Parathyroid Hormone Preparations Research Articles

Role of teriparatide in fracture healing: A prospective study

Background: The biology of bone repair and healing is interesting and a huge number of studies have been done in this aspect with the potential technologies for enhancing fracture-healing. Teriparatide an anabolic agent a recombinant parathyroid hormone preparation that activates osteoblastic bone formation has been used in the treatment of postmenopausal osteoporosis. It has also been reported to be effective in accelerating the rate of fracture healing. The conclusion that PTH as an effective anabolic therapy for the enhancement of bone repair after fracture has been studied in animals and very few case studies. This prospective study was conducted to determine and establish the role of recombinant teriparatide in fracture healing in delayed union fractures, osteoporotic fractures, periprosthetic fractures. Materials and Methods: The study was done on 20 patients with fractures selected on the basis of inclusion and exclusion criteria at Shashwat hospital, Pune during the period of September 2011 to September 2015. All patients were administered 20ug of teriparatide injections daily. The injections were given for 2 - 4 months depending on type of fracture and the time required for radiological evidence of union. Radiographs were taken every 4 weeks to see for callus and fracture union. The primary endpoint of fracture healing was taken as the time of cortical bridging in three of four cortices Results: The fractures treated with 20ug of teriparatide exhibited early signs of union with abundant callus formation significant decrease in radiological union time and early rehabilitation of patients in all three groups. Conclusions: This study shows that the daily systemic administration of recombinant teriparatide enhances fracture-healing by increasing bone mineral content, density and strength. It also produces a sustained anabolic effect throughout the remodeling phase of fracture-healing. It also emphasizes that teriparatide is a relatively safe drug. Economical compared to other options of fracture healing like bone grafting, second surgery and is less invasive and has better patient compliance.

Development and validation of a novel cell-based assay for potency determination of human parathyroid hormone (PTH)

Parathyroid hormone (PTH) is the primary regulator of serum calcium homeostasis and plays a major role in bone metabolism. Its actions are mediated via the PTH1 receptor (PTH1R) resulting in adenylate cyclase activation and consequently production of cyclic adenosine mono-phosphate (cAMP). The latter stimulates cellular metabolic pathways. This study describes the development, validation and applications of a novel cell-based potency assay for PTH using HEK293 cells over-expressing PTH1R. PTH concentration-dependent cAMP formation in these cells was quantitatively analyzed employing time-resolved fluorescence technology (TR-FRET). The optimized assay was precise, reproducible and exhibited a high sensitivity to PTH with a limit of quantification in the low picogram range. The potencies of differently manufactured PTH1–34 peptides, as well as a full-length variant (PTH1–84), were all accurately measured. Since PTH activity is inhibited by neutralizing antibodies against PTH, the assay was adapted to detect and measure neutralizing antibodies in human serum. Thus, applications of this novel cell-based PTH potency assay were extended to immunogenicity testing of PTH preparations in non-clinical and clinical settings.

Validation of a simple RP-HPLC method developed for the quantification of meta-cresol in parathyroid hormones formulation

Aim: To develop and validate a rapid and sensitive reverse phase high performance liquid chromatography (RP-HPLC) method with UV detection for quantification of meta-cresol (m-cresol) in pharmaceutical preparation of parathyroid hormone (1–34) (PTH). Materials and Methods: Chromatography was performed on a Jupiter RP C-18 (4.6mm ID x 250mmL, porosity 300Å, particle size 5μm) with a guard column (reversed- phase C18 column of 4.6mm ID x 12.5mmL, porosity 300Å, particle size 5μm) using a mobile phase containing 0.1% TFA in 60% methanol with isocratic program at 1.0mL/ min flow rate. Detection was carried out at 217nm. The method was validated as per ICH guidelines for linearity (correlation coefficient = 0.99), range, accuracy, precision, and robustness (n = 9 during accuracy parameter whereas n =15 during linearity and range parameter and n = 6 during repeatability). Robustness was confirmed by considering two factors; âge effect of the mobile phase and test sample and with different columns during method development. Results : The method was linear over the concentration range of 75–120μg/mL. The precision of the method in terms of relative standard deviation was evaluated from intra- and inter-day replicate injections of system suitability standards of m-cresol using different equipment and different columns. Components of within-and between-batch variances were found to be below 2% (n = 30) and 3%, respectively, which constituted an acceptable level of variation. Retention time was found to be about 5.2min and 10.9min for m-cresol and PTH, respectively. Conclusion:The developed method thus has the potential of being useful for routine quality control of m-cresol.

The effects of excipients and particle engineering on the biophysical stability and aerosol performance of parathyroid hormone (1-34) prepared as a dry powder for inhalation.

Pulmonary delivery of therapeutic peptides and proteins has many advantages including high relative bioavailability, rapid systemic absorption and onset of action and a non-invasive mode of administration which improves patient compliance. In this study, we investigated the effect of spray-drying (SD) and spray freeze-drying processes on the stability and aerosol performance of parathyroid hormone (PTH) (1-34) microparticles. In this study, the stabilisation effect of trehalose (a non-reducing sugar) and Brij 97 (a non-ionic surfactant) on spray-dried PTH particles was assessed using analytical techniques including circular dichroism (CD), fluorescence spectroscopy, modulated differential scanning calorimetry and an in vitro bioactivity assay. Physical characterisation also included electron microscopy, tap density measurement and laser light diffraction. The aerosol aerodynamic performance of the formulations was assessed using the Andersen cascade impactor. Based on these studies, a formulation for spray freeze-drying was selected and the effects of the two particle engineering techniques on the biophysical stability and aerosol performance of the resulting powders was determined. CD, fluorescence spectroscopy and bioactivity data suggest that trehalose when used alone as a stabilising excipient produces a superior stabilising effect than when used in combination with a non-ionic surfactant. This highlights the utility of CD and fluorescence spectroscopy studies for the prediction of protein bioactivity post-processing. Therefore, a method and formulation suitable for the preparation of PTH as a dry powder was developed based on spray-drying PTH with trehalose as a stabiliser with the bioactivity of SD PTH containing trehalose being equivalent to that of unprocessed PTH.

Inter-method variability in PTH measurement: Implication for the care of CKD patients

The National Kidney Foundation/Kidney-Dialysis Outcome Quality Initiative guidelines recommend to maintain the serum intact parathyroid hormone (PTH) concentration between 150 and 300 ng/l in chronic kidney disease (CKD) stage 5 patients. As these limits were derived from studies that used the Allegro intact PTH assay, we aimed to evaluate whether they were applicable to other PTH assays. We compared the PTH concentrations measured with 15 commercial immunoassays in 47 serum pools from dialysis patients, using the Allegro intact PTH assay as the reference. We also evaluated the recovery of graded amounts of synthetic 1-84 and 7-84 PTH added separately to a serum pool. Although the assays were highly correlated, the concentrations differed from one assay to another. The median bias between the tested assays and the Allegro intact PTH assay ranged from -44.9 to 123.0%. When the PTH concentrations were 150 or 300 ng/l with the Allegro intact PTH assay, they ranged with other assays from 83 to 323 ng/l and from 160 to 638 ng/l, respectively. The tested assays recognized 7-84 PTH with various cross-reactivities, whereas a given amount of 1-84 PTH was recovered differently by these assays. We found important inter-method variability in PTH results owing to both antibody specificity and standardization reasons. The unacceptable consequence is that opposite therapeutic attitudes may be reached in a single patient depending on the PTH assay used. We propose to use assay-specific decision limits for CKD patients, or to apply a correcting factor to the PTH results obtained with a given assay.

The use of intermittent human parathyroid hormone as a treatment for osteoporosis.

Parathyroid hormone (PTH), given intermittently, is an anabolic agent. PTH has been demonstrated to increase bone mass and reduce vertebral and nonvertebral fractures, and has been approved for use in the US and Europe. PTH is a genetically engineered 34 amino acid protein with the designation teriparatide (recombinant DNA origin) or recombinant human PTH 1-34. A recombinant DNA preparation with all 84 amino acids of the native PTH molecule is in clinical trials. These PTH preparations are self-administered daily injections, and it is approved for women and men at high risk for fractures, including patients with prevalent fractures, low bone mass, and multiple risk factors. PTH is likely to be used most frequently in patients who fracture on therapy, but can be used in high-risk treatment-naïve patients. Previous treatment with alendronate appears to impair the anabolic response of PTH preparations. Patients who have Paget's disease, prior radiation therapy to the skeleton, as well as children and young adults with open epiphyses, are at higher risk for osteosarcoma and should not be given PTH. Patients with hypercalcemia and hyperparathyroidism also should not receive the drug.

Synthetic carboxyl-terminal fragments of parathyroid hormone (PTH) decrease ionized calcium concentration in rats by acting on a receptor different from the PTH/PTH-related peptide receptor.

Even if the carboxyl-terminal (C-) fragments/intact (I-) PTH ratio is tightly regulated by the ionized calcium (Ca(2+)) concentration in humans and animals, in health and in disease, the physiological roles of C-PTH fragments and of the C-PTH receptor remain elusive. To explore these issues, we studied the influence of synthetic C-PTH peptides of various lengths on Ca(2+) concentration and on the calcemic response to human (h) PTH-(1-34) and hPTH-(1-84) in anesthetized thyroparathyroidectomized (TPTX) rats. We also looked at the capacity of these PTH preparations to react with the PTH/PTHrP receptor and with a receptor for the carboxyl (C)-terminal portion of PTH (C-PTH receptor) in rat osteosarcoma cells, ROS 17/2.8. The Ca(2+) concentration was reduced by 0.19 +/- 0.03 mmol/liter over 2 h in all TPTX groups. Infusion of solvent over 2 more h had no further effect on the Ca(2+) concentration (-0.01 +/- 0.01 mmol/liter), whereas infusion of hPTH-(7-84) or a fragment mixture [10% hPTH-(7-84) and 45% each of hPTH-(39-84) and hPTH-(53-84)] 10 nmol/h further decreased the Ca(2+) concentration by 0.18 +/- 0.02 (P<0.001) and 0.07+/-0.04 mmol/liter (P< 0.001), respectively. Infusion of hPTH-(1-84) or hPTH-(1-34) (1 nmol/h) increased the Ca(2+) concentration by 0.16 +/- 0.03 (P < 0.001) and 0.19 +/- 0.03 mmol/liter (P < 0.001), respectively. Adding hPTH-(7-84) (10 nmol/h) to these preparations prevented the calcemic response and maintained Ca(2+) concentrations equal to or below levels observed in TPTX animals infused with solvent alone. Adding the fragment mixture (10 nmol/h) to hPTH-(1-84) did not prevent a normal calcemic response, but partially blocked the response to hPTH-(1-34), and more than 3 nmol/h hPTH-(7-84) prevented it. Both hPTH-(1-84) and hPTH-(1-34) stimulated cAMP production in ROS 17/2.8 clonal cells, whereas hPTH-(7-84) was ineffective in this respect. Both hPTH-(1-84) and hPTH-(1-34) displaced (125)I-[Nle(8,18),Tyr(34)]hPTH-(1-34) amide from the PTH/PTHrP receptor, whereas hPTH-(7-84) had no such influence. Both hPTH-(1-84) and hPTH-(7-84) displaced (125)I-[Tyr(34)]hPTH-(19-84) from the C-PTH receptor, the former preparation being more potent on a molar basis, whereas hPTH-(1-34) had no effect. These results suggest that C-PTH fragments, particularly hPTH-(7-84), can influence the Ca(2+) concentration negatively in vivo and limit in such a way the calcemic responses to hPTH-(1-84) and hPTH-(1-34) by interacting with a receptor different from the PTH/PTHrP receptor, possibly a C-PTH receptor.

Effect of Excess Parathyroid Hormone on Human Bone Marrow Fibroblasts

Patients with uremia have excess levels of parathyroid hormone (PTH). It has been reported that excess PTH is associated with bone marrow fibrosis. The present study was undertaken to examine the in vitro effect of intact 1-34 PTH and the active 1-34 N-terminal fragment on human bone-marrow-derived fibroblasts. Proliferation of fibroblasts was not stimulated by PTH in concentrations present in uremia (5-30 U/ml) nor by uremic sera which contained either high or low PTH levels. The lack of stimulation contrasted with the activity of both PTH preparations in other systems, i.e., inhibitory to erythroid colonies and stimulatory to heart muscle cells.

Effect of biological activity of PTH on its peripheral metabolism in the rat

Previous studies from our laboratory have characterized the peripheral metabolism of parathyroid hormone (PTH) in the dog using radioimmunoassay techniques following injection or infusion of biologically active, bovine PTH preparations. Other investigators have used biologically-inactive labelled PTH and interpreted their results as representative of the normal physiological processes. Since oxidized inactive PTH does not bind to PTH receptors and since we have found substantial differences between the tissue uptake of active and inactive PTH preparations, it is possible that results obtained with inactive PTH preparations may not totally represent the normal metabolism of PTH. Therefore, we performed studies in rats to compare the disappearance of immunoreactive PTH (i-PTH) from plasma following injection of active or inactive syn b-PTH 1-34. The maneuvers of bilateral ureteral ligation and bilateral nephrectomy were utilized to characterize the sites of tissue uptake of i-PTH. The results obtained indicate that inactive syn b-PTH 1-34 has a significantly slower disappearance from plasma than biologically active syn b-PTH 1-34. Reduction of glomerular filtration by acute bilateral ureteral ligation decreased the disappearance of oxidized PTH more than active PTH. Thus, the results indicate a major dependence on glomerular filtration for the removal of inactive syn b-PTH 1-34. The demonstration that the peripheral metabolism of active and inactive syn b-PTH 1-34 is not identical suggests that studies of the metabolism of inactive PTH preparations do not accurately reflect that of biologically active PTH.

PARATHYROID HORMONE INHIBITS HUMAN PLATELET FUNCTION

Several inhibitors of platelet function have been found in blood from uraemic patients. It has been suggested that parathyroid hormone (PTH) is a major uraemic toxin underlying many manifestations of the uraemic syndrome. Two different preparations of PTH strongly inhibited human platelet aggregation and secretion induced in vitro by different stimuli. The data suggest one possible pathway for the participation of excess PTH in the pathogenesis of the platelet dysfunction and bleeding tendency in uraemia.

The Effect of Parathyroid Hormone on Erythropoiesis in Serum-Free Cultures of Fetal Mouse Liver Cells

AbstractThe effects of parathyroid hormone (PTH) on erythropoiesis in serum-free cultures of fetal mouse liver cells were investigated to determine if PTH might, at least partially, be responsible for the anemias of uremia, hyperparathyroidism, and spaceflight. Both crude and pure preparations of PTH were employed with similar effects. PTH at concentrations 10-100 times “normal” produced a dose-dependent stimulation of erythropoiesis which was only observed in the complete absence of exogenous erythropoietin (Ep). Although the responses to both PTH and Ep were calcium-dependent pharmacological analysis of the PTH log-dose/log-response relationship showed the PTH effect to be markedly different from that of Ep. At concentrations approximately 240 times normal PTH inhibited both endogenous and Ep-mediated heme synthesis. These concentrations are considerably greater than those reported in uremia, hyperparathyroidism, or during spaceflight casting doubt on the hypothesis that PTH is directly responsible for ...

Hypotensive actions of parathyroid hormone preparations in vertebrates

Hypotensive actions of parathyroid hormone preparations in vertebrates

Hypotensive action of parathyroid hormone preparations on rats and dogs.

Bovine parathyroid extract and two commercial preparations containing the first 34 amino acids of synthetic bovine parathyroid hormone [bPTH-(1-34)]produced dose-related hypotension in anesthetized rats. Dogs were 10 times more sensitive to the two bPTH-(1-34) preparations than were rats. Propranolol, phentolamine, atropine, and promethazine did not affect the hypotensive action of bPTH-(1-34) in rats and dogs. bPTH-(1-34) decreased perfusion pressure in rat hindlimbs perfused in situ with Ringer's solution and was a vasodilator in dog kidneys perfused in vitro with Ringer's solution. Helical strips of rabbit aorta were also relaxed by bPTH-(1-34). We conclude that the direct vasodilatory action of bPTH preparations represents an intrinsic property of parathyroid hormone and that the hypotensive effect of this hormone is produced by part or all of the first NH2-terminal 34 amino acids.

Response of human and chick kidney adenylate cyclase to biologically active synthetic fragments of human parathyroid hormone

The 34-amino acid NH 2-terminal fragment of human parathyroid hormone synthesized according to the sequence described by Niall et al. (1) is approximately 140 times more potent than the fragment synthesized according to Brewer et al (2) in activating human renal cortex adenylate cyclase. The potencies of the two peptides, relative to the effect of MRC standard bovine parathyroid hormone preparation 67 342 in this system, were 5600 ± 600 (S.E.M.) units/mg and 40 ± 5 units/mg respectively. The potencies of the more active peptide and the corresponding bovine parathyroid hormone sequence were similar in this system and also in assays based upon the production of cyclic AMP by chick kidney both in vivo and in vitro .

Parathyroid hormone preparations, salmon calcitonin, and urine flow in the South American lungfish, Lepidosiren paradoxa.

In the South American lungfish, Lepidosiren paradoxa, injections of salmon calcitonin or bovine parathyroid extract have no effect on serum calcium levels. However, salmon calcitonin elicits a distinct diuresis. Crude or purified parathyroid extract or synthetic parathyroid hormone (1-34 fraction) produce a distinct antidiuresis.

Further studies on acetamidination as a technique for preparation of a biologically valid 3-H-labeled tracer for parathyroid hormone.

The technique of acetamidination of amino groups in parathyroid hormone (PTH) for the purpose of preparation of a tritiated, biologically valid tracer for this hormone, has been more extensively studied. It was found that eight of the ten amino groups in PTH are readily reactive but that two appear unreactive. The kinetics of labeling suggest that preparations of PTH which are labeled to this level of 80% of theoretical consist of a homogeneous population of molecules in which each PTH molecule contains eight tritiated acetamidino groups. There is no question of the presence of unlabeled hormone in such preparations. Eighty per cent labeled acetamidino-PTH is identical, qualitatively and quantitatively, in its biological activity with native PTH as shown by three accepted bioassays: serum calcium elevation, urine phosphate excretion, and activation of kidney cortex plasma membrane adenylate cyclase in vitro. The dose-response relationships are identical for labeled and native hormone in all three systems. Tritiated acetamidino-PTH tends to lose biological activity on storage but full activity can be regained by reduction of the hormone with excess cysteine. However, cysteine reduction conducted at 80 degrees for 2 hours causes some loss of tritium from the hormone. Therefore, dithiothreitol reduction at room temperature was utilized to maintain biological activity. Eighty per cent acetamidino-PTH can be further purified by ion exchange chromatography on carboxymethylcellulose using a continuous gradient of sodium acetate in 8 M urea. Such chromatography reveals the presence of isohormones in both cold and radioactive PTH. Co-chromatography of tritiated acetamidino-PTH with a cold isohormone of PTH shows that while the isohormones can be separated from one another, the acetamidino derivatives of each isohormone elute in a virtually identical position with their parent unlabeled PTH molecule.

A bioassay for parathyroid hormone based on hormonal inhibition of CO2 production from citrate in mouse calvarium.

An in vitro method has been developed as bioassay for parathyroid hormone. It is based on the inhibition by the hormone of 14CO2 yield produced by the metabolism of 14C-citric acid in mouse calvarial organ culture system. The log-dose response plot was linear over the range of 0.0025 to 0.15 μ/ml. The method is capable of detecting parathyroid hormone when net bone resorption (calcium release to the medium) is inhibited by removal of serum from the culture medium or by addition to the system of thyrocalcitonin, or high concentrations of phosphate. Moreover, the response of the system to parathyroid hormone was essentially unaffected by a wide range of calcium in the culture medium. Inactive components in crude parathyroid hormone preparations were inactive in this system as was peroxide-inactivated parathyroid hormone. The response of the system is related to events associated with bone resorption, since agents such as dibutyryl cyclic AMP, 25-hydroxycholecalciferol, vitamin A and heparin which are effective in this process, also produced an inhibition of 14CO2 yield. (Endocrinology89: 1425, 1971)

Mode of action of parathyroid hormone and cyclic adenosine 3',5'-monophosphate on renal tubular phosphate reabsorption in the dog.

To evaluate the effects of parathyroid hormone and cyclic adenosine monophosphate on proximal tubular sodium and phosphate reabsorption, micropuncture studies were performed on dogs that received a highly purified preparation of parathyroid hormone (PTH), dibutyryl cyclic 3',5'-adenosine monophosphate (cyclic AMP), 5'-AMP, and saline. PTH resulted in a 30-40% inhibition of sodium and phosphate reabsorption in the proximal tubule unassociated with a rise in either total kidney or single nephron glomerular filtration rate (GFR). The bulk of the phosphate rejected proximally was excreted in the final urine while sodium excretion rose minimally despite the marked proximal inhibition, consistent with the presence of reabsorptive sites in the distal nephron for sodium but not phosphate. The infusion of dibutyryl cyclic AMP either systemically or directly into the renal artery inhibited proximal sodium and phosphate reabsorption in the absence of changes in either total kidney or single nephron GFR, resembling the effects of PTH quantitatively and qualitatively. In contrast, another adenine nucleotide, 5'-AMP, did not inhibit the reabsorption of either sodium or phosphate. These observations support the thesis that renal effects of PTH are mediated via stimulation of renal cortical adenyl cyclase. The infusion of a moderate saline load, 25 ml/kg, also produced a similar inhibition of proximal tubular fractional sodium and phosphate reabsorption with a marked phosphaturia but only minimal natriuresis. Thus, changes in sodium and phosphate reabsorption occur in parallel in the proximal tubule when sodium reabsorption is inhibited either with volume expansion or with administration of "specific" phosphaturic agents such as PTH or cyclic AMP. These data are consistent with the thesis that phosphate reabsorption is dependent upon proximal tubular sodium reabsorption wherein the phosphaturic effect of PTH might be the result of a primary inhibition of proximal tubular sodium reabsorption mediated by adenyl cyclase stimulation.

Effect of parathyroid hormone and vitamin A on the retention of radiostrontium in the rat.

THE experimental data about the influence of parathyroid hormone on the excretion of radiostrontium are ambiguous. Some workers have reported that there is no effect1–5, others that there is a diminished retention6–10, and still others claim that retention of radiostrontium is enhanced under the influence of parathyroid hormone11. These discrepancies may be accounted for in a number of ways. Thus the results indicating a positive influence have only been obtained by measuring the urinary excretion of radiostrontium; it is understandable that this slight effect should not be evident in the comparatively insensitive direct determination of radiostrontium in the whole body or the skeleton. Moreover, the capacity of commercial preparations of parathyroid hormone to mobilize calcium from the bone may vary. Finally, most studies so far have been concerned with the administration of parathyroid hormone a comparatively short time after the incorporation of radiostrontium; experiments with calcium-45, however, indicate that the parathyroid influences calcium in the deeper bone areas which are not closely in contact with the easily exchangeable pool12,13. It follows that the time of administration of parathyroid hormone may affect the results of experiments.

Importance of the thyroid gland in the prevention of hypercalcemia in rats.

The role of the thyroid and parathyroid glands in counteracting the hypercalcemic action of exogenous parathyroid hormone was examined. Acutely parathyroidectomized, thyroparathyroidectomized, and sham-operated rats were injected either with commercial parathyroid extract or with partially purified parathyroid hormone preparation. The rats were bled 5 or 6 hr later. The serum calcium was slightly higher in the parathyroidectomized rats injected with the parathyroid hormone preparations than in the sham-operated rats injected with control solution, but never higher than in the shamoperated rats treated with hormone. In thyroparathyroidectomized rats injected with parathyroid hormone preparations the serum calcium rose sharply (3-5 mg of calcium/100 ml of serum) and the increase was significantly higher than in the other hormone-treated groups. These results provide additional evidence that the thyroid gland is important in the prevention of experimental hypercalcemia in rats, presumably by secreting thyroc...