EOS789, a novel pan-phosphate transporter inhibitor, is effective for the treatment of chronic kidney disease–mineral bone disorder
Authors: Yoshinori Tsuboi, Shuichi Ohtomo, Yasuhiro Ichida, Hitoshi Hagita, Kazuharu Ozawa, Manami Iida, Shunsuke Nagao, Hisashi Ikegami, Tadakatsu Takahashi, Naoshi Horiba
Journal: Kidney International (2020)

Chronic kidney disease is characterized as impaired renal function along with the imbalance and dysregulation of mineral metabolism; recognized as chronic kidney disease-mineral and bone disorder. Hyperphosphatemia, characterized by altered phosphate homeostasis along with elevated fibroblast growth factor-23 and intact parathyroid hormone, is such an alteration of mineral metabolism. We discovered a novel inhibitor, EOS789, that interacts with several sodium-dependent phosphate transporters (NaPi-IIb, PiT-1, and PiT-2) known to contribute to intestinal phosphate absorption. This inhibitor dose-dependently increased the fecal phosphorus excretion rate and inversely decreased the urinary phosphorus excretion rate in normal rats, suggesting inhibition of intestinal phosphorus absorption. In rats with adenine-induced hyperphosphatemia, EOS789 markedly decreased the serum phosphate, fibroblast growth factor-23, and intact parathyroid hormone below values found in normal control rats. Notably, this pan-phosphate transporter inhibitor exhibited a more potent effect on serum phosphate than a NaPi-IIb-selective inhibitor in rats with hyperphosphatemia indicating that PiT-1 and PiT-2 play important roles in intestinal phosphate absorption. Moreover, in a long-term study, EOS789 sustained the suppression of serum phosphorus in parallel with fibroblast growth factor-23 and intact parathyroid hormone and ameliorated ectopic calcification of the thoracic aorta. Additionally, EOS789 treatment also ameliorated kidney deterioration in rats with progressive kidney injury, probably due to the strict phosphate control. Thus, EOS789 has potent efficacy against hyperphosphatemia and its complications and could provide a significant benefit to patients who are ineffectively treated with phosphate binders. Chronic kidney disease is characterized as impaired renal function along with the imbalance and dysregulation of mineral metabolism; recognized as chronic kidney disease-mineral and bone disorder. Hyperphosphatemia, characterized by altered phosphate homeostasis along with elevated fibroblast growth factor-23 and intact parathyroid hormone, is such an alteration of mineral metabolism. We discovered a novel inhibitor, EOS789, that interacts with several sodium-dependent phosphate transporters (NaPi-IIb, PiT-1, and PiT-2) known to contribute to intestinal phosphate absorption. This inhibitor dose-dependently increased the fecal phosphorus excretion rate and inversely decreased the urinary phosphorus excretion rate in normal rats, suggesting inhibition of intestinal phosphorus absorption. In rats with adenine-induced hyperphosphatemia, EOS789 markedly decreased the serum phosphate, fibroblast growth factor-23, and intact parathyroid hormone below values found in normal control rats. Notably, this pan-phosphate transporter inhibitor exhibited a more potent effect on serum phosphate than a NaPi-IIb-selective inhibitor in rats with hyperphosphatemia indicating that PiT-1 and PiT-2 play important roles in intestinal phosphate absorption. Moreover, in a long-term study, EOS789 sustained the suppression of serum phosphorus in parallel with fibroblast growth factor-23 and intact parathyroid hormone and ameliorated ectopic calcification of the thoracic aorta. Additionally, EOS789 treatment also ameliorated kidney deterioration in rats with progressive kidney injury, probably due to the strict phosphate control. Thus, EOS789 has potent efficacy against hyperphosphatemia and its complications and could provide a significant benefit to patients who are ineffectively treated with phosphate binders. see commentary on page 273 see commentary on page 273 Translational StatementIn current clinical practice, oral phosphate binders are used to treat hyperphosphatemia, but many patients avoid taking them because of the high administration volume and gastrointestinal side effects. Thus, a novel therapy for hyperphosphatemia is eagerly needed. EOS789, a pan-phosphate transporter inhibitor, is able to inhibit intestinal phosphate absorption with mechanisms of action that are different from phosphate binders. Our nonclinical data suggest that EOS789 could become a novel therapy for patients with hyperphosphatemia that frees them from the pill burden of traditional phosphate binders. The therapeutic potential of EOS789 has been investigated in clinical studies (ClinicalTrials.gov number NCT02965053, JAPIC ID JapicCTI-152992). In current clinical practice, oral phosphate binders are used to treat hyperphosphatemia, but many patients avoid taking them because of the high administration volume and gastrointestinal side effects. Thus, a novel therapy for hyperphosphatemia is eagerly needed. EOS789, a pan-phosphate transporter inhibitor, is able to inhibit intestinal phosphate absorption with mechanisms of action that are different from phosphate binders. Our nonclinical data suggest that EOS789 could become a novel therapy for patients with hyperphosphatemia that frees them from the pill burden of traditional phosphate binders. The therapeutic potential of EOS789 has been investigated in clinical studies (ClinicalTrials.gov number NCT02965053, JAPIC ID JapicCTI-152992). Chronic kidney disease (CKD) is not only characterized as impaired renal function, but it also comprises the imbalance and dysregulation of mineral metabolism, recognized as chronic kidney disease–mineral and bone disorder (CKD-MBD). In patients with CKD or end-stage renal failure (ESRD), the amount of phosphate excreted from kidneys is decreased or abolished, which can imbalance phosphate levels in blood and lead to hyperphosphatemia. Hyperphosphatemia is characterized by dysregulation in phosphate homeostasis along with an elevation of fibroblast growth factor 23 (FGF-23) and intact parathyroid hormone (PTH). It is common in patients with CKD and ESRD, and multiple studies show that it is an important risk factor not only for secondary hyperparathyroidism but also for cardiovascular events—via vascular calcification—and mortality.1Staude H. Jeske S. Schmitz K. et al.Cardiovascular risk and mineral bone disorder in patients with chronic kidney disease.Kidney Blood Press Res. 2013; 37: 68-83Crossref PubMed Scopus (28) Google Scholar, 2Slatopolsky E. Brown A. Dusso A. Role of phosphorus in the pathogenesis of secondary hyperparathyroidism.Am J Kidney Dis. 2001; 37: 54-57Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 3Mathew S. Tustison K.S. Sugatani T. et al.The mechanism of phosphorus as a cardiovascular risk factor in CKD.J Am Soc Nephrol. 2008; 19: 1092-1105Crossref PubMed Scopus (201) Google Scholar, 4Kestenbaum B. Sampson J.N. Rudser K.D. et al.Serum phosphate levels and mortality risk among people with chronic kidney disease.J Am Soc Nephrol. 2005; 16: 520-528Crossref PubMed Scopus (926) Google Scholar, 5Block G.A. Klassen P.S. Lazarus J.M. et al.Mineral metabolism, mortality, and morbidity in maintenance hemodialysis.J Am Soc Nephrol. 2004; 15: 2208-2218Crossref PubMed Scopus (2170) Google Scholar Phosphate in blood is strictly regulated by being absorbed in the small intestine and excreted from kidneys.6Bergwitz C. Jüppner H. Regulation of phosphate homeostasis by PTH, vitamin D, and FGF23.Annu Rev Med. 2010; 61: 91-104Crossref PubMed Scopus (454) Google Scholar Phosphate is known to be absorbed mainly in the upper small intestine via both passive and active mechanisms, and the sodium-dependent phosphate transporters NaPi-IIb (solute carrier family 34 member 2, SLC34A2), PiT-1 (SLC20A1), and PiT-2 (SLC20A2) are reported to be involved in intestinal active phosphate absorption.7Wagner C.A. Hernando N. Forster I.C. et al.The SLC34 family of sodium-dependent phosphate transporters.Pflugers Arch. 2014; 466: 139-153Crossref PubMed Scopus (102) Google Scholar, 8Forster I.C. Hernando N. Biber J. et al.Phosphate transport kinetics and structure-function relationships of SLC34 and SLC20 proteins.Curr Top Membr. 2012; 70: 313-356Crossref PubMed Scopus (46) Google Scholar In clinical practice, oral phosphate binders, dietary instruction, and hemodialysis to remove phosphorus are used to treat hyperphosphatemia. The currently available phosphate binders, which include metal-based compounds (e.g., calcium carbonate and lanthanum carbonate) and polymer-based compounds (e.g., sevelamer carbonate), are widely used in treatment; however, many patients have low compliance because of the high administration volume, the accumulation of metal in tissues, and gastrointestinal side effects.9Carfagna F. Del Vecchio L. Pontoriero G. et al.Current and potential treatment options for hyperphosphatemia.Expert Opin Drug Saf. 2018; 17: 597-607Crossref PubMed Scopus (17) Google Scholar, 10Sonikian M. Papachristou E. Goumenos D.S. Optimal use of phosphate binders in chronic kidney disease.Expert Opin Pharmacother. 2013; 14: 2521-2532Crossref PubMed Scopus (7) Google Scholar, 11Covic A. Rastogi A. Hyperphosphatemia in patients with ESRD: assessing the current evidence linking outcomes with treatment adherence.BMC Nephrol. 2013; 14: 153Crossref PubMed Scopus (47) Google Scholar Several novel drugs to treat hyperphosphatemia have been clinically evaluated, such as sodium/hydrogen exchanger 3 inhibitor tenapanor, nicotinamide, and NaPi-IIb inhibitor ASP3325. Tenapanor significantly decreased serum phosphorus levels in patients with ESRD,12Block G.A. Rosenbaum D.P. Yan A. Chertow G.M. Efficacy and safety of tenapanor in patients with hyperphosphatemia receiving maintenance hemodialysis: a randomized phase 3 trial.J Am Soc Nephrol. 2019; 30: 641-652Crossref PubMed Scopus (54) Google Scholar but 8.3% of these patients discontinued the drug due to diarrhea, which was the most common adverse event for tenapanor. Nicotinamide lowered serum phosphorus in clinical studies, but the treatment discontinuation rate was also high due to adverse events, such as thrombocytopenia.13Lenglet A. Liabeuf S. El Esper N. et al.Efficacy and safety of nicotinamide in haemodialysis patients: the NICOREN study.Nephrol Dial Transplant. 2017; 32: 870-879Crossref PubMed Scopus (0) Google Scholar Finally, ASP3325 failed to reduce serum phosphorus in patients with ESRD.14Larsson T.E. Kameoka C. Nakajo I. et al.NPT-IIb inhibition does not improve hyperphosphatemia in CKD.Kidney Int Rep. 2017; 3: 73-80Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar From the viewpoint of safety and effectiveness, a novel therapy for hyperphosphatemia is still eagerly needed. Here we identified EOS789, which is a novel pan-inhibitor of NaPi-IIb, PiT-1, and PiT-2. We evaluated the in vitro inhibitory activity of EOS789 on sodium-dependent phosphate transporters; its in vivo mechanisms of action; and its effects on hyperphosphatemia, ectopic calcification, and kidney dysfunction in rats. The chemical structure of EOS789 was shown in Figure 1. EOS789 dose-dependently inhibited human or rat NaPi-IIb, PiT-1, and PiT-2 transporters reported to play a role in intestinal phosphate absorption7Wagner C.A. Hernando N. Forster I.C. et al.The SLC34 family of sodium-dependent phosphate transporters.Pflugers Arch. 2014; 466: 139-153Crossref PubMed Scopus (102) Google Scholar, 8Forster I.C. Hernando N. Biber J. et al.Phosphate transport kinetics and structure-function relationships of SLC34 and SLC20 proteins.Curr Top Membr. 2012; 70: 313-356Crossref PubMed Scopus (46) Google Scholar (Table 1). The Lineweaver-Burk plot analysis showed that EOS789 inhibited phosphate uptake in a noncompetitive manner for NaPi-IIb, PiT-1, and PiT-2 (Figure 2). EOS789 also inhibited phosphate uptake in normal rat intestinal brush border membrane vesicles. The 50% inhibitory concentration value was 3.1 ± 0.3 μmol/l.Table 1The in vitro IC50 values of EOS789 or NaPi-IIb inhibitor against human or rat NaPi-IIb, PiT-1, and PiT-2TransporterEOS789NaPi-IIb inhibitorHuman NaPi-IIb6.80.092Human PiT-11.5>30Human PiT-21.7>30Rat NaPi-IIb3.90.097Rat PiT-11.917.1Rat PiT-21.728.2The 50% inhibitory concentration (IC50) value (μmol/l) of EOS789 or NaPi-IIb inhibitor for phosphate transporters. The experiments were performed using CHO-K1 cells expressing human or rat sodium-dependent phosphate transporters NaPi-IIb, PiT-1, or PiT-2. Data represent means. Open table in a new tab Figure 2Inhibition of human sodium-dependent phosphate transporters (a) NaPi-IIb, (b) PiT-1, and (c) PiT-2 by EOS789. The experiments were performed using CHO-K1 cells expressing human NaPi-IIb, PiT-1, or PiT-2. The Lineweaver-Burk plots were created by plotting the inverse velocity (1/v) as a function of the inverse of the substrate concentration (1/[S]).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The 50% inhibitory concentration (IC50) value (μmol/l) of EOS789 or NaPi-IIb inhibitor for phosphate transporters. The experiments were performed using CHO-K1 cells expressing human or rat sodium-dependent phosphate transporters NaPi-IIb, PiT-1, or PiT-2. Data represent means. EOS789 also inhibited human or rat NaPi-IIa and NaPi-IIc (Supplementary Table S1), which are known to play a central role in renal phosphate reabsorption.5Block G.A. Klassen P.S. Lazarus J.M. et al.Mineral metabolism, mortality, and morbidity in maintenance hemodialysis.J Am Soc Nephrol. 2004; 15: 2208-2218Crossref PubMed Scopus (2170) Google Scholar In contrast, the 50% inhibitory concentration values of EOS789 against human sodium-glucose linked transporter 1 and organic anion transporter 1 were >30 μmol/l (Supplementary Table S1). To clarify the gastrointestinal absorption (Fa value) of EOS789, the plasma radioactivity concentrations after the i.v. and oral administration of [14C]EOS789 were evaluated in normal rats. The average value of the area under the concentration-time curve from zero to infinity (AUCinf) was 31,900 ng eq. × h/ml in rats on i.v. 2 mg/kg of EOS789 and 22,000 ng eq. × h/ml in rats on oral 20 mg/kg of EOS789. The Fa value was 6.9%, indicating that EOS789 has low gastrointestinal absorption. To understand the mechanism of action of EOS789 in vivo, fecal phosphorus excretion was evaluated in rats on EOS789 with 33PO4. EOS789 dose-dependently enhanced fecal 33P excretion and decreased the urinary 33P excretion rate and serum 33P radioactivity, suggesting that EOS789 suppresses intestinal phosphorus absorption via the inhibition of intestinal phosphate transporters (Figure 3). To clarify the effect of EOS789 on hyperphosphatemia, we evaluated its ability to lower the serum phosphorus in rats with adenine-induced hyperphosphatemia. The serum phosphorus concentration of the disease control (DC) group was increased significantly to 9.7 mg/dl compared with the normal control (NC) group (6.1 mg/dl). EOS789 dose-dependently decreased the serum phosphorus concentration (Figure 4a). EOS789 also dose-dependently decreased the serum FGF-23 and intact PTH concentration (Figure 4b and c). Serum calcium was increased in rats only on 0.5% EOS789 (Supplementary Figure S1). Any digestive problems, such as diarrhea and constipation, were not observed. To understand the potency of the pan-phosphate transporter inhibition of EOS789, we evaluated EOS789 and a NaPi-IIb inhibitor (Table 1) in adenine-induced hyperphosphatemia rats. EOS789 dose-dependently decreased serum phosphorus concentration, increased fecal phosphorus excretion, and decreased urinary phosphorus excretion (Figure 5). In contrast, the NaPi-IIb inhibitor had only a limited effect on these parameters (Figure 5). Of particular note, the effects of 1.0% EOS789 on serum phosphate, fecal phosphorus excretion, and urinary phosphorus excretion were significantly stronger than those of a 1.0% NaPi-IIb inhibitor despite having the same dose amount (Figure 5). These data clearly suggest that EOS789 inhibits intestinal phosphate absorption more potently than NaPi-IIb inhibitors, resulting in a greater reduction of serum phosphate. Any digestive problems, such as diarrhea and constipation, were not observed even in rats on 1.0% EOS789. To elucidate the long-term effects of EOS789 on CKD-MBD progression, we evaluated the effects of EOS789 in chronic anti-Thy1.1 nephritis rats. Serum phosphate, FGF-23, and intact PTH levels were significantly elevated in the DC group (Figure 6). At the end of the study, the 0.3% of EOS789 dosing significantly suppressed serum phosphate, FGF-23, and intact PTH levels. On the other hand, the 0.1% of EOS789 did not decrease serum FGF-23 and intact PTH levels in spite of serum phosphate reduction, suggesting insufficient phosphate control in rats on 0.1% of EOS789. EOS789 did not affect the serum calcium level (Supplementary Figure S2). Aortic calcification was evaluated by measuring calcium content in dried aorta. The aortic calcium content in the DC group significantly increased compared with the NC group, and 0.3% of EOS789 markedly suppressed this increase (Figure 7). Pictures representing von Kossa staining are shown in Figure 7. The histopathologic calcium deposition was observed in 4 of 19 in the DC group and 4 of 20 in the 0.1% EOS789 group, but in none in the 0.3% EOS789 group. Serum creatinine gradually increased in the DC group during the study period, and 0.3% of EOS789 significantly decreased this in parallel with the phosphate-related factors shown above (Figure 8). Severe glomerulosclerosis, degeneration of tubules, and tubulointerstitial fibrosis were observed in the DC group and EOS789 treatment significantly ameliorated these histopathologic changes (Figure 8, Table 2).Table 2Histopathologic evaluation of glomerulosclerosis, degeneration of tubules, and tubulointerstitial fibrosisNCDCEOS 0.1%EOS 0.3%Glomerulosclerosis0.00 ± 0.002.60 ± 0.04aP < 0.0001 vs. NC group (Welch test).2.45 ± 0.04bP < 0.025, dP < 0.005, and cP < 0.0001 vs. DC group (parametric Williams test) at the end of the study.2.32 ± 0.05cP < 0.025, dP < 0.005, and cP < 0.0001 vs. DC group (parametric Williams test) at the end of the study.Degeneration of tubules0.00 ± 0.002.54 ± 0.06aP < 0.0001 vs. NC group (Welch test).2.45 ± 0.042.23 ± 0.06dP < 0.025, dP < 0.005, and cP < 0.0001 vs. DC group (parametric Williams test) at the end of the study.Tubulointerstitial fibrosis0.00 ± 0.002.39 ± 0.09aP < 0.0001 vs. NC group (Welch test).2.21 ± 0.092.01 ± 0.07dP < 0.025, dP < 0.005, and cP < 0.0001 vs. DC group (parametric Williams test) at the end of the study.DC, disease control; EOS, EOS789; NC, normal control.Data represent mean ± SE.a P < 0.0001 vs. NC group (Welch test).b P < 0.025, dP < 0.005, and cP < 0.0001 vs. DC group (parametric Williams test) at the end of the study. Open table in a new tab DC, disease control; EOS, EOS789; NC, normal control. Data represent mean ± SE. Transforming growth factor beta 1 (TGF-β1) is a key factor in the development of kidney injury.15López-Hernández F.J. López-Novoa J.M. Role of TGF-β in chronic kidney disease: an integration of tubular, glomerular and vascular effects.Cell Tissue Res. 2012; 347: 141-154Crossref PubMed Scopus (222) Google Scholar The mRNA expressions of TGF-β1 were increased in the DC group, and these increases were significantly suppressed by EOS789 (Figure 9). Also increased in the DC group were the gene expressions of extracellular matrix, that is, collagen type 1 α1 and collagen type 3 α1, and α-smooth muscle actin, which is recognized as common marker of myofibroblast.16Duffield J.S. Lupher M. Thannickal V.J. et al.Host responses in tissue repair and fibrosis.Annu Rev Pathol. 2013; 8: 241-276Crossref PubMed Scopus (402) Google Scholar EOS789 significantly decreased these as well (Figure 9). To the best of our knowledge, this is the first study to report on the potency of a pan-phosphate transporter inhibitor. Here, we demonstrated that EOS789 strongly inhibits sodium-dependent phosphate transporters belonging to the SLC20 family or SLC34 family and markedly ameliorates hyperphosphatemia, its complications, vascular calcification, and kidney dysfunction. It is thought that phosphate is absorbed via both passive and active mechanisms in intestine, but their contributions still remain unknown. The observation that EOS789 treatment returned the serum phosphorus level to normal range without diarrhea in adenine-induced hyperphosphatemia rats suggests that active transport contributes much more highly to intestinal phosphorus absorption. Moreover, based on the superior phosphate-lowering effect of EOS789 compared with the NaPi-IIb inhibitor, not only NaPi-IIb but also PiT-1 and PiT-2 play important roles in active mechanisms. This may be able to explain results from the previous clinical study in which the NaPi-IIb inhibitor ASP3325 failed to reduce serum phosphorus level in patients with ESRD.14Larsson T.E. Kameoka C. Nakajo I. et al.NPT-IIb inhibition does not improve hyperphosphatemia in CKD.Kidney Int Rep. 2017; 3: 73-80Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar On the other hand, nicotinamide, which has an inhibitory potency against NaPi-IIb, decreased serum phosphate levels in patients with hyperphosphatemia.13Lenglet A. Liabeuf S. El Esper N. et al.Efficacy and safety of nicotinamide in haemodialysis patients: the NICOREN study.Nephrol Dial Transplant. 2017; 32: 870-879Crossref PubMed Scopus (0) Google Scholar, 17Eto N. Miyata Y. Ohno H. Yamashita T. Nicotinamide prevents the development of hyperphosphataemia by suppressing intestinal sodium-dependent phosphate transporter in rats with adenine-induced renal failure.Nephrol Dial Transplant. 2005; 20: 1378-1384Crossref PubMed Scopus (112) Google Scholar Taken together, phosphate seems to be actively absorbed at least in part by NaPi-IIb, but its contribution may not be big in humans and rats. In kidney, phosphate reabsorption is assumed to be unaffected by EOS789 administration, although EOS789 potently inhibits NaPi-IIa and NaPi-IIc in vitro, both of which play a central role in renal phosphate transportation. Because EOS789 is barely absorbed in intestine and the absorbed EOS789 is almost completely excreted into feces (data not shown), the concentration of EOS789 in kidney does not seem sufficient to inhibit renal phosphate transporters. In fact, urinary 33P excretion was dose-dependently decreased by EOS789 treatment, rather than increased, in our 33P rat study. Thus, we understand the intestinal mechanism by which EOS789 lowers blood phosphate. EOS789 exhibits unique multi-inhibitory activity against at least 2 distinct sodium-dependent phosphate transporter families, the SLC20A family (PiT-1 and PiT-2) and SLC34A family (NaPi-IIa, NaPi-IIb, and NaPi-IIc). Unfortunately, the binding site of EOS789 to these transporters still remains unknown because the 3-dimensional structures of NaPi-IIb, PiT-1, and PiT-2 have not been elucidated yet. It is well known that PiT-1and -2 and NaPi-IIb preferentially transport monovalent and divalent inorganic phosphate, respectively, and the protein structures are considered to be different between 2 families.7Wagner C.A. Hernando N. Forster I.C. et al.The SLC34 family of sodium-dependent phosphate transporters.Pflugers Arch. 2014; 466: 139-153Crossref PubMed Scopus (102) Google Scholar, 8Forster I.C. Hernando N. Biber J. et al.Phosphate transport kinetics and structure-function relationships of SLC34 and SLC20 proteins.Curr Top Membr. 2012; 70: 313-356Crossref PubMed Scopus (46) Google Scholar On the other hand, considering that EOS789 is a noncompetitive inhibitor, it might work as an allosteric inhibitor of NaPi-IIb, PiT-1, and PiT-2 proteins. In other words, NaPi-IIb, PiT-1, and PiT-2 might share a similar structure associated with phosphate transport, and EOS789 might bind to that common structure and inhibit their transport. It will be of great interest to clarify the inhibition mechanism of EOS789. The targets of EOS789—NaPi-IIa/b/c and PiT-1 and -2—are widely expressed in human body, and the systemic inhibition of their functions potentially affects phosphate metabolism. The pulmonary alveolar microlithiasis by NaPi-IIb mutation and familial idiopathic basal ganglia calcification with PiT-2 mutation are widely known.18Ferreira J.B. Pimentel L. Keasey M.P. et al.First report of a de novo mutation at SLC20A2 in a patient with brain calcification.J Mol Neurosci. 2014; 54: 748-751Crossref PubMed Scopus (28) Google Scholar, 19Castellana G. Castellana G. Gentile M. et al.Pulmonary alveolar microlithiasis: review of the 1022 cases reported worldwide.Eur Respir Rev. 2015; 24: 607-620Crossref PubMed Scopus (67) Google Scholar It is recently reported that PiT-2 may play a role in bone development and its deletion leads to decreased MBD.20Yamada S. Wallingford M.C. Borgeia S. et al.Loss of PiT-2 results in abnormal bone development and decreased bone mineral density and length in mice.Biochem Biophys Res Commun. 2018; 495: 553-559Crossref PubMed Scopus (13) Google Scholar However, in fact, no toxicological effects in lung, brain, or bone were observed in rats and dogs on EOS789 in repeated oral dose toxicity studies lasting up to 13 weeks (data not shown). Even though the systemic inhibition of NaPi-IIb and PiT-2 has a potential risk of ectopic calcification and decreased bone mineral density, EOS789 is expected to avoid these safety risks due to its very low fractional absorption. Potential toxicity due to nonspecific action of EOS789 should be also discussed. For in vitro profiling, EOS789 was evaluated at 10 μmol/l in a total of 136 in vitro pharmacological assays in BioPrint panels (Eurofins Panlabs, Inc. St. Charles, MO).21Bendels S. Bissantz C. Fasching B. et al.Safety screening in early drug discovery: an optimized assay panel.J Pharmacol Toxicol Methods. 2019; 99: 106609Crossref PubMed Scopus (15) Google Scholar The only targets that EOS789 inhibited >50% were the sigma and L type Ca2+ channels (data not shown). The in vitro transporter uptake assays also indicated that EOS789 is a multi-, but selective, inhibitor against sodium-dependent phosphate transporters (Table 1 and Supplementary Table S1). Actually, no toxicological findings that may cause safety issues in humans were observed in any examined organs at expected pharmacological dose ranges in repeated-dose toxicological studies in rats and dogs, although potential effects on gastrointestinal motor function were noted in a pharmacology study in rats (data not shown). Considering these results, the safety risk due to nonspecific action of EOS789 seems to be low. Circulating FGF-23 progressively increases during CKD, and this is recognized to exert initial adaptive responses against hyperphosphatemia.22Scialla J.J. Wolf M. Roles of phosphate and fibroblast growth factor 23 in cardiovascular disease.Nat Rev Nephrol. 2014; 10: 268-278Crossref PubMed Scopus (134) Google Scholar, 23Isakova T. Wahl P. Vargas G.S. et al.Fibroblast growth factor 23 is elevated before parathyroid hormone and phosphate in chronic kidney disease.Kidney Int. 2011; 79: 1370-1378Abstract Full Text Full Text PDF PubMed Scopus (878) Google Scholar Many studies indicate that increased circulating FGF-23 is associated with increased risk of adverse outcomes, including all-cause mortality in patients with CKD and who are on hemodialysis.23Isakova T. Wahl P. Vargas G.S. et al.Fibroblast growth factor 23 is elevated before parathyroid hormone and phosphate in chronic kidney disease.Kidney Int. 2011; 79: 1370-1378Abstract Full Text Full Text PDF PubMed Scopus (878) Google Scholar, 24Gutiérrez O.M. Mannstadt M. Isakova T. et al.Fibroblast growth factor 23 and mortality among patients undergoing hemodialysis.N Engl J Med. 2008; 359: 584-592Crossref PubMed Scopus (1377) Google Scholar, 25Isakova T. Xie H. Yang W. et al.Fibroblast growth factor 23 and risks of mortality and end-stage renal disease in patients with chronic kidney disease.JAMA. 2011; 305: 2432-2439Crossref PubMed Scopus (789) Google Scholar In our chronic anti-Thy1.1 nephritis rats study, serum FGF-23 was dramatically elevated in the late phase of disease, and notably, EOS789 markedly suppressed serum FGF-23 by strictly regulating phosphate. In this way, EOS789 is expected to protect patients suffering from CKD or ESRD from hyper-FGF-23–related disorders, such as left ventricular hypertrophy and congestive heart failure. Secondary hyperparathyroidism is among the complications in CKD and ESRD and causes renal osteodystrophy.18Ferreira J.B. Pimentel L. Keasey M.P. et al.First report of a de novo mutation at SLC20A2 in a patient with brain calcification.J Mol Neurosci. 2014; 54: 748-751Crossref PubMed Scopus (28) Google Scholar The CKD-related hormonal disturbances, such as hyperphosphatemia and calcitriol deficiency, are implicated in the development of secondary hyperparathyroidism.23Isakova T. Wahl P. Vargas G.S. et al.Fibroblast growth factor 23 is elevated before parathyroid hormone and phosphate in chronic kidney disease.Kidney Int. 2011; 79: 1370-1378Abstract Full Text Full Text PDF PubMed Scopus (878) Google Scholar, 26Babayev R. Nickolas T.L. Bone disorders in chronic kidney disease: an update in diagnosis and management.Semin Dial. 2015; 28: 645-653Crossref PubMed Scopus (24) Google Scholar In our study, EOS789 significantly suppressed the elevation of serum-intact PTH level in parallel with serum phosphorus level. The serum calcitriol level was only slightly recovered both in adenine-induced hyperphosphatemia rats and chronic anti-Thy1.1 nephritis rats on EOS789, but not significantly (data not shown). The PTH-lowering effect of EOS789 is achieved mainly by strict phosphate control, and this may help patients with CKD and ESRD who