Radioimmunoassays for the 28–48 region of parathyroid hormone detect intact hormone but not hormone fragments

Summary Metabolism of parathyroid hormone (PTH) is known to involve cleavage in the 28-48 region. With the goal of establishing a radioimmunoassay specific for the intact PTH molecule, we have tested the hypothesis that assays specific for the 28-48 region might fail to recognize the hormone fragments arising in vivo. Antisera against native bovine PTH (bPTH) contain antibody species directed at the 28-48 region of bPTH, since (a) 5 of 6 such antisera showed inhibition of binding of¹²⁵I-labeled bPTH-(1-84) by unlabeled synthetic bPTH-(28-48), and (b) 9 of 9 such antisera bound¹²⁵I-labeled bPTH-(28-48). The latter radioligand yields assays specific for the 28-48 region; 5 of 5 such assays recognized intact bPTH-(1-84), but none recognized bPTH-(1-34), bPTH-(37-84), or both fragments together. Binding of¹²⁵I-bPTH-(28-48) to anti-bPTH sera was not inhibited by intact human PTH (hPTH). Likewise, only 1 of 6 antisera against native hPTH would bind¹²⁵I-bPTH-(28-48) or show inhibition of¹²⁵I-bPTH-(1-84) binding by unlabeled bPTH-(28-48). This poor immunological cross-reactivity between the 28-48 regions of bPTH and hPTH implies a major conformational transition between the two hormones in the 40–47 region of the molecule where the primary structure differs at 5 of 8 positions. A 28-48 specific assay employing an antiserum raised against both bPTH and hPTH did recognize both bPTH-(1-84) and hPTH-(1-84), but not bPTH-(1-34), hPTH-(1-34), bPTH-(37-84), or hPTH-(44-68). Thus, 6 different 28-48 region-specific assays have been shown to recognize intact PTH but not hormone fragments resembling those thought to be formed in vivo.     

Relative sensitivity of kidney and bone to the amino-terminal fragment b-PTH (1–30) of native bovine parathyroid hormone: Implications for assessment of bioactivity of parathyroid hormone fragments in vivo and in vitro

Summary Adenylate cyclase activation in renal cortical membranes is widely used to assess the potency of parathyroid hormone (PTH) and hormonal fragments. Recent studies, however, suggest that the structural requirements for biological activity of PTH in bone may differ from those in kidney. In isolated perfused canine bone, cyclic AMP production is markedly increased by syn-b-PTH (1–34) whereas intact b-PTH (1–84) has minimal effect. Furthermore, a potent inhibitor of PTH stimulated adenylate cyclase activity, Nle⁸Nle¹⁸Tyr³⁴ syn-b-PTH (3–34) NH₂, devoid of biological activity in kidney, is an agonist in isolated bone. The present studies examine the effects in bone and kidney of the amino-terminal fragment b-PTH (1–30), prepared by cleavage of intact b-PTH (1–84) by Cathepsin Bin vitro. In basolateral canine renal cortical membranes, b-PTH (1–30) was less active than syn-b-PTH (1–34) in its ability to stimulate adenylate cyclase. Half maximal stimulation of adenylate cyclase activity by b-PTH (1–30) was 50 nM compared with 2 nM for syn-b-PTH (1–34). Maximum enzyme activation by b-PTH (1–30) reached only 50% of the activation by syn-b-PTH (1–34). Addition of Gpp(NH)p (1 mM) increased the affinities for both peptides. The relative difference in potency, however, remained unchanged. In contrast, in isolated bone, b-PTH (1–30) and syn-b-PTH (1–34) were equipotent in increasing cyclic AMP production. These data provide further evidence for differences in the structural requirements for PTH activity in bone and kidney and suggest that bioassays of PTH and PTH fragments in kidney may not accurately reflect the effects of PTH in bone.     

Parathyroid hormone receptors in human dermal fibroblasts: structural and functional characterization

Previous studies have established the presence of parathyroid hormone (PTH)-sensitive adenylate cyclase activity in cultured human skin fibroblasts. The present study was undertaken to identify and quantitate PTH receptors directly in such cells. Human dermal fibroblast cell line CRL 1564 was found to possess specific binding sites for [125I]PTH(1-34). These sites bound PTH selectively; bovine and human PTH(1-34) and PTH(1-84) competed for [125I]PTH(1-34) binding sites, whereas the unrelated peptides calcitonin, insulin, AVP, angiotensin II, and ACTH(1-24) were inactive even at micromolar concentrations. Competitive binding experiments demonstrated the presence of binding site heterogeneity. These data fit a "two-site" model (p less than 0.001) in which one binding component has high affinity (Kd = 2.5 ng/ml = 0.6 nM) and low capacity (10(4) sites/cell) while the other has low affinity (Kd = 5.9 micrograms/ml = 1.5 microM) and high capacity (greater than 10(7) sites/cell). Similar high- and low-affinity [125I]bPTH(1-34) binding sites were seen also in CRL 1564 membranes containing a PTH-responsive adenylate cyclase. The Kd of the high-affinity sites was identical to the concentration of unlabeled bPTH(1-34) (4.2 ng/ml = 1.0 nM) required to half-maximally elevated cyclic AMP in CRL 1564 cells. Affinity labeling of specific PTH binding sites revealed the presence of multiple components with Mrs of 85, 70, 40, 33, and 23 kD on SDS-PAGE. Competition experiments did not disclose structurally discrete high- and low-affinity sites. Thus, structurally homologous PTH receptors in human skin fibroblasts apparently can assume two affinity states: (i) a high-affinity state coupled to adenylate cyclase and (ii) a low-affinity state that may represent uncoupled receptors.     

Tissue deposition and metabolism of125I-labeled synthetic amino-terminal parathyroid hormone bPTH(1–34)

Summary The tissue deposition and metabolism of¹²⁵I-labeled synthetic amino-terminal parathyroid hormone, bPTH(1–34), were studied in rats. In comparison with the intact hormone molecule bPTH(1–84), the synthetic fragment was (a) cleared more rapidly from serum; (b) degraded more rapidly in peripheral tissues; (c) deposited to a greater extent in kidney; (d) deposited to a much smaller extent in liver. In bone, the accumulation of total radioactivity was approximately the same with both labeled hormones. The possible physiological significance of these patterns of distribution and metabolism is discussed.     

Solubilization of functional receptors for parathyroid hormone and parathyroid hormone-related peptide from clonal rat osteosarcoma cells,ROS17/2.8

Summary ROS17/2.8 cells, a cell line derived from a rat osteosarcoma, have abundant receptors for parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP). A particulate membrane fraction was prepared from these cells and it was solubilized using relatively mild conditions with digitonin (0.25%), a nonionic detergent. When radioligands of both PTH and PTHrP were incubated with this membrane fraction in the absence of any protease inhibitor at 15°C, approximately 75% of these radioligands were degraded within 2 hours. This degradative activity was inhibited more effectively by bacitracin than by any of several other protease inhibitors tested. The digitonin-solubilized PTH/PTHrP receptors were radiolabeled in the presence of bacitracin using radioiodinated [Tyr³⁶]PTHrP(1-36) amide (PTHrP(1-36) and N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), as cross-linker. When an aliquot of the reaction solution was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography, a broad band was observed that had an apparent molecular size of 90,000 daltons (M_r=90 kD). This band was no longer seen when the binding was conducted in the presence of 10^-6 M of unlabeled PTHrP(1-36), and it was decreased in density when binding was conducted in the presence of 10^-6 M of unlabeled [Nle^8,18, Tyr³⁴] bovine PTH(1-34) amide (NlePTH). The solubilized receptors retained their capacity to bind the radioligand after partial purification by wheat-germ agglutinin affinity-chromatography. The use of relatively mild detergent conditions thus offers a means to solubilize receptors that retain their capacity to bind PTH and PTHrP.     

Efficacy of whole PTH assay and 1-84/non-(1-84) PTH ratio in patients on hemodialysis or undergoing parathyroidectomy

For measurement of parathyroid hormone (PTH), the intact PTH (I-PTH) assay is generally used. However, I-PTH assay apparently detects PTH 7-84 fragments, in addition to PTH 1-84. The whole PTH (W-PTH) assay exhibits no cross-reactivity with PTH 7-84. Ratio of PTH 1-84 to non-(1-84) PTH has been proposed as a non-invasive indicator of bone turnover in patients with end-stage renal disease (ESRD). We evaluated the efficacy of W-PTH assay and 1-84/non-(1-84) PTH ratio in hemodialysis patients and patients who had undergone parathyroidectomy. PTH levels were measured using W-PTH and I-PTH assays in 138 hemodialysis patients, 27 healthy controls and 10 patients who were scheduled to undergo parathyroidectomy for secondary hyperparathyroidism. Alkaline phosphatase, bone alkaline phosphatase and intact osteocalcin were also measured for comparison with 1-84/non-(1-84) PTH ratio. W-PTH was strongly correlated with I-PTH in both groups, and with all three bone metabolic markers in hemodialysis patients. In hemodialysis patients, both PTH and bone metabolic markers were significantly lower in the subgroup with a PTH ratio < 1.0 than in the subgroup with a PTH ratio > or = 1.0. However, PTH ratio exhibited no significant correlation with bone metabolism markers. PTH ratio was higher after parathyroidectomy than before. W-PTH and I-PTH assays offer identical indicators of bone metabolism in ESRD patients. However, 1-84/non-(1-84) PTH ratio may be of limited diagnostic use regarding bone turnover if a cut-off value of 1.0 is used. PTH 1-84 may be secreted relatively more than non-(1-84) PTH after parathyroidectomy, due to decreases in serum calcium. As the W-PTH assay allows easy calculation of non-(1-84) PTH by subtracting the I-PTH value, this assay contributes to identification of the function of non-(1-84) PTH fragments in various conditions.     

Binding of the carboxyl-terminal fragments of human parathyroid hormone to rat osteoblastic cells ROS 17/2.8

We have recently demonstrated that the carboxyl-terminal (C-terminal) PTH fragments increase or decrease alkaline phosphatase activity in dexamethasone-treated ROS 17/2.8 cells, depending on the length of deletion of amino-terminal amino acids of the PTH molecule, and interact with amino-terminal (N-terminal) PTH fragment [Acta Endocrinol 128:367]. In the present study, we examined individual and combined inhibitory effects of N-terminal and a series of truncated C-terminal PTH fragments [PTH (1-34), (35-84), (53-84) and (71-84)] on the binding of intact PTH molecule [PTH (1-84)] to ROS 17/2.8 cells. The C-terminal PTH fragments, as well as the N-terminal PTH fragment, partially inhibited the binding of [³⁵S]-labeled PTH (1-84) to the cells. The inhibitory effect of C-terminal PTH fragments was reduced along with the deletion of aminoterminal amino acids of the PTH molecule, but still retained in the shortest C-terminal PTH fragment, PTH (71-84). When added together, PTH (1-34) reinforced the inhibitory effect of each C-terminal PTH fragment. The combination of PTH (1-34) and the complementary C-terminal PTH fragment, PTH (35-84), resulted in inhibition of [³⁵S] PTH (1-84) binding to the level obtained by addition of the same concentration of unlabeled PTH (1-84).     

Formation and serum disappearance of fragments of parathyroid hormone in the infused dog

Summary Radioiodinated parathyroid hormone [¹²⁵I-PTH(1-84)] was infused into intact dogs. At various times venous samples were chromatographed to determine the levels of intact hormone (1-84) and large fragments in the circulation. Both bioactive (electrolytically iodinated) and inactive (chloramine-T-labeled) preparations were used. In both instances, plateau concentrations of intact hormone and of large metabolite(s) were quickly reached. After cessation of infusion the levels of intact hormone and metabolite(s) quickly declined. Clearly, in the dog, the peripheral formation and disappearance of large fragments of exogenous PTH occur at rates comparable to the clearance of the intact hormone itself.     

The reversed ratio of 1-84 PTH (whole PTH)/intact PTH in a patient on hemodialysis associated with primary hyperparathyroidism

Intact PTH measures not only 1-84 PTH, but also other fragments such as 7-84 PTH. Lately, a measurement of 1-84 PTH has been available as whole PTH assay and the ratio of whole PTH/intact PTH is considered to be between 0.5 and 0.7 in patients on hemodialysis. Therefore, intact PTH should be higher than whole PTH. We present a 57-year-old male with chronic renal failure on hemodialysis whose whole PTH was higher than intact PTH (the reversed ratio of whole PTH/intact PTH). He showed one enlarged parathyroid gland by an ultrasonic test, CT examination and RI subtraction study. After this gland was removed by surgery, the ratio of whole PTH/intact PTH normalized. The size of the resected gland was 22 x 15 x 11 mm. The histologic examination revealed adenoma. This indicates that, if patients with chronic renal failure showed the reversed ratio of whole PTH/intact PTH, the possibility that they could have primary hyperparathyroidism in addition to secondary hyperparathyroidism should be considered.     

Circulating 1-84 PTH and large C-terminal PTH fragment levels in uremia

Background. The so-called intact parathyroid hormone (iPTH) assay detects large C-terminal PTH fragments that are lacking several N-terminal amino acid residues in addition to 1-84 PTH molecules.Methods. Blood samples were obtained from 65 predialysis patients (male, 35; female, 30) and 109 dialysis patients (male, 73; female, 36). The plasma 1-84 PTH levels were determined by a specific immunoradiometric assay (IRMA).Results. The ratio of 1-84 PTH/iPTH did not show a significant correlation with glomerular filtration rate (GFR) among patients with a GFR of more than 80ml/min, while it showed a positive correlation with GFR among patients with a GFR of less than 80ml/min. The ratio of 1-84 PTH/iPTH demonstrated a significant tendency to decrease in the order of patients with normal renal function (0.928 ± 0.182), those with renal dysfunction (0.836 ± 0.186; P < 0.05 vs patients with GFR > 80ml/min [i.e., normal renal function]), and those with maintenance hemodialysis (0.618 ± 0.123; P < 0.01 vs patients with GFR > 80ml/min). The plasma levels of 1-84 PTH and conventional iPTH showed a close correlation in dialysis patients. Neither 1-84 PTH levels nor secondary parameters calculated from them showed a better correlation with bone metabolic markers than iPTH levels.Conclusions. Circulating large C-terminal PTH fragment levels are increased in uremic patients. However, this noninvasive study failed to demonstrate the superiority of the specific 1-84 assay compared with the conventional iPTH assay to evaluate bone metabolism.     

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, Gln¹⁰,Arg¹¹,Trp¹⁴,Lys²⁶]‐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.     

Tissue deposition and metabolism of 125I-labeled synthetic amino-terminal parathyroid hormone bPTH(1-34)

The tissue deposition and metabolism of 125I-labeled synthetic amino-terminal parathyroid hormone, bPTH(1-34), were studied in rats. In comparison with the intact hormone molecule bPTH(1-84), the synthetic fragment was (a) cleared more rapidly from serum; (b) degraded more rapidly in peripheral tissues; (c) deposited to a greater extent in kidney; (d) deposited to a much smaller extent in liver. In bone, the accumulation of total radioactivity was approximately the same with both labeled hormones. The possible physiological significance of these patterns of distribution and metabolism is discussed.     

New PTH assays and renal osteodystrophy

Parathyroid hormone (PTH) levels have been used instead of bone histomorphometric analysis in renal failure, but the assessment of tetracycline-labeled bone biopsy remains the most reliable method to diagnose the different subtypes of renal osteodystrophy. The availability of the first-generation immunometric PTH assay (1^st PTH-IMA) allowed the distinction between the different types of renal bone diseases. However, 1^st PTH-IMA not only detects the intact hormone PTH(1–84), but also additional PTH truncated fragments. A second-generation immunometric PTH assay (2^nd PTH-IMA) recognizes only PTH(1–84) and possible PTH fragments that are truncated at the carboxyl-terminus, but not PTH(7–84). In addition, whether assessment of the ratio PTH(1–84) and amino-terminally truncated PTH(1–84) fragments is a better predictor of bone turnover remains controversial. An initial study using the 2^nd PTH-IMA suggested that the ratio between PTH(1–84) and amino-terminally truncated PTH(1–84) fragments more accurately predicts bone turnover in adult patients treated with hemodialysis. However, subsequent studies using the Scantibodies assay have failed to better predict the underlying bone disease in adults undergoing maintenance hemodialysis. Furthermore, a different 2^nd PTH-IMA (Immutopics) with similar, but not identical, in vitro characteristics did not show a superior predictive value of the ratio in pediatric patients treated with peritoneal dialysis. Although the 2^nd PTH-IMA may provide important new insights into the physiology of parathyroid gland function, at present, measurement of PTH using either 1^st or 2^nd PTH-IMAs provides similar accuracy for predicting bone turnover in patients treated with dialysis. Thus, the current data do not yet support the claim that 2^nd PTH-IMAs provide an advantage over 1^st PTH-IMAs for the diagnosis of the different subtypes of renal bone diseases.     

Differential effects of intermittent PTH(1-34) and PTH(7-34) on bone microarchitecture and aortic calcification in experimental renal failure

PTH(1-84) and PTH(7-84) are elevated in chronic kidney disease (CKD). These peptides, as their shorter analogs PTH(1-34) and PTH(7-34) both promote PTH receptor (PTH1R) internalization but only PTH(1-34) and PTH(1-84) activate the receptor. Here, we examined the effects of intermittent administration of PTH(1-34) and PTH(7-34) on mineral ion metabolism, bone architecture, and vascular calcification in rats with experimental CKD. CKD with or without parathyroidectomy (PTX) was established by 5/6 nephrectomy (NPX) in rats. Animals were divided into 4 groups: Sham PTX+ sham NPX (Sham); PTX+ sham NPX (PTX); Sham PTX+NPX (NPX); PTX+NPX (PTX/NPX). Rats were treated with single daily doses of 40 microg/kg PTH(1-34), PTH(7-34), or vehicle. Creatinine was higher in NPX and Ca lower in PTX and PTX/NPX groups than in Sham or NPX rats. Plasma phosphate was higher in PTX, NPX and PTX/NPX than in Sham rats. PTH(1-34) was more hypercalcemic than PTH(7-34) in PTX rats. Fractional bone volume in rats treated with PTH(1-34) increased significantly in all groups compared to that of vehicle treatment. In addition, trabecular number, thickness and volumetric bone density increased in rats treated with PTH(1-34). In contrast, PTH(1-34) diminished vascular calcification. Bone and renal PTH1R mRNA expression was reduced as much or more in PTX/NPX rats as in NPX alone, whereas PTH(7-34) had no effect on PTH1R expression. Renal but not bone PTH1R mRNA increased in response to PTH(1-34). These findings suggest that PTH(1-34) exerts greater hypercalcemic and anabolic effects in parathyroidectomized and/or nephrectomized rats than does PTH(7-34). There was no evidence for significant bone or vascular actions of PTH(7-34). We conclude that PTH(1-34) protects against vascular calcification and bone demineralization in experimental renal failure.     

Intact PTH assay overestimates true 1-84 PTH levels after maxacalcitol therapy in dialysis patients with secondary hyperparathyroidism.

BACKGROUND: Although the so-called intact parathyroid hormone (iPTH) assay detects not only true 1-84 PTH (1-84PTH) but also large C-terminal PTH fragments, it remains inconclusive whether the 1-84PTH assay is more useful in clinical practice. Previous studies have shown that the results of these two PTH assays in dialysis patients are closely correlated. METHODS: Chronic dialysis patients whose plasma iPTH levels were >400 pg/ml were selected for inclusion in the present study. Following a 4 week wash-out time during which all vitamin D administration was halted, maxacalcitol was intravenously injected at the end of dialysis sessions three times per week for 24 weeks, at an initial dosage of 10 micro g. RESULTS: Ninety-seven patients with secondary hyperparathyroidism were included in our analysis. Their serum calcium levels were elevated from the start levels while phosphate levels remained unchanged. The plasma 1-84PTH levels constantly declined throughout the 24 weeks. Although the patients' plasma 1-84PTH and iPTH levels were closely correlated with each other both at the beginning of the study and after 24 weeks of maxacalcitol therapy, the ratio of 1-84PTH/iPTH consistently decreased throughout the study period (P<0.01). The changes in the ratio were significantly correlated with changes in serum calcium levels. CONCLUSIONS: Twenty-four weeks of intravenous maxacalcitol injection therapy significantly reduced the 1-84PTH/iPTH ratio. Estimated 1-84PTH levels from iPTH levels using a conversion formula obtained before the treatment were 21.0+/-20.4% higher than measured 1-84PTH levels after the therapy. Thus, iPTH measurement has a potential risk to overestimate 1-84PTH levels when evaluating the efficacy of maxacalcitol therapy in dialysis patients with secondary hyperparathyroidism.     

Skeletal adenylate cyclase: Effect of Mg2+, Ca2+, and PTH

Summary Plasma membranes were prepared from mineralized guinea pig bone in order to study Mg²⁺ and Ca²⁺ modulation of skeletal adenylate cyclase. Plasma membrane preparation was accomplished by crushing the bone in liquid N₂ and subsequent multiple washings in buffer containing EGTA to remove all Ca²⁺ prior to adenylate cyclase assay. Skeletal adenylate cyclase was found to be dependent on GTP and Mg²⁺ and responsive to bovine 1-34 PTH. Ca²⁺ caused a competitive inhibition of Mg²⁺-activated skeletal adenylate cyclase. The apparent KaMg was 1.9±0.3 in the presence of 0.2 μM Ca²⁺ but increased to a mean of 7.2±1.3 in the presence of 5.0 μM Ca²⁺. Analysis of the Ca²⁺ inhibition curves at concentrations from .05 μM-1.0 mM were consistent with the presence of two Ca²⁺ inhibition sites, one with an apparent Ki of 1–2 μM and the other with an apparent Ki of approximately 500 μM. Lowering the Mg²⁺ concentration increased the contribution of the high affinity Ca²⁺ binding site to the overall Ca²⁺ inhibition, and raising the Mg²⁺ concentration had the opposite effect. While bPTH 1-34 enhanced adenylate cyclase activity, it did not increase the affinity of Mg²⁺ for skeletal adenylate cyclase nor did it alter the K_iCa or the pattern of Ca²⁺ inhibition. These data may explain the skeletal resistance to PTH during Mg deficiency. A fall in the intracellular Mg would render the adenylate cyclase more susceptible to inhibition by the prevailing intracellular Ca²⁺ concentration. Since PTH does not appear to modulate either Mg²⁺ activation or Ca²⁺ inhibition of skeletal adenylate cyclase, cAMP-mediated PTH induction of bone resorption would be impaired, and hypocalcemia would occur.     

Differing effects of PTH 1–34, PTH 1–84, and zoledronic acid on bone microarchitecture and estimated strength in postmenopausal women with osteoporosis: An 18‐month open‐labeled observational study using HR‐pQCT

Whereas the beneficial effects of intermittent treatment with parathyroid hormone (PTH) (intact PTH 1–84 or fragment PTH 1–34, teriparatide) on vertebral strength is well documented, treatment may not be equally effective in the peripheral skeleton. We used high‐resolution peripheral quantitative computed tomography (HR‐pQCT) to detail effects on compartmental geometry, density, and microarchitecture as well as finite element (FE) estimated integral strength at the distal radius and tibia in postmenopausal osteoporotic women treated with PTH 1–34 (20 µg sc daily, n = 18) or PTH 1–84 (100 µg sc daily, n = 20) for 18 months in an open‐label, nonrandomized study. A group of postmenopausal osteoporotic women receiving zoledronic acid (5 mg infusion once yearly, n = 33) was also included. Anabolic therapy increased cortical porosity in radius (PTH 1–34 32 ± 37%, PTH 1–84 39 ± 32%, both p < 0.001) and tibia (PTH 1–34 13 ± 27%, PTH 1–84 15 ± 22%, both p < 0.001) with corresponding declines in cortical density. With PTH 1–34, increases in cortical thickness in radius (2.0 ± 3.8%, p < 0.05) and tibia (3.8 ± 10.4%, p < 0.01) were found. Trabecular number increased in tibia with both PTH 1–34 (4.2 ± 7.1%, p < 0.05) and PTH 1–84 (5.3 ± 8.3%, p < 0.01). Zoledronic acid did not impact cortical porosity at either site but increased cortical thickness (3.0 ± 3.5%, p < 0.01), total (2.7 ± 2.5%, p < 0.001) and cortical density (1.5 ± 2.0%, p < 0.01) in tibia as well as trabecular volume fraction in radius (2.5 ± 5.1%, p < 0.05) and tibia (2.2 ± 2.2%, p < 0.01). FE estimated bone strength was preserved, but not increased, with PTH 1–34 and zoledronic acid at both sites, whereas it decreased with PTH 1–84 in radius (?2.8 ± 5.8%, p < 0.05) and tibia (–3.9 ± 4.8%, p < 0.001). Conclusively, divergent treatment‐specific effects in cortical and trabecular bone were observed with anabolic and zoledronic acid therapy. The finding of decreased estimated strength with PTH 1–84 treatment was surprising and warrants confirmation. © 2013 American Society for Bone and Mineral Research.     

Abaloparatide at the Same Dose Has the Same Effects on Bone as PTH (1-34) in Mice

Abaloparatide, a novel analog of parathyroid hormone-related protein (PTHrP 1–34), became in 2017 the second osteoanabolic therapy for the treatment of osteoporosis. This study aims to compare the effects of PTH (1-34), PTHrP (1-36), and abaloparatide on bone remodeling in male mice. Intermittent daily subcutaneous injections of 80 μg/kg/d were administered to 4-month-old C57Bl/6J male mice for 6 weeks. During treatment, mice were followed by DXA-Piximus to assess changes in bone mineral density (BMD) in the whole body, femur, and tibia. At either 4 or 18 hours after the final injection, femurs were harvested for μCT analyses and histomorphometry, sera were assayed for bone turnover marker levels, and tibias were separated into cortical, trabecular, and bone marrow fractions for gene expression analyses. Our results showed that, compared with PTH (1-34), abaloparatide resulted in a similar increase in BMD at all sites, whereas no changes were found with PTHrP (1-36). With both PTH (1-34) and abaloparatide, μCT and histomorphometry analyses revealed similar increases in bone volume associated with an increased trabecular thickness, in bone formation rate as shown by P1NP serum level and in vivo double labeling, and in bone resorption as shown by CTX levels and osteoclast number. Gene expression analyses of trabecular and cortical bone showed that PTH (1-34) and abaloparatide led to different actions in osteoblast differentiation and activity, with increased Runx2, Col1A1, Alpl, Bsp, Ocn, Sost, Rankl/Opg, and c-fos at different time points. Abaloparatide seems to generate a faster response on osteoblastic gene expression than PTH (1-34). Taken together, abaloparatide at the same dose is as effective as PTH (1-34) as an osteoanabolic, with an increase in bone formation but also an increase in bone resorption in male mice. © 2019 American Society for Bone and Mineral Research.     

Identification and characterization of parathyroid hormone receptors in rat renal cortical plasma membranes using radioligand binding

Parathyroid hormone (PTH) receptors have been described in renal tissue from several species, but not in the rat. In this study, radioligand binding techniques were used to identify and characterize PTH receptors in rat kidney cortical membranes. The sulfur-free PTH analog [Nle8,18Tyr34]bovine PTH-(1-34)amide was iodinated using the iodogen method. This ligand was suitable for use in identifying PTH receptors in canine renal membranes, but not rat renal membranes. Synthetic, unsubstituted rat PTH-(1-34) was iodinated using the milder, lactoperoxidase technique and was purified by HPLC on a C8 column. [125I]rat PTH-(1-34) bound rapidly to both rat and dog renal membranes. At 22 degrees C reaction reached steady state within 20 minutes, and this level was maintained for at least 3 h. Specific binding was routinely greater than 90% for rat kidney and greater than 95% for dog kidney. Similar results were obtained at 4 degrees C with a longer time required to attain steady state (approximately 45 minutes). Binding was reversible as demonstrated by dissociation of bound ligand after either infinite dilution or displacement with excess nonradioactive PTH. Binding was saturable and of high affinity (rat kidney: Bmax = 2.3 pmol/mg protein, Kd = 3.1 nM, dog kidney: Bmax = 2.1 pmol/mg protein, Kd = 3.7 nM). Rat renal cortical adenylate cyclase activity was stimulated by rat PTH in a dose-dependent manner with an EC50 of 4 nM, a value in good agreement with the binding data. This study demonstrates the feasibility of identifying and characterizing parathyroid hormone receptors in rat renal cortical plasma membranes using radioligand binding techniques.     

ZP2307, a novel, cyclic PTH(1-17) analog that augments bone mass in ovariectomized rats

Daily injections of human parathyroid hormone (1-34), hPTH(1-34), provide a highly effective treatment option for severe osteoporosis. However, PTH analogs shorter than 28 amino acids do not retain any bone augmenting potential. Here, we present ZP2307 ([Ac₅c¹, Aib³, Leu⁸, Gln¹⁰, Har¹¹, Ala¹², Trp¹⁴, Asp¹⁷]PTH(1-17)-NH₂), a novel, chemically modified and cyclized hPTH(1-17) analog, that augments bone mass in ovariectomized, osteopenic rats. Subcutaneous administration of this structurally constrained, K¹³-D¹⁷ side-chain-to-side-chain cyclized peptide reversed bone loss and increased bone mineral density (BMD) up to or above baseline levels in rat long bones and vertebrae. Highly significant effects of ZP2307 were achieved at doses of 40–320 nmol/kg. Micro-CT and histomorphometric analyses showed that ZP2307 improved quantitative and qualitative parameters of bone structure. Biomechanical testing of rat femora confirmed that ZP2307 dramatically increased bone strength. Over a broad maximally effective dose range (40–160 nmol/kg) ZP2307 did not increase serum concentrations of ionized free calcium above normal levels. Only at the highest dose (320 nmol/kg) ZP2307 induced hypercalcemic calcium levels in the ovariectomized rats.To our knowledge ZP2307 is the smallest PTH peptide analog known to exert augmentation of bone. Our findings suggest that ZP2307 has the potential to effectively augment bone mass over a broad dose range without a concomitant increase in the serum concentration of ionized free calcium above the normal range.