patients with hemodialysis. Cinacalcet improved the achievement of target serum PTH and Ca levels and helped drastically reduce the number of parathyroidectomies. However, cinacalcet has side effects involving the gastrointestinal tract, such as nausea and vomiting, which makes it difficult to increase the dose and may result in reduced compliance. Evocalcet has been developed to improve defects of cinacalcet for management of SHPT. Evocalcet acts as an allosteric modulator of CaR, just like cinacalcet. However, its metabolic pathway is different from that of cinacalcet. The metabolism of evocalcet by cytochrome P450 is very low, so evocalcet has higher bioavailability. As a result, its pharmacologically effective dose for the inhibition of PTH secretion is lower than that of cinacalcet. Evocalcet had less of an effect on the gastrointestinal tract than cinacalcet because of the reduced dose required. In a clinical trial with a randomized, double-blind, head-to-head comparison study, it was also confirmed that the incidence of gastrointestinal-related adverse events was lower in the evocalcet group than in the cinacalcet group. Evocalcet may thus be a potent option for the management of SHPT.

This ad hoc analysis of a previously conducted phase 3 head-to-head comparison study of evocalcet and cinacalcet in secondary hyperparathyroidism patients undergoing maintenance hemodialysis evaluated the efficacy and safety of combined once-daily oral evocalcet and intravenous vitamin D receptor activator treatment stratified by weekly vitamin D receptor activator dose (117, 45, and 91 patients in no, low [< 1.5 μg], and high [≥ 1.5 μg] dose groups, respectively). Effects of vitamin D receptor activator were assessed on the basis of intact parathyroid hormone, corrected calcium, phosphorus, and fibroblast growth factor-23 levels; percent changes from baseline; proportions of patients who achieved target intact parathyroid hormone, corrected calcium, and phosphorus at Weeks 28–30; and adverse drug reactions. Intact parathyroid hormone, corrected calcium, phosphorus, and fibroblast growth factor-23 levels decreased in all groups; phosphorus and fibroblast growth factor-23 levels remained high in the high dose group. In the low and high dose groups, greater proportions of patients achieved the corrected calcium target compared with the no dose group (p = 0.043). Ratios of intact-to-C-terminal fibroblast growth factor-23 decreased in all groups. In low and high dose groups, hypocalcemia was less common than in the no dose group (p = 0.014). Evocalcet with concomitant vitamin D receptor activator demonstrated benefits such that more patients achieved the corrected calcium target and exhibited decreased fibroblast growth factor-23 synthesis; the incidence of hypocalcemia also decreased. Clinical trial registration: ClinicalTrials.gov (NCT02549391) and JAPIC (JapicCTI-153013).

The calcium-sensing receptor is an important treatment target for secondary hyperparathyroidism (SHPT) in patients undergoing dialysis. In addition to vitamin D receptor activator, cinacalcet has recently been widely used for SHPT management, and the significant suppression of parathyroid hormone (PTH) with better control of serum calcium and phosphorus has been reported. However, low adherence and insufficient dose escalation mainly due to frequent gastrointestinal adverse events, still remain as major issues. To overcome these unmet needs, we have developed a new oral calcimimetic agent evocalcet, which has recently been approved by the Pharmaceutical Affairs Act in Japan.PubMed was searched from inception until April 2020 with the word evocalcet to summarize the development of this new calcimimetic agent, its pharmacokinetics, and the results of clinical trials, along with an overview of the differences among calcimimetic agents. This review also includes the management of SHPT with a focus on calcimimetics.Evocalcet evoked fewer gastrointestinal-related adverse events while suppressing PTH at a lower dose than cinacalcet. These data suggest evocalcet may contribute to better adherence and sufficient dose escalation in patients with SHPT. Whether or not evocalcet improves clinical outcomes remains to be elucidated.

Calcimimetics represent a class of pharmacological agents that activate the extracellular calcium receptor. The receptor is expressed at high levels in the parathyroid glands, thyroidal C-cells, and the kidney. Calcimimetics can suppress parathyroid functions and decrease the circulating levels of parathyroid hormone. Because of these pharmacological effects, calcimimetics can control abnormalities of the parathyroid glands in hyperparathyroidism. The emergence of calcimimetics has advanced clinical practice to manage hyperparathyroidism and related hypercalcemia. This article summarizes the basic characteristics of calcimimetics and then specifically refers to three calcimimetics used clinically: cinacalcet hydrochloride, evocalcet, and etelcalcetide hydrochloride.

The calcium-sensing receptor (CaSR) plays an important role in sensing extracellular calcium ions and regulating parathyroid hormone secretion by parathyroid gland cells, and the receptor is a suitable target for the treatment of hyperparathyroidism. Cinacalcet hydrochloride is a representative CaSR agonist which widely used for the hyperparathyroidism. However, it has several issues to clinical use, such as nausea/vomiting and strong inhibition of CYP2D6. We tried to improve these issues of cinacalcet for a new pharmaceutical agent as a preferable CaSR agonist. Optimization from cinacalcet resulted in the identification of pyrrolidine compounds and successfully led to the discovery of evocalcet as an oral allosteric CaSR agonist. Evocalcet, which exhibited highly favorable profiles such as CaSR agonistic activity and good DMPK profiles, will provide a novel therapeutic option for secondary hyperparathyroidism.

Calcimimetics allosterically activate the calcium receptor (CaR) and inhibit the secretion of parathyroid hormone (PTH). Cinacalcet hydrochloride (cinacalcet) has been approved as the first calcimimetic drug for the treatment of secondary hyperparathyroidism (SHPT) in patients with hemodialysis. Cinacalcet improved the achievement of target serum PTH and Ca levels and helped drastically reduce the number of parathyroidectomies. However, cinacalcet has side effects involving the gastrointestinal tract, such as nausea and vomiting, which makes it difficult to increase the dose and may result in reduced compliance. Evocalcet has been developed to improve defects of cinacalcet for management of SHPT. Evocalcet acts as an allosteric modulator of CaR, just like cinacalcet. However, its metabolic pathway is different from that of cinacalcet. The metabolism of evocalcet by cytochrome P450 is very low, so evocalcet has higher bioavailability. As a result, its pharmacologically effective dose for the inhibition of PTH secretion is lower than that of cinacalcet. Evocalcet had less of an effect on the gastrointestinal tract than cinacalcet because of the reduced dose required. In a clinical trial with a randomized, double-blind, head-to-head comparison study, it was also confirmed that the incidence of gastrointestinal-related adverse events was lower in the evocalcet group than in the cinacalcet group. Evocalcet may thus be a potent option for the management of SHPT.

Evocalcet is a novel calcimimetic agent with fewer gastrointestinal (GI) adverse effects compared to cinacalcet. Although it is thought that cinacalcet induces GI side effects through the direct stimulation of the calcium receptor (CaR) expressed in the GI tract, the differences in the direct stimulatory effects of these two drugs on the GI tract have not been reported. In this study, we analyzed the difference in the GI effects of these two calcimimetic agents using miniature pigs by detecting vagus nerve stimulation after oral administration of the agents. Although cinacalcet induced vomiting in miniature pigs, evocalcet never induced emetic symptoms. A significant increase in the vagus nerve action potentials was observed after the administration of cinacalcet. Although the increase of that after the administration of evocalcet was mild and not significant in comparison to that in the vehicle group, it was not significantly different from the vagus nerve action potentials after cinacalcet treatment.

Background Elevated parathyroid hormone (PTH) levels in secondary hyperparathyroidism (SHPT) lead to vascular calcification, which is associated with cardiovascular events and mortality. Increased PTH production is caused by the excessive proliferation of parathyroid gland cells, which is accelerated by abnormal mineral homeostasis. Evocalcet, an oral calcimimetic agent, inhibits the secretion of PTH from parathyroid gland cells and has been used for the management of SHPT in dialysis patients. We observed the effects of evocalcet on ectopic calcification and parathyroid hyperplasia using chronic kidney disease (CKD) rats with SHPT. Methods CKD rats with SHPT induced by adenine received evocalcet orally for 5 weeks. The calcium and inorganic phosphorus content in the aorta, heart and kidney was measured. Ectopic calcified tissues were also assessed histologically. To observe the effects on the proliferation of parathyroid gland cells, parathyroid glands were histologically assessed in CKD rats with SHPT induced by 5/6 nephrectomy (Nx) after receiving evocalcet orally for 4 weeks. Results Evocalcet prevented the increase in calcium and inorganic phosphorus content in the ectopic tissues and suppressed calcification of the aorta, heart and kidney in CKD rats with SHPT by reducing the serum PTH and calcium levels. Evocalcet suppressed the parathyroid gland cell proliferation and reduced the sizes of parathyroid cells in CKD rats with SHPT. Conclusions These findings suggest that evocalcet would prevent ectopic calcification and suppress parathyroid hyperplasia in patients with SHPT.

Cinacalcet HCl (cinacalcet) is a calcimimetic compound, which suppresses parathyroid (PTH) hormone secretion from parathyroid glands in both primary hyperparathyroidism (PHPT) and secondary hyperparathyroidism (SHPT). We previously reported the suppressive effect of cinacalcet on PTH secretion in vivo in a PHPT model mouse, in which parathyroid-targeted overexpression of the cyclin D1 oncogene caused chronic biochemical hyperparathyroidism and parathyroid cell hyperplasia. Although cinacalcet suppressed parathyroid cell proliferation in SHPT in 5/6-nephrectomized uremic rats, its effect on PHPT has not yet been determined. In this study, the effect of cinacalcet on parathyroid cell proliferation was analyzed in PHPT mice. Cinacalcet (1 mg/g) was mixed into the rodent diet and orally administrated to 80-week-old PHPT mice for 10 days before death. 5-Bromo-2'-deoxyuridine (BrdU, 6 mg/day) was infused by an osmotic pump for 5 days before death, followed by immunostaining of the thyroid-parathyroid complex using an anti-BrdU antibody to estimate parathyroid cell proliferation. Compared to untreated PHPT mice, cinacalcet significantly suppressed both serum calcium and PTH. The proportion of BrdU-positive cells to the total cell number in the parathyroid glands increased considerably in untreated PHPT mice (9.5 ± 3.1%) compared to wild-type mice (0.7 ± 0.1%) and was significantly suppressed by cinacalcet (1.2 ± 0.2%). Cinacalcet did not affect apoptosis in the parathyroid cells of PHPT mice. These data suggest that cinacalcet suppressed both serum PTH levels and parathyroid cell proliferation in vivo in PHPT.

Cinacalcet hydrochloride (cinacalcet), an oral calcimimetic agent has been widely used for the management of secondary hyperparathyroidism (SHPT) in chronic kidney disease (CKD). In sharp contrast to vitamin D receptor activators, cinacalcet suppresses SHPT without inducing hypercalcemia or hyperphosphatemia. Nevertheless, some patients remain refractory to SHPT with this agent, as the dose cannot be sufficiently increased due to gastrointestinal symptoms. In order to resolve this issue, we have developed a newly synthesized calcimimetic agent, evocalcet (MT-4580/KHK7580). In a rat model of CKD induced by 5/6 nephrectomy, oral administration of evocalcet efficiently suppressed the secretion of parathyroid hormone (PTH). With regard to the gastro-intestinal effects, cinacalcet induced a significant delay in gastric emptying in rats, while evocalcet did no marked effects on it. Evocalcet also demonstrated the less induction of emesis compared to cinacalcet in common marmosets. The pharmacological effects of evocalcet were observed at lower doses because of its higher bioavailability than cinacalcet, which may have contributed to the reduced GI tract symptoms. In addition, evocalcet showed no substantial direct inhibition of any CYP isozymes in in vitro liver microsome assay, suggesting a better profile in drug interactions than cinacalcet that inhibits cytochrome P450 (CYP) 2D6. These findings suggest that evocalcet can be a better alternative to cinacalcet, an oral calcimimetic agent, with a wider safety margin.

X-linked hypophosphatemia (XLH), characterized by renal phosphate wasting, is the most common cause of vitamin D-resistant rickets. It has been postulated that some phosphaturic factor plays a causative role in XLH and its murine homolog, the Hyp mouse. Fibroblast growth factor 23 (FGF23) is a physiological phosphaturic factor; its circulatory level is known to be high in most patients with XLH and Hyp mice, suggesting its pathophysiological role in this disease. To test this hypothesis, we treated Hyp mice with anti-FGF23 antibodies to inhibit endogenous FGF23 action. A single injection of the antibodies corrected the hypophosphatemia and inappropriately normal serum 1,25-dihydroxyvitamin D. These effects were accompanied by increased expressions of type IIa sodium-phosphate cotransporter and 25-hydroxyvitamin-D-1alpha-hydroxylase and a suppressed expression of 24-hydroxylase in the kidney. Repeated injections during the growth period ameliorated the rachitic bone phenotypes typically observed in Hyp mice, such as impaired longitudinal elongation, defective mineralization, and abnormal cartilage development. Thus, these results indicate that excess actions of FGF23 underlie hypophosphatemic rickets in Hyp mice and suggest a novel therapeutic potential of the FGF23 antibodies for XLH.

細胞外液中のカルシウムイオン（Ca2+）濃度（［Ca2+］e）のわずか数%の変化を副甲状腺が鋭敏に感知し，副甲状腺ホルモン（PTH）分泌が速やかに変動する．その結果，腎，骨，腸管でのCa出納が制御され，［Ca2+］eの恒常性が維持されている．この副甲状腺が有する［Ca2+］eの感知機構の本体として，1993年にクローニングされたのがカルシウム受容体（CaR）であり，7回膜貫通Gタンパク共役型受容体（GPCR）の構造をとる．副甲状腺細胞表面上のCaRにポジティブ アロステリック モジュレーター（positive allosteric modulator）として作用し，［Ca2+］eが上昇した場合と同様に，PTH分泌を強力に抑制する薬理作用を有する化合物をCaR作動薬（calcimimetics）と称する．シナカルセト塩酸塩（シナカルセト）は，現在，臨床使用されている唯一のcalcimimeticsであり，慢性腎不全の合併症の一つで，過剰なPTH分泌の亢進状態で特徴付けられる二次性副甲状腺機能亢進症（2HPT）の治療薬として開発された．2HPTを呈する慢性腎不全ラットを用いた薬効薬理試験において，血中PTH低下，Ca低下，副甲状腺細胞増殖抑制，線維性骨炎発症抑制，骨密度・骨強度低下抑制，血管石灰化発症抑制などの薬理作用が認められている．また，透析施行中の2HPT患者において，血中PTH低下，Ca低下，リン（P）低下，骨代謝マーカー低下といったミネラル代謝異常に対する改善作用のみならず，生活の質（QOL）の改善や心血管系疾患が原因での入院イベントの発生低下などの有用性も認められている．シナカルセトは，維持透析下の2HPTを適応症として，本邦では2007年10月に製造販売承認を受け，2008年1月より，レグパラ®錠（25 mg・75 mg）として臨床使用されている．その優れた血中PTH低下作用に加えて，既存の2HPT治療薬であるビタミンD（VD）製剤の副作用（血中Ca，P上昇作用）とは相反する作用を有するという点で，2HPT治療のいわば特効薬として期待されている．また，薬理学的見地からも，無機イオンであるCa2+を内因性リガンドとする受容体を標的とする点や，GPCRに対する世界初のアロステリック モジュレーターであるという点において，特筆すべき薬剤であるといえる．

Clinical studies have been performed to determine the effect of cinacalcet HCl (cinacalcet), an allosteric modulator of the calcium-sensing receptor (CaR), on primary hyperparathyroidism (PHPT) and secondary hyperparathyroidism of uremia (SHPT). However, no in vitro studies on human parathyroid cells have been reported to date. In this study, the inhibitory effect of cinacalcet on PTH secretion was analyzed in primary cultured parathyroid cells obtained from patients. The investigation involved three PHPT and three SHPT patients subjected to therapeutic parathyroidectomy. Notably, all SHPT patients were resistant to intravenous vitamin D analogue therapy. Removed parathyroid tumors were used for immunohistochemistry and parathyroid cell primary culture. Immunohistochemical analyses revealed diminished expression of CaR and vitamin D receptor (VDR) in all parathyroid tumors. PTH secretion from cultured parathyroid cells of PHPT and SHPT patients was suppressed by extracellular Ca2+ and cinacalcet in a dose-dependent manner. Rates of suppression of PTH secretion in PHPT and SHPT by cinacalcet (1000 nmol/l) were 61% +/- 21% and 61% +/- 19%, respectively. Cinacalcet demonstrates significant potency in the suppression of PTH secretion in primary cultured human parathyroid cells in vitro, despite reduced levels of the target protein, CaR. Data from this in vitro analysis support the clinical application of cinacalcet in PHPT and SHPT therapy.

Fibrous dysplasia (FD) patients sometimes suffer from concomitant hypophosphatemic rickets/osteomalacia, resulting from renal phosphate wasting. It was recently reported that FD tissue in the patients with McCune-Albright syndrome (MAS) expressed fibroblast growth factor-23 (FGF-23), which is now known to be as a pathogenic phosphaturic factor in patients with oncogenic osteomalacia and X-linked hypophosphatemic rickets. Since it remains controversial whether serum phosphate levels are influenced by FGF23 expressions in FD tissue, isolated FD patients without MAS syndrome were examined for the relationship between FGF23 expressions, circulating levels of FGF-23 and phosphate to negate the effects of MAS-associated endocrine abnormalities on serum phosphate. Eighteen paraffin embedded FD tissues and 2 frozen tissues were obtained for the study. Sixteen of 18 isolated FD tissues were successfully analyzed GNAS gene, which exhibited activated mutations observed in MAS. Eight of 16 FD tissues, which exhibited GNAS mutations, revealed positive staining for FGF-23. These evidence indicate that postzygotic activated mutations of GNAS is necessary for the FD tissue formation by mosaic distribution of mutated osteogenic cell lineage, but is not sufficient to elevate FGF23 expression causing generalized osteomalacia with severe renal phosphate wasting. The expression level of FGF23 in isolated FD tissue with hypophosphatemic osteomalacia determined by real-time PCR was abundant close to the levels in OOM tumors. Osteoblasts/osteocytes in woven bone were predominant source of circulating FGF-23 in FD tissues by immunohistochemistry. A negative correlation of the intensity of FGF-23 staining with serum inorganic phosphate levels indicated that the expression of FGF23 in focal FD tissues could be a prominent determinant of serum phosphate levels in isolated FD patient. These data provide novel insights into the regulatory mechanism of serum inorganic phosphate levels in isolated FD patients and extend the notion that FGF-23 originating from FD tissue may cause hypophosphatemia not only in isolated FD patients but also in the patients with MAS syndrome.

Although serum calcium (Ca2+) concentration regulates the generation of amino-terminally (N-terminally) truncated forms of human PTH (hPTH) degraded from (1-84)hPTH, no studies have yet reported whether the parathyroid gland itself is responsible for this process.Our objective was to determine the site of N-terminal truncation and its roles in PTH metabolism in parathyroid cells in vitro.The effect of extracellular Ca2+ concentration was examined on N-terminal truncation in primary cultured parathyroid cells. The parathyroid glands were obtained from the patients with primary and uremia-associated secondary hyperparathyroidisms who underwent therapeutic parathyroidectomies.The N-terminally truncated fragments were detectable with commercially available intact PTH (I-PTH) assays, but not with the bio-intact PTH (Bio-PTH) assay, which detected only the (1-84)hPTH. HPLC revealed that generation of N-terminally truncated fragments detectable by I-PTH increased with extracellular Ca2+ concentration. Suppression of PTH secretion by increasing the extracellular Ca2+ concentration was more evident with the Bio-PTH assay than with the I-PTH assay for both cultured parathyroid cells prepared from parathyroid adenomas and uremia-associated secondary hyperparathyroidism. The Bio-PTH/I-PTH ratio, which is the ratio of (1-84)hPTH to the sum of (1-84)hPTH and N-terminally truncated fragments, decreased in response to increases in extracellular Ca2+.These findings suggest that the N-terminal truncation is regulated by extracellular Ca2+ concentration and works to suppress the generation of (1-84)hPTH in parathyroid cells.

Cinacalcet HCl, an allosteric modulator of the calcium-sensing receptor (CaR), has recently been approved for the treatment of secondary hyperparathyroidism in patients with chronic kidney disease on dialysis, due to its suppressive effect on parathyroid hormone (PTH) secretion. Although cinacalcet’s effects in patients with primary and secondary hyperparathyroidism have been reported, the crucial relationship between the effect of calcimimetics and CaR expression on the parathyroid glands requires better understanding. To investigate its suppressive effect on PTH secretion in primary hyperparathyroidism, in which hypercalcemia may already have stimulated considerable CaR activity, we investigated the effect of cinacalcet HCl on PTH-cyclin D1transgenic mice (PC2 mice), a model of primary hyperparathyroidism with hypo-expression of CaR on their parathyroid glands. A single administration of 30 mg/kg body weight (BW) of cinacalcet HCl significantly suppressed serum calcium (Ca) levels 2 h after administration in 65- to 85-week-old PC2 mice with chronic biochemical hyperparathyroidism. The percentage reduction in serum PTH was significantly correlated with CaR hypo-expression in the parathyroid glands. In older PC2 mice (93–99 weeks old) with advanced hyperparathyroidism, serum Ca and PTH levels were not suppressed by 30 mg cinacalcet HCl/kg. However, serum Ca and PTH levels were significantly suppressed by 100 mg/kg of cinacalcet HCl, suggesting that higher doses of this compound could overcome severe hyperparathyroidism. To conclude, cinacalcet HCl demonstrated potency in a murine model of primary hyperparathyroidism in spite of any presumed endogenous CaR activation by hypercalcemia and hypo-expression of CaR in the parathyroid glands.

Disorders of mineral and bone metabolism have been implicated as a risk factor in the high mortality in patients with chronic kidney disease (CKD). Hyperphosphatemia, disorders of vitamin D metabolism and secondary hyperparathyroidism of uremia (SHPT) are therapeutic targets in these patients to improve the mortality. Animal models for CKD are indispensable and uremic rats produced by 5/6-nephrectomies are one of the most useful animal models for the development of new therapeutic agents. As there are limitations of uremic rats such as short lifespan and less severity of secondary hyperparathyroidism distinct from CKD patients on maintenance hemodialysis, the development of new model animals is expected.This review discusses the molecular pathogenesis of hyperfunctioning parathyroid diseases and the applications of animal models exhibiting hyperparathyroidisms in the aspect of the development of new therapeutics.PTH-cyclin D1 transgenic mice, with parathyroid-targeted overexpression of cyclin D1 oncogene, not only developed abnormal parathyroid cell proliferation but, notably, also developed biochemical hyperparathyroidism with characteristic abnormalities in bone. The mice exhibit age-dependent development of biochemical hyperparathyroidism, which enables testing of the drug precisely. In addition, the mice develop parathyroid cell hyperplasia, followed by monoclonal expansion, which is observed in refractory SHPT patients.

The calcium-sensing receptor (CaR) on the parathyroid cell surface senses ionized calcium concentration in the extracellular fluid and regulates minute-to-minute parathyroid hormone (PTH) secretion. Synthetic allosteric modulators of CaR have been developed, and one of the positive modulators (calcimimetics) is cinacalcet HCl or cinacalcet. Cinacalcet increases the sensitivity of CaR to be activated by extracellular calcium, and thus suppresses PTH release. Cinacalcet is an effective treatment for secondary hyperparathyroidism (SHPT) in patients with uremia on hemodialysis. In this review, based on basic experiments using cinacalcet, we postulate the beneficial effects of cinacalcet on hyperfunctioning parathyroid diseases.

High serum parathyroid hormone levels are associated with vascular calcification. Cinacalcet is a calcimimetic agent that inhibits parathyroid hormone secretion and is used to treat patients with secondary hyperparathyroidism. Here we measured the effects of oral cinacalcet on calcification of the aorta and heart in rats with a remnant kidney (5/6 nephrectomy) model of uremia that were fed a high-phosphate diet containing lactose to accelerate the process of aortic calcification. Alizarin red staining showed that the smooth muscle in the aortic arch of rats with a remnant kidney was calcified. The tissue levels of calcium and phosphorus in the aorta and hearts of these rats were significantly increased compared to sham-operated rats. Expression of the osteoblastic lineage genes osteocalcin, osteopontin and runt-related gene 2 were also increased in the aorta of these rats. Cinacalcet suppressed these calcification-related changes by reducing serum parathyroid hormone, calcium, phosphorus, and the calcium-phosphorus product. Parathyroidectomy also suppressed calcification in this model. We suggest that cinacalcet inhibits calcification of the aorta and heart in uremic patients with secondary hyperparathyroidism by decreasing serum parathyroid hormone levels.

Fibroblast growth factor-23 (FGF-23) is a circulating factor regulating phosphate and vitamin D homeostasis. Phosphate intake and an administration of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) increase circulating FGF-23 levels, and elevated FGF-23 prevents hyperphosphatemia and vitamin D toxicity by hyperphosphaturia and suppression of circulating 1,25(OH)(2)D level, comprising a feedback loop to maintain phosphate and vitamin D homeostasis. Excess secretion of parathyroid hormone (PTH), another phosphaturic factor, elevates the serum calcium level in primary hyperparathyroidism. PTH also elevates circulating FGF-23 level, which cooperatively enhances the phosphaturia, resulting in hypophosphatemia. The circulating FGF-23 level increases as renal function declines in chronic kidney disease (CKD), and it exhibits significant and positive correlations with serum phosphate, calcium, and PTH in CKD patients on maintenance hemodialysis, suggesting that these parameters regulate circulating FGF-23 level. Tight associations between circulating FGF-23 and calcium levels were observed both in patients with primary hyperparathyroidism and in CKD patients on maintenance hemodialysis, suggesting the role of serum calcium on FGF-23 regulatory mechanisms. FGF-23 may have a physiological role in preventing tissue damage such as ectopic calcifications, partly via suppressing the serum calcium x phosphate product by accelerating urinary phosphate excretion and suppressing vitamin D activation.

The importance of fibroblast growth factor 23 (FGF-23) in the pathogenesis of phosphate wasting disorders has been established, but controversy remains about how parathyroid hormone (PTH), which also stimulates urinary phosphate excretion, regulates the circulating level of FGF-23. We found that the serum FGF-23 concentration was higher in PTH-cyclin D1 transgenic mice, a model of primary hyperparathyroidism, than in wild-type mice. The serum FGF-23 concentration was significantly and directly correlated with serum PTH and calcium, and inversely correlated with phosphate levels in 90- to 118-week-old mice (all P < 0.005). Quantitative real-time reverse-transcriptase PCR revealed abundant expression of fgf23 in bone, especially in calvaria. The fgf23 expression in calvaria was significantly higher in the transgenic mice compared to the wild-type mice, and correlated well with serum FGF-23 levels. There was a direct correlation between the expression of fgf23 and the expression of osteocalcin and ALP, suggesting that activation of osteoblasts is important in the regulation of FGF-23. Serum FGF-23 levels decreased in the transgenic mice after parathyroidectomy. In conclusion, PTH plays a major role in the regulation of serum FGF-23 level in primary hyperparathyroidism, likely via activation of osteoblasts in bone.

Magnesium is the second most abundant cation in bone and has been recognized as one of essential elements of bone. It is estimated that 1300g of calcium, 14g of magnesium, and 600g of phosphorus exist in bones of a 70-kg human adult.1 Basic structure of bone is composed of hydroxyapatite [Ca5(OH)(PO4)3], but Newman proposed that magnesium is incorporated in bone crystal in the following formula: [Ca9 ++(H2O+)2(PO4 ≡)6(OH−)2][Ca++ · Mg0.3 ++ · Na0.3 + · CO3 − · Cit0.3].2

In a previous study, we identified several structurally unrelated scaffolds of the Rho kinase inhibitor using pharmacophore information obtained from the results of a high-throughput screening and structural information from a homology model of Rho kinase. 1H-Indazole is one of the candidate scaffolds on which a new series of potent Rho kinase inhibitors could be developed. In this study, the detailed structure-activity relationship of 1H-indazole analogues was studied. During this study, we found that the cell-free enzyme inhibitory potential of Rho kinase inhibitors having the 1H-indazole scaffold did not necessarily correlate with their inhibitory potential toward the chemotaxis of cultured cells. The choice of the linker substructure was shown to be an important factor for the 1H-indazole analogues to inhibit the chemotaxis of cells. Optimization of the 1H-indazole inhibitors with respect to the in vitro inhibition of monocyte chemotaxis induced by MCP-1 was carried out. The inhibitory potential was improved both in the cell-free enzyme assay and in the chemotaxis assay.

Objective: While the importance of fibroblast growth factor (FGF)-23 is established in phosphate-wasting disorders, little is known about the mechanisms regulating its circulating level. To investigate the role of parathyroid hormone (PTH) and calcium in FGF-23 metabolism, we examined plasma FGF-23 levels in patients with primary hyperparathyroidism (PHPT). Patients and methods: Fifty patients with PHPT and 52 controls were employed in this study. Plasma was obtained from 18 PHPT patients who underwent parathyroidectomy (PTX) on the first postoperative morning without vitamin D administration. Time-course samples were also obtained from 5 of 18 PTX patients without vitamin D analogs or calcium administration. The expression of Fgf23 on resected parathyroid glands was analyzed by reverse transcription (RT)–PCR and immunohistochemistry. Results: FGF-23 was significantly elevated in PHPT patients compared with controls. FGF-23 levels were significantly correlated positively with serum corrected calcium and intact PTH levels, and negatively with creatinine clearance and inorganic phosphate, among which creatinine clearance and corrected calcium were independently associated factors. In 18 PTX patients, postoperative FGF-23 levels were significantly decreased compared with preoperative levels. Corrected-calcium levels were significantly decreased 1 h after PTX, and this was followed by a reduction in plasma FGF-23 levels in time-course study. In addition, postoperative FGF-23 levels in 18 PTX patients were significantly correlated with corrected calcium, consistent with a role of serum calcium as one of the major regulators of FGF-23. The absence of Fgf23 expression in parathyroid glands indicated that the parathyroid glands were not major sources of circulating FGF-23. Conclusions: Serum calcium may regulate circulating FGF-23 levels in PHPT.

Cardiovascular disease is one of the largest cause of mortality in maintenance hemodialysis patients. Vascular calcifications frequently encountered in hemodialysis patients. In advanced uremic secondary hyperparathyroidism model mice, vascular calcifications were observed in their aorta, by Kossa staining. This mouse model may be a useful model to study vascular calcification in maintenance hemodialysis patients.

Serum PTH (7-84) is accumulated in patients with secondary hyperparathyroidism. It is also known that serum calcium (Ca) increases the generation of N-terminally truncated forms of parathyroid hormone (PTH). In this study, we examined whether accumulation of PTH (7-84) fraction is a parathyroid glandular origin or not by using primary cultured parathyroid cells from patients with primary and secondary hyperparathyroidism. The Bio-PTH/I-PTH ratio, indicating the ratio of PTH (1-84) to the sum of (1-84) PTH and N-terminally truncated fragment, was suppressed by increase in extracellular Ca2+ concentration for both cultured parathyroid cells prepared from parathyroid adenomas and uremia-associated secondary hyperparathyroidism. There is no difference between the ratios in primary and secondary hyperparathyroidism. These findings suggest that N-terminal truncation is regulated by extracellular Ca2+ concentration in parathyroid cells, but accumulation of PTH (7-84) fragments in patients with secondary hyperparathyroidism is mainly caused by uremia.

The calcium receptor (CaR), which regulates Ca(2+) homeostasis, senses Mg(2+) and participates in Mg(2+) metabolism mainly at the parathyroid gland and kidney as well as Ca(2+) metabolism. In the parathyroid gland, Mg(2+) suppresses parathyroid hormone (PTH) secretion by the activation of CaR, and regulates the sensitivity of CaR by its unique effect. In the kidney, the activation of CaR inhibits Mg(2+) reabsorption directly or indirectly, mainly at the downstream of loop of Henle.

Several structurally unrelated scaffolds of the Rho kinase inhibitor were designed using pharmacophore information obtained from the results of a high-throughput screening and structural information from a homology model of Rho kinase. A docking simulation using the ligand-binding pocket of the Rho kinase model helped to comprehensively understand and to predict the structure-activity relationship of the inhibitors. This understanding was useful for developing new Rho kinase inhibitors of higher potency and selectivity. We identified several potent platforms for developing the Rho kinase inhibitors, namely, pyridine, 1H-indazole, isoquinoline, and phthalimide.

Atsuya Takami, Masayuki Iwakubo, , Yuji Okada, Takehisa Kawata, Hideharu Odai, Nobuaki Takahashi, Kazutoshi Shindo, Kaname Kimura, Yoshimichi Tagami, Mika Miyake, Kayoko Fukushima, Masaki Inagaki, Mutsuki Amano, Kozo Kaibuchi and Hiroshi Iijima* Pharmaceutical Research Laboratories, Kirin Brewery Co. Ltd, 3 Miyhara-cho, Takasaki-shi, Gunma 370-1295, Japan Central Laboratory for Key Technology, Kirin Brewery Co. Ltd, 1-13-5 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan Laboratory of Biochemistry, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya, Aichi 464-8681, Japan Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, 65 Maizuru-machi, Showa-ku, Nagoya, Aichi 466-8500, Japan