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The Calcilytic Agent NPS 2143 Rectifies Hypocalcemia in a Mouse Model With an Activating Calcium-Sensing Receptor (CaSR) Mutation: Relevance to Autosomal Dominant Hypocalcemia Type 1 (ADH1).

Hannan FM, Walls GV, Babinsky VN, Nesbit MA, Kallay E, Hough TA, Fraser WD, Cox RD, Hu J, Spiegel AM, Thakker RV - Endocrinology (2015)

Bottom Line: Negative allosteric CaSR modulators, known as calcilytics, have been shown to normalize the gain-of-function associated with ADH-causing CaSR mutations in vitro and represent a potential targeted therapy for ADH1.Wild-type (Leu723) and Nuf mutant (Gln723) CaSRs were expressed in HEK293 cells, and the effect of NPS 2143 on their intracellular calcium responses was determined by flow cytometry.Intraperitoneal injection of NPS 2143 in Nuf mice led to significant increases in plasma calcium and PTH without elevating urinary calcium excretion.

View Article: PubMed Central - PubMed

Affiliation: Academic Endocrine Unit (F.M.H., G.V.W., V.N.B., M.A.N., E.K., R.V.T.), Radcliffe Department of Medicine, Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, OX3 7LJ, United Kingdom; Medical Research Council (MRC) Mammalian Genetics Unit and Mary Lyon Centre (T.A.H., R.D.C.), MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire, OX11 0RD, United Kingdom; Department of Medicine (W.D.F.), Norwich Medical School, University of East Anglia, Norwich, NR4 7TJ, United Kingdom; Laboratory of Bioorganic Chemistry (J.H.), National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892; and Albert Einstein College of Medicine (A.M.S.), Bronx, New York 10461.

ABSTRACT
Autosomal dominant hypocalcemia type 1 (ADH1) is caused by germline gain-of-function mutations of the calcium-sensing receptor (CaSR) and may lead to symptomatic hypocalcemia, inappropriately low serum PTH concentrations and hypercalciuria. Negative allosteric CaSR modulators, known as calcilytics, have been shown to normalize the gain-of-function associated with ADH-causing CaSR mutations in vitro and represent a potential targeted therapy for ADH1. However, the effectiveness of calcilytic drugs for the treatment of ADH1-associated hypocalcemia remains to be established. We have investigated NPS 2143, a calcilytic compound, for the treatment of ADH1 by in vitro and in vivo studies involving a mouse model, known as Nuf, which harbors a gain-of-function CaSR mutation, Leu723Gln. Wild-type (Leu723) and Nuf mutant (Gln723) CaSRs were expressed in HEK293 cells, and the effect of NPS 2143 on their intracellular calcium responses was determined by flow cytometry. NPS 2143 was also administered as a single ip bolus to wild-type and Nuf mice and plasma concentrations of calcium and PTH, and urinary calcium excretion measured. In vitro administration of NPS 2143 decreased the intracellular calcium responses of HEK293 cells expressing the mutant Gln723 CaSR in a dose-dependent manner, thereby rectifying the gain-of-function associated with the Nuf mouse CaSR mutation. Intraperitoneal injection of NPS 2143 in Nuf mice led to significant increases in plasma calcium and PTH without elevating urinary calcium excretion. These studies of a mouse model with an activating CaSR mutation demonstrate NPS 2143 to normalize the gain-of-function causing ADH1 and improve the hypocalcemia associated with this disorder.

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Related in: MedlinePlus

Plasma adjusted calcium, PTH, phosphate, and creatinine concentrations after ip injection of either NPS 2143 or drug vehicle. Plasma concentrations of (A–C) adjusted calcium, (D–F) PTH, (G–I) phosphate, and (J–L) creatinine were measured in untreated (U) mice, or 1, 4, or 24 hours after mice were given an ip bolus injection of control (C) or drug (D) solutions (n = 4–14 for all groups). $, P < .001 for a comparison between untreated (U) Nuf/+ or Nuf/Nuf mice and respective untreated wild-type mice. **, P < .01 and ***, P < .001 for a comparison between respective wild-type, Nuf/+, or Nuf/Nuf mice given control (C) or drug (D) solutions. †, P < .05 and ‡, P < .01 for a comparison between wild-type, Nuf/+, or Nuf/Nuf mice given control (C) or drug (D) solutions and respective untreated (U) mice.
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Figure 3: Plasma adjusted calcium, PTH, phosphate, and creatinine concentrations after ip injection of either NPS 2143 or drug vehicle. Plasma concentrations of (A–C) adjusted calcium, (D–F) PTH, (G–I) phosphate, and (J–L) creatinine were measured in untreated (U) mice, or 1, 4, or 24 hours after mice were given an ip bolus injection of control (C) or drug (D) solutions (n = 4–14 for all groups). $, P < .001 for a comparison between untreated (U) Nuf/+ or Nuf/Nuf mice and respective untreated wild-type mice. **, P < .01 and ***, P < .001 for a comparison between respective wild-type, Nuf/+, or Nuf/Nuf mice given control (C) or drug (D) solutions. †, P < .05 and ‡, P < .01 for a comparison between wild-type, Nuf/+, or Nuf/Nuf mice given control (C) or drug (D) solutions and respective untreated (U) mice.

Mentions: The in vitro studies revealed that NPS 2143 was effective in rectifying the gain-of-function of the mutant Gln723 CaSR, and we therefore pursued studies to determine whether ip injection of the calcilytic compound NPS 2143 could improve the hypocalcemia associated with Nuf mice. Untreated Nuf/+ and Nuf/Nuf mice were significantly hypocalcemic and hyperphosphatemic (P < .001), and had significantly reduced plasma PTH concentrations when compared with age-matched wild-type mice (Figure 3). No significant alterations were noted in plasma concentrations of sodium, potassium, creatinine, and alkaline phosphatase activity between wild-type and affected Nuf mice (Table 2 and Supplemental Tables 1 and 2). Intraperitoneal bolus administration of NPS 2143 resulted in a significant (P < .001) rise in plasma calcium concentrations at 1 hour after injection in wild-type, Nuf/+, and Nuf/Nuf mice (Table 2, Supplemental Tables 1 and 2, and Figure 3, A–C). Thus, NPS 2143 successfully improved the hypocalcemia associated with Nuf/+ and Nuf/Nuf mice compared with mice given the drug vehicle alone or untreated mice. At 4 hours after NPS 2143 administration, plasma calcium values remained significantly elevated in wild-type and affected Nuf/+ mice compared with respective untreated mice (Figure 3, A and B), and at 24 hours, plasma calcium concentrations decreased to levels that were not significantly different from mice given the drug vehicle alone or untreated mice (Figure 3, A–C). Furthermore, NPS 2143 treatment resulted in a marked rise in plasma PTH concentrations in wild-type and affected Nuf mice at 1 hour (Figure 3, D–F). PTH concentrations decreased to baseline values at 24 hours after NPS 2143 administration (Figure 3, D–F). Treatment with NPS 2143 did not significantly alter plasma phosphate concentrations in wild-type or affected Nuf mice compared with control group mice (Table 2, Supplemental Tables 1 and 2, and Figure 3, G–I). Indeed, administration of NPS 2143 or the drug vehicle alone was associated with significant increases in plasma phosphate in wild-type and affected Nuf mice at 1 and 4 hours compared with respective untreated mice (Figure 3, G–I), and these increases in plasma phosphate were accompanied by elevations in plasma urea and creatinine concentrations (Table 2 and Figure 3, J–L), which may indicate dehydration leading to renal impairment. NPS 2143 administration was otherwise well tolerated and not associated with adverse effects. A single ip dose of NPS 2143 had no significant effect on urinary calcium parameters such as 24-hour urinary calcium excretion, calcium to creatinine ratio, or CCCR (Table 3 and Supplemental Tables 3 and 4).


The Calcilytic Agent NPS 2143 Rectifies Hypocalcemia in a Mouse Model With an Activating Calcium-Sensing Receptor (CaSR) Mutation: Relevance to Autosomal Dominant Hypocalcemia Type 1 (ADH1).

Hannan FM, Walls GV, Babinsky VN, Nesbit MA, Kallay E, Hough TA, Fraser WD, Cox RD, Hu J, Spiegel AM, Thakker RV - Endocrinology (2015)

Plasma adjusted calcium, PTH, phosphate, and creatinine concentrations after ip injection of either NPS 2143 or drug vehicle. Plasma concentrations of (A–C) adjusted calcium, (D–F) PTH, (G–I) phosphate, and (J–L) creatinine were measured in untreated (U) mice, or 1, 4, or 24 hours after mice were given an ip bolus injection of control (C) or drug (D) solutions (n = 4–14 for all groups). $, P < .001 for a comparison between untreated (U) Nuf/+ or Nuf/Nuf mice and respective untreated wild-type mice. **, P < .01 and ***, P < .001 for a comparison between respective wild-type, Nuf/+, or Nuf/Nuf mice given control (C) or drug (D) solutions. †, P < .05 and ‡, P < .01 for a comparison between wild-type, Nuf/+, or Nuf/Nuf mice given control (C) or drug (D) solutions and respective untreated (U) mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4541614&req=5

Figure 3: Plasma adjusted calcium, PTH, phosphate, and creatinine concentrations after ip injection of either NPS 2143 or drug vehicle. Plasma concentrations of (A–C) adjusted calcium, (D–F) PTH, (G–I) phosphate, and (J–L) creatinine were measured in untreated (U) mice, or 1, 4, or 24 hours after mice were given an ip bolus injection of control (C) or drug (D) solutions (n = 4–14 for all groups). $, P < .001 for a comparison between untreated (U) Nuf/+ or Nuf/Nuf mice and respective untreated wild-type mice. **, P < .01 and ***, P < .001 for a comparison between respective wild-type, Nuf/+, or Nuf/Nuf mice given control (C) or drug (D) solutions. †, P < .05 and ‡, P < .01 for a comparison between wild-type, Nuf/+, or Nuf/Nuf mice given control (C) or drug (D) solutions and respective untreated (U) mice.
Mentions: The in vitro studies revealed that NPS 2143 was effective in rectifying the gain-of-function of the mutant Gln723 CaSR, and we therefore pursued studies to determine whether ip injection of the calcilytic compound NPS 2143 could improve the hypocalcemia associated with Nuf mice. Untreated Nuf/+ and Nuf/Nuf mice were significantly hypocalcemic and hyperphosphatemic (P < .001), and had significantly reduced plasma PTH concentrations when compared with age-matched wild-type mice (Figure 3). No significant alterations were noted in plasma concentrations of sodium, potassium, creatinine, and alkaline phosphatase activity between wild-type and affected Nuf mice (Table 2 and Supplemental Tables 1 and 2). Intraperitoneal bolus administration of NPS 2143 resulted in a significant (P < .001) rise in plasma calcium concentrations at 1 hour after injection in wild-type, Nuf/+, and Nuf/Nuf mice (Table 2, Supplemental Tables 1 and 2, and Figure 3, A–C). Thus, NPS 2143 successfully improved the hypocalcemia associated with Nuf/+ and Nuf/Nuf mice compared with mice given the drug vehicle alone or untreated mice. At 4 hours after NPS 2143 administration, plasma calcium values remained significantly elevated in wild-type and affected Nuf/+ mice compared with respective untreated mice (Figure 3, A and B), and at 24 hours, plasma calcium concentrations decreased to levels that were not significantly different from mice given the drug vehicle alone or untreated mice (Figure 3, A–C). Furthermore, NPS 2143 treatment resulted in a marked rise in plasma PTH concentrations in wild-type and affected Nuf mice at 1 hour (Figure 3, D–F). PTH concentrations decreased to baseline values at 24 hours after NPS 2143 administration (Figure 3, D–F). Treatment with NPS 2143 did not significantly alter plasma phosphate concentrations in wild-type or affected Nuf mice compared with control group mice (Table 2, Supplemental Tables 1 and 2, and Figure 3, G–I). Indeed, administration of NPS 2143 or the drug vehicle alone was associated with significant increases in plasma phosphate in wild-type and affected Nuf mice at 1 and 4 hours compared with respective untreated mice (Figure 3, G–I), and these increases in plasma phosphate were accompanied by elevations in plasma urea and creatinine concentrations (Table 2 and Figure 3, J–L), which may indicate dehydration leading to renal impairment. NPS 2143 administration was otherwise well tolerated and not associated with adverse effects. A single ip dose of NPS 2143 had no significant effect on urinary calcium parameters such as 24-hour urinary calcium excretion, calcium to creatinine ratio, or CCCR (Table 3 and Supplemental Tables 3 and 4).

Bottom Line: Negative allosteric CaSR modulators, known as calcilytics, have been shown to normalize the gain-of-function associated with ADH-causing CaSR mutations in vitro and represent a potential targeted therapy for ADH1.Wild-type (Leu723) and Nuf mutant (Gln723) CaSRs were expressed in HEK293 cells, and the effect of NPS 2143 on their intracellular calcium responses was determined by flow cytometry.Intraperitoneal injection of NPS 2143 in Nuf mice led to significant increases in plasma calcium and PTH without elevating urinary calcium excretion.

View Article: PubMed Central - PubMed

Affiliation: Academic Endocrine Unit (F.M.H., G.V.W., V.N.B., M.A.N., E.K., R.V.T.), Radcliffe Department of Medicine, Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, OX3 7LJ, United Kingdom; Medical Research Council (MRC) Mammalian Genetics Unit and Mary Lyon Centre (T.A.H., R.D.C.), MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire, OX11 0RD, United Kingdom; Department of Medicine (W.D.F.), Norwich Medical School, University of East Anglia, Norwich, NR4 7TJ, United Kingdom; Laboratory of Bioorganic Chemistry (J.H.), National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892; and Albert Einstein College of Medicine (A.M.S.), Bronx, New York 10461.

ABSTRACT
Autosomal dominant hypocalcemia type 1 (ADH1) is caused by germline gain-of-function mutations of the calcium-sensing receptor (CaSR) and may lead to symptomatic hypocalcemia, inappropriately low serum PTH concentrations and hypercalciuria. Negative allosteric CaSR modulators, known as calcilytics, have been shown to normalize the gain-of-function associated with ADH-causing CaSR mutations in vitro and represent a potential targeted therapy for ADH1. However, the effectiveness of calcilytic drugs for the treatment of ADH1-associated hypocalcemia remains to be established. We have investigated NPS 2143, a calcilytic compound, for the treatment of ADH1 by in vitro and in vivo studies involving a mouse model, known as Nuf, which harbors a gain-of-function CaSR mutation, Leu723Gln. Wild-type (Leu723) and Nuf mutant (Gln723) CaSRs were expressed in HEK293 cells, and the effect of NPS 2143 on their intracellular calcium responses was determined by flow cytometry. NPS 2143 was also administered as a single ip bolus to wild-type and Nuf mice and plasma concentrations of calcium and PTH, and urinary calcium excretion measured. In vitro administration of NPS 2143 decreased the intracellular calcium responses of HEK293 cells expressing the mutant Gln723 CaSR in a dose-dependent manner, thereby rectifying the gain-of-function associated with the Nuf mouse CaSR mutation. Intraperitoneal injection of NPS 2143 in Nuf mice led to significant increases in plasma calcium and PTH without elevating urinary calcium excretion. These studies of a mouse model with an activating CaSR mutation demonstrate NPS 2143 to normalize the gain-of-function causing ADH1 and improve the hypocalcemia associated with this disorder.

Show MeSH
Related in: MedlinePlus