Homozygous Ser-1 to Pro-1 mutation in parathyroid hormone identified in hypocalcemic patients results in secretion of a biologically inactive pro-hormone.

Like other secreted peptides, nascent parathyroid hormone (PTH) is synthesized with a pre- and a pro-sequence (25 and 6 amino acids, respectively). These precursor segments are sequentially removed in parathyroid cells before packaging into secretory granules. Three patients from two unrelated families who presented during infancy with symptomatic hypocalcemia were found to have a homozygous serine (S) to proline (P) change affecting the first amino acid of the mature PTH. Unexpectedly, biological activity of synthetic [P1]PTH(1-34) was indistinguishable from that of unmodified [S1]PTH(1-34). However, in contrast to conditioned medium from COS-7 cells expressing prepro[S1]PTH(1-84), medium from cells expressing prepro[P1]PTH(1-84) failed to stimulate cAMP production despite similar PTH levels when measured by an intact assay that detects PTH(1-84) and large amino-terminally truncated fragments thereof. Analysis of the secreted, but inactive PTH variant led to the identification of pro[P1]PTH(-6 to +84). Synthetic pro[P1]PTH(-6 to +34) and pro[S1]PTH(-6 to +34) had much less bioactivity than the corresponding PTH(1-34) analogs. Unlike pro[S1]PTH(-6 to +34), pro[P1]PTH(-6 to +34) was resistant to cleavage by furin suggesting that the amino acid variant impairs preproPTH processing. Consistent with this conclusion, plasma of patients with the homozygous P1 mutation had elevated proPTH levels, as determined with an in-house assay specific for pro[P1]PTH(-6 to +84). In fact, a large fraction of PTH detected by the commercial intact assay represented the secreted pro[P1]PTH. In contrast, two commercial biointact assays that use antibodies directed against the first few amino acid residues of PTH(1-84) for capture or detection failed to detect pro[P1]PTH.

Evaluation and Management of Primary Hyperparathyroidism: Summary Statement and Guidelines from the Fifth International Workshop.

The last international guidelines on the evaluation and management of primary hyperparathyroidism (PHPT) were published in 2014. Research since that time has led to new insights into epidemiology, pathophysiology, diagnosis, measurements, genetics, outcomes, presentations, new imaging modalities, target and other organ systems, pregnancy, evaluation, and management. Advances in all these areas are demonstrated by the reference list in which the majority of listings were published after the last set of guidelines. It was thus, timely to convene an international group of over 50 experts to review these advances in our knowledge. Four Task Forces considered: 1. Epidemiology, Pathophysiology, and Genetics; 2. Classical and Nonclassical Features; 3. Surgical Aspects; and 4. Management. For Task Force 4 on the Management of PHPT, Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) methodology addressed surgical management of asymptomatic PHPT and non-surgical medical management of PHPT. The findings of this systematic review that applied GRADE methods to randomized trials are published as part of this series. Task Force 4 also reviewed a much larger body of new knowledge from observations studies that did not specifically fit the criteria of GRADE methodology. The full reports of these 4 Task Forces immediately follow this summary statement. Distilling the essence of all deliberations of all Task Force reports and Methodological reviews, we offer, in this summary statement, evidence-based recommendations and guidelines for the evaluation and management of PHPT. Different from the conclusions of the last workshop, these deliberations have led to revisions of renal guidelines and more evidence for the other recommendations. The accompanying papers present an in-depth discussion of topics summarized in this report. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Evaluation and Management of Hypoparathyroidism Summary Statement and Guidelines from the Second International Workshop.

This clinical practice guideline addresses the prevention, diagnosis, and management of hypoparathyroidism (HypoPT) and provides evidence-based recommendations. The HypoPT task forces included four teams with a total of 50 international experts including representatives from the sponsoring societies. A methodologist (GG) and his team supported the taskforces and conducted the systematic reviews. A formal process following the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) methodology and the systematic reviews provided the structure for seven of the guideline recommendations. The task force used a less structured approach based on narrative reviews for 20 non-GRADEd recommendations. Clinicians may consider postsurgical HypoPT permanent if it persists for >12 months after surgery. To predict which patients will not develop permanent postsurgical HypoPT, we recommend evaluating serum PTH within 12 to 24 hours post total thyroidectomy (strong recommendation, moderate quality evidence). PTH > 10 pg/mL (1.05 pmol/L) virtually excludes long-term HypoPT. In individuals with nonsurgical HypoPT, genetic testing may be helpful in the presence of a positive family history of nonsurgical HypoPT, in the presence of syndromic features, or in individuals younger than 40 years. HypoPT can be associated with complications, including nephrocalcinosis, nephrolithiasis, renal insufficiency, cataracts, seizures, cardiac arrhythmias, ischemic heart disease, depression, and an increased risk of infection. Minimizing complications of HypoPT requires careful evaluation and close monitoring of laboratory indices. In patients with chronic HypoPT, the panel suggests conventional therapy with calcium and active vitamin D metabolites as first-line therapy (weak recommendation, low-quality evidence). When conventional therapy is deemed unsatisfactory, the panel considers the use of PTH. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Management of Primary Hyperparathyroidism.

Since the last international guidelines were published in 2014 on the evaluation and management of primary hyperparathyroidism (PHPT), new information has become available with regard to evaluation, diagnosis, epidemiology, genetics, classical and nonclassical manifestations, surgical and nonsurgical approaches, and natural history. To provide the most current summary of these developments, an international group, consisting of over 50 experts in these various aspects of PHPT, was convened. This paper provides the results of the task force that was assigned to review the information on the management of PHPT. For this task force on the management of PHPT, two questions were the subject of systematic reviews using the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) methodology. The full report addressing surgical and nonsurgical management of PHPT, utilizing the GRADE methodology, is published separately in this series. In this report, we summarize the results of that methodological review and expand them to encompass a much larger body of new knowledge that did not specifically fit the criteria of the GRADE methodology. Together, both the systematic and narrative reviews of the literature, summarized in this paper, give the most complete information available to date. A panel of experts then considered the last set of international guidelines in light of the newer data and assessed the need for their revision. This report provides the evidentiary background to the guidelines report. In that report, evidence from all task forces is synthesized into a summary statement and revised guidelines for the evaluation and management of PHPT. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Actions of Parathyroid Hormone Ligand Analogues in Humanized PTH1R Knockin Mice.

Rodent models are commonly used to evaluate parathyroid hormone (PTH) and PTH-related protein (PTHrP) ligands and analogues for their pharmacologic activities and potential therapeutic utility toward diseases of bone and mineral ion metabolism. Divergence, however, in the amino acid sequences of rodent and human PTH receptors (rat and mouse PTH1Rs are 91% identical to the human PTH1R) can lead to differences in receptor-binding and signaling potencies for such ligands when assessed on rodent vs human PTH1Rs, as shown by cell-based assays in vitro. This introduces an element of uncertainty in the accuracy of rodent models for performing such preclinical evaluations. To overcome this potential uncertainty, we used a homologous recombination-based knockin (KI) approach to generate a mouse (in-host strain C57Bl/6N) in which complementary DNA encoding the human PTH1R replaces a segment (exon 4) of the murine PTH1R gene so that the human and not the mouse PTH1R protein is expressed. Expression is directed by the endogenous mouse promoter and hence occurs in all biologically relevant cells and tissues and at appropriate levels. The resulting homozygous hPTH1R-KI (humanized) mice were healthy over at least 10 generations and showed functional responses to injected PTH analog peptides that are consistent with a fully functional human PTH1R in target bone and kidney cells. The initial evaluation of these mice and their potential utility for predicting behavior of PTH analogues in humans is reported here.

Optimization of PTH/PTHrP Hybrid Peptides to Derive a Long-Acting PTH Analog (LA-PTH).

Prolonged signaling at the parathyroid hormone receptor 1 (PTHR1) correlates with the capacity of a ligand to bind to a G protein-independent receptor conformation (R0). As long-acting PTH (LA-PTH) ligands hold interest as potential treatments for hypoparathyroidism (HP), we explored the structural basis in the ligand for stable R0 binding and prolonged cAMP signaling. A series of PTH/PTHrP hybrid analogs were synthesized and tested for actions in vitro and in vivo. Of the series, [Ala1,3,12,Gln10,Arg11,Trp14]-PTH(1-14)/PTHrP(15-36) (M-PTH/PTHrP) bound with high affinity to R0, induced prolonged cAMP responses in UMR106 rat osteoblast-derived cells, and induced the most prolonged increases in serum calcium (sCa) in normal rats. Daily s.c. injection of M-PTH/PTHrP into thyroparathyroidectomized (TPTX) rats, a model of HP, normalized sCa without raising urine Ca. In contrast, oral alfacalcidol, a widely used treatment for HP, normalized sCa, but induced frank hypercalciuria. M-PTH/PTHrP exhibited low solubility in aqueous solutions of neutral pH; however, replacement of Leu18, Phe22, and His26 with the less hydrophobic residues, Ala, Ala, and Lys, at those respective positions markedly improved solubility while maintaining bioactivity. Indeed, we recently showed that the resultant analog [Ala18,22,Lys26]-M-PTH/PTHrP or LA-PTH, effectively normalizes sCa in TPTX rats and mediates prolonged actions in monkeys. These studies provide useful information for optimizing PTH and PTHrP ligand analogs for therapeutic development. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

An Inverse Agonist Ligand of the PTH Receptor Partially Rescues Skeletal Defects in a Mouse Model of Jansen's Metaphyseal Chondrodysplasia.

Jansen's metaphyseal chondrodysplasia (JMC) is a rare disease of bone and mineral ion physiology that is caused by activating mutations in PTHR1. Ligand-independent signaling by the mutant receptors in cells of bone and kidney results in abnormal skeletal growth, excessive bone turnover, and chronic hypercalcemia and hyperphosphaturia. Clinical features further include short stature, limb deformities, nephrocalcinosis, and progressive losses in kidney function. There is no effective treatment option available for JMC. In previous cell-based assays, we found that certain N-terminally truncated PTH and PTHrP antagonist peptides function as inverse agonists and thus can reduce the high rates of basal cAMP signaling exhibited by the mutant PTHR1s of JMC in vitro. Here we explored whether one such inverse agonist ligand, [Leu11 ,dTrp12 ,Trp23 ,Tyr36 ]-PTHrP(7-36)NH2 (IA), can be effective in vivo and thus ameliorate the skeletal abnormalities that occur in transgenic mice expressing the PTHR1-H223R allele of JMC in osteoblastic cells via the collagen-1α1 promoter (C1HR mice). We observed that after 2 weeks of twice-daily injection and relative to vehicle controls, the IA analog resulted in significant improvements in key skeletal parameters that characterize the C1HR mice, because it reduced the excess trabecular bone mass, bone marrow fibrosis, and levels of bone turnover markers in blood and urine. The overall findings provide proof-of-concept support for the notion that inverse agonist ligands targeted to the mutant PTHR1 variants of JMC can have efficacy in vivo. Further studies of such PTHR1 ligand analogs could help open paths toward the first treatment option for this debilitating skeletal disorder. © 2019 American Society for Bone and Mineral Research.

Prolonged Pharmacokinetic and Pharmacodynamic Actions of a Pegylated Parathyroid Hormone (1-34) Peptide Fragment.

Polyethylene glycol (PEG) addition can prolong the pharmacokinetic and pharmacodynamic actions of a bioactive peptide in vivo, in part by impeding rates of glomerular filtration. For parathyroid hormone (PTH) peptides, pegylation could help in exploring the actions of the hormone in the kidney; e.g., in dissecting the relative roles that filtered versus blood-borne PTH play in regulating phosphate transport. It could also lead to potential alternate forms of treatment for hypoparathyroidism. We thus synthesized the fluorescent pegylated PTH derivative [Lys13 (tetramethylrhodamine {TMR}), Cys35 (PEG-20,000 Da)]PTH(1-35) (PEG-PTHTMR ) and its non-pegylated counterpart [Lys13 (TMR), Cys35 ]PTH(1-35) (PTHTMR ) and assessed their properties in cells and in mice. In PTHR1-expressing HEK-293 cells, PEG-PTHTMR and PTHTMR exhibited similar potencies for inducing cAMP signaling, whereas when injected into mice, the pegylated analog persisted much longer in the circulation (>24 hours versus ∼ 1 hour) and induced markedly more prolonged calcemic and phosphaturic responses than did the non-pegylated control. Fluorescence microscopy analysis of kidney sections obtained from the injected mice revealed much less PEG-PTHTMR than PTHTMR on the luminal brush-border surfaces of renal proximal tubule cells (PTCs), on which PTH regulates phosphate transporter function, whereas immunostained phosphorylated PKA substrate, a marker of cAMP signaling, was increased to similar extents for the two ligands and for each, was localized to the basolateral portion of the PTCs. Pegylation of a bioactive PTH peptide thus led to prolonged pharmacokinetic/pharmacodynamic properties in vivo, as well as to new in vivo data that support a prominent role for PTH action at basolateral surfaces of renal proximal tubule cells. © 2016 American Society for Bone and Mineral Research.

Management of Hypoparathyroidism: Present and Future.

CONTEXT: Conventional management of hypoparathyroidism has focused upon maintaining the serum calcium with oral calcium and active vitamin D, often requiring high doses and giving rise to concerns about long-term consequences including renal and brain calcifications. Replacement therapy with PTH has recently become available. This paper summarizes the results of the findings and recommendations of the Working Group on Management of Hypoparathyroidism. EVIDENCE ACQUISITION: Contributing authors reviewed the literature regarding physiology, pathophysiology, and nutritional aspects of hypoparathyroidism, management of acute hypocalcemia, clinical aspects of chronic management, and replacement therapy of hypoparathyroidism with PTH peptides. PubMed and other literature search engines were utilized. EVIDENCE SYNTHESIS: Under normal circumstances, interactions between PTH and active vitamin D along with the dynamics of calcium and phosphorus absorption, renal tubular handing of those ions, and skeletal responsiveness help to maintain calcium homeostasis and skeletal health. In the absence of PTH, the gastrointestinal tract, kidneys, and skeleton are all affected, leading to hypocalcemia, hyperphosphatemia, reduced bone remodeling, and an inability to conserve filtered calcium. Acute hypocalcemia can be a medical emergency presenting with neuromuscular irritability. The recent availability of recombinant human PTH (1-84) has given hope that management of hypoparathyroidism with the missing hormone in this disorder will provide better control and reduced needs for calcium and vitamin D. CONCLUSIONS: Hypoparathyroidism is associated with abnormal calcium and skeletal homeostasis. Control with calcium and active vitamin D can be a challenge. The availability of PTH (1-84) replacement therapy may usher new opportunities for better control with reduced supplementation requirements.

Management of Hypoparathyroidism: Summary Statement and Guidelines.

OBJECTIVE: Hypoparathyroidism is a rare disorder characterized by hypocalcemia and absent or deficient PTH. This report presents a summary of current information about epidemiology, presentation, diagnosis, clinical features, and management and proposes guidelines to help clinicians diagnose, evaluate, and manage this disorder. PARTICIPANTS: Participants in the First International Conference on the Management of Hypoparathyroidism represented a worldwide constituency with acknowledged interest and expertise in key basic, translational, and clinical aspects of hypoparathyroidism. Three Workshop Panels were constituted to address questions for presentation and discussion at the Conference held in Florence, Italy, May 7-9, 2015. At that time, a series of presentations were made, followed by in-depth discussions in an open forum. Each Workshop Panel also met in closed sessions to formulate the three evidence-based reports that accompany this summary statement. An Expert Panel then considered this information, developed summaries, guidelines, and a research agenda that constitutes this summary statement. EVIDENCE: Preceding the conference, each Workshop Panel conducted an extensive literature search as noted in the individual manuscripts accompanying this report. All presentations were based upon the best peer-reviewed information taking into account the historical and current literature. CONSENSUS PROCESS: This report represents the Expert Panel's synthesis of the conference material placed in a context designed to be relevant to clinicians and those engaged in cutting-edge studies of hypoparathyroidism. CONCLUSIONS: This document not only provides a summary of our current knowledge but also places recent advances in its management into a context that should enhance future advances in our understanding of hypoparathyroidism.

Pharmacodynamic Actions of a Long-Acting PTH Analog (LA-PTH) in Thyroparathyroidectomized (TPTX) Rats and Normal Monkeys.

Hypoparathyroidism is a disease of chronic hypocalcemia and hyperphosphatemia due to a deficiency of parathyroid hormone (PTH). PTH and analogs of the hormone are of interest as potential therapies. Accordingly, we examined the pharmacological properties of a long-acting PTH analog, [Ala(1,3,12,18,22) , Gln(10) ,Arg(11) ,Trp(14) ,Lys(26) ]-PTH(1-14)/PTHrP(15-36) (LA-PTH) in thyroparathyroidectomized (TPTX) rats, a model of HP, as well as in normal monkeys. In TPTX rats, a single intravenous administration of LA-PTH at a dose of 0.9 nmol/kg increased serum calcium (sCa) and decreased serum phosphate (sPi) to near-normal levels for longer than 48 hours, whereas PTH(1-34) and PTH(1-84), each injected at a dose 80-fold higher than that used for LA-PTH, increased sCa and decreased sPi only modestly and transiently (<6 hours). LA-PTH also exhibited enhanced and prolonged efficacy versus PTH(1-34) and PTH(1-84) for elevating sCa when administered subcutaneously (s.c.) into monkeys. Daily s.c. administration of LA-PTH (1.8 nmol/kg) into TPTX rats for 28 days elevated sCa to near normal levels without causing hypercalciuria or increasing bone resorption markers, a desirable goal in the treatment of hypoparathyroidism. The results are supportive of further study of long-acting PTH analogs as potential therapies for patients with hypoparathyroidism. © 2016 American Society for Bone and Mineral Research.

Guidelines for the management of asymptomatic primary hyperparathyroidism: summary statement from the Fourth International Workshop.

OBJECTIVE: Asymptomatic primary hyperparathyroidism (PHPT) is routinely encountered in clinical practices of endocrinology throughout the world. This report distills an update of current information about diagnostics, clinical features, and management of this disease into a set of revised guidelines. PARTICIPANTS: Participants, representing an international constituency, with interest and expertise in various facets of asymptomatic PHPT constituted four Workshop Panels that developed key questions to be addressed. They then convened in an open 3-day conference September 19-21, 2013, in Florence, Italy, when a series of presentations and discussions addressed these questions. A smaller subcommittee, the Expert Panel, then met in closed session to reach an evidence-based consensus on how to address the questions and data that were aired in the open forum. EVIDENCE: Preceding the conference, each question was addressed by a relevant, extensive literature search. All presentations and deliberations of the Workshop Panels and the Expert Panel were based upon the latest information gleaned from this literature search. CONSENSUS PROCESS: The expert panel considered all the evidence provided by the individual Workshop Panels and then came to consensus. CONCLUSION: In view of new findings since the last International Workshop on the Management of Asymptomatic PHPT, guidelines for management have been revised. The revised guidelines include: 1) recommendations for more extensive evaluation of the skeletal and renal systems; 2) skeletal and/or renal involvement as determined by further evaluation to become part of the guidelines for surgery; and 3) more specific guidelines for monitoring those who do not meet guidelines for parathyroid surgery. These guidelines should help endocrinologists and surgeons caring for patients with PHPT. A blueprint for future research is proposed to foster additional investigation into issues that remain uncertain or controversial.

Critical role of parathyroid hormone (PTH) receptor-1 phosphorylation in regulating acute responses to PTH.

Agonist-induced phosphorylation of the parathyroid hormone (PTH) receptor 1 (PTHR1) regulates receptor signaling in vitro, but the role of this phosphorylation in vivo is uncertain. We investigated this role by injecting "knock-in" mice expressing a phosphorylation-deficient (PD) PTHR1 with PTH ligands and assessing acute biologic responses. Following injection with PTH (1-34), or with a unique, long-acting PTH analog, PD mice, compared with WT mice, exhibited enhanced increases in cAMP levels in the blood, as well as enhanced cAMP production and gene expression responses in bone and kidney tissue. Surprisingly, however, the hallmark hypercalcemic and hypophosphatemic responses were markedly absent in the PD mice, such that paradoxical hypocalcemic and hyperphosphatemic responses were observed, quite strikingly with the long-acting PTH analog. Spot urine analyses revealed a marked defect in the capacity of the PD mice to excrete phosphate, as well as cAMP, into the urine in response to PTH injection. This defect in renal excretion was associated with a severe, PTH-induced impairment in glomerular filtration, as assessed by the rate of FITC-inulin clearance from the blood, which, in turn, was explainable by an overly exuberant systemic hypotensive response. The overall findings demonstrate the importance in vivo of PTH-induced phosphorylation of the PTHR1 in regulating acute ligand responses, and they serve to focus attention on mechanisms that underlie the acute calcemic response to PTH and factors, such as blood phosphate levels, that influence it.

A short history of parathyroid hormone, its biological role, and pathophysiology of hormone excess.

Research on parathyroid hormone (PTH) over the preceding century was an exciting but sometimes confusing tale, with steady advances yet long periods of stalled progress, angry debates, and missed opportunities. There were sometimes fierce debates about the function of the parathyroids. These were finally resolved by 1925 when a potent biologic extract useful for testing in animals was finally made by Collip, and the role of PTH in calcium metabolism was established unequivocally. In the decades that followed, the pathophysiology of hormone excess (severe bone loss and other symptoms) was elucidated. Diagnosis can now be made with high reliability, even in the absence of clinical manifestation. The modern clinical profile of asymptomatic hyperparathyroidism is best described as a disorder in which there are neither signs nor symptoms traditionally associated with hypercalcemia or PTH excess.

Benefits and risks of bisphosphonate therapy for osteoporosis.

CONTEXT: There has been considerable concern recently in the scientific and lay media regarding the benefits vs. the risks of bisphosphonates for the treatment of osteoporosis. Risks include possible associations with osteonecrosis of the jaw (ONJ) and atypical femur fractures. In this perspective, we review the use of bisphosphonates for the treatment of osteoporosis, including an objective assessment of the risks vs. the benefits of these drugs. EVIDENCE ACQUISITION: Authors' knowledge of the field and results of focused literature searches are presented. EVIDENCE SYNTHESIS: Bisphosphonates have proven efficacy in the prevention of bone loss and in the reduction of fractures in postmenopausal women and men with established osteoporosis. Although bisphosphonates, at doses used to treat osteoporosis, may be associated with an increased risk of ONJ and atypical femur fractures, many more fractures are prevented by the use of these drugs compared to the relatively low risk of these complications. Although oral bisphosphonates are associated with upper gastrointestinal side effects and iv bisphosphonates with acute phase reactions, the association of bisphosphonate use with esophageal cancer and atrial fibrillation is not well supported by current data. CONCLUSIONS: Bisphosphonates have been proven to prevent fractures in patients with established osteoporosis or those who are at high risk of fracture. In contrast, the incidence of major complications associated with bisphosphonate use, such as ONJ and atypical femur fractures, is very low.