Human Parathyroid Hormone Fragment Research Articles

P8.033: Teriparatide in Kidney Transplant Patients With Bisphosphonate-Resistent Osteoporosis

Introduction: Bisphosphonates increase Bone mineral density (BMD) by suppressing bone turnover inhibiting osteoclastic function with risk of inducing adynamic bone disease. Teriparatide is a fragment of the natural human parathyroid hormone (PTH) consisting of the first 34 amino acids counting from the N-terminus end of the natural PTH. Is anabolic drugs stimulate bone formation by increasing osteoblast activity and regulates both bone formation and resorption reducing the risk of vertebral and nonvertebral fractures. Arm: Teriparatide (Parathormone 1-34) treatment in renal trasplant patients with bisphosphonate-resistent osteoporosis.Metods: observational describe study of cases series of 10 kidney transplant patients with bone fragility without response to prolonged treatment with bisphosphonates (greater than 3 years). They were treated with daily subcutaneous injections of 20 microg teriparatide (PTH 1-34) by 18 months. All the patients had 6 months of bisphosphonate wash out. Measurements: we investigated bone turnover markers and effect of teriparatide over BMD of the femoral neck and lumbar spine as well also fractures, graft rejection, cardiovascular events, death and drug related adverse event. Results: 10 patients 9 women, mean age 64.4±14.15 years, BMI 24.3 ±2.64, etiology 3GMN, 3 PKD, 3 unknown, 1 NDBT, HD 9, preempive 1, transplant Time 10.12±5.21 years, 1 preemptive, 8 DBD and 1 LRD, Induction: 8 thymoglobulin, 2 basiliximab, Maintenance 5 belatacept/mycophenolate/MTP, 2 rapamune/mycophenolate/MTP, 3 tacrolimus/mycophenolate/MTP, 6 patients with fractures prior to treatment. Mean of treatment with bisphosphonates 4.1±1.79 years. It is observed at 18 months of treatment with 1-34 PTH increased levels of bone alkaline phosphatase (13.26 UI/L ± 5.75 vs 17.90 ± 7.28, p = 0.045), with NS changes in the rest of the analytes (Table 1) and increase of lumbar bone density of 11.27% (0.8816 gr/cm2 (TS-2.6) vs 0.9941 gr/cm2 (TS-1.38)) and femoral neck of 7.07% (0.6752 gr/cm2(TS-2.5) vs 0.7230 gr/cm2(TS-2.19)). 2 patients presented wrist fractures during treatment with low response(lumbar BMD) to treatment. No cardiovascular events, rejections, deaths were reported. Adverse Events: 1 patient presented hypotension during the first month of treatment. Conclusion: In this group of patients, and adequate response to teriparatide treatment was observed however our limitation were small number of patients and bone biopsy prior teriparatide treatment.

Quantification of Anti-Osteoporotic Anabolic Peptide in Stealth Lipid Nanovesicles Through Validated RP-HPLC Method.

Teriparatide is a recombinant fragment of human parathyroid hormone, a potent osteoanabolic agent used for osteoporosis. The present study endeavored to develop a simple, rapid, and reliable reverse phase-high performance liquid chromatography (RP-HPLC) method for the determination of teriparatide in pegylated lipid nanovesicles for rapid formulation development/optimization. A rapid RP-HPLC-based analytical method was developed for the quantification of teriparatide in pegylated lipid nanovesicles. The method was optimized on a Waters XBridge C18 (4.6 × 150 mm, 10 μm) column with a mobile phase consisting of 0.1% formic acid in water and acetonitrile both in a linear gradient program. In the method, a short run time of 9 min was achieved at a flow rate of 1.0 mL/min with an injection volume of 50 µL at a detection wavelength of 210 nm. The developed method was validated according to the ICH Q2 (R2) guideline. The method was applied for the quantification of teriparatide in prepared pegylated lipid nanovesicles. Teriparatide encapsulated pegylated lipid nanovesicles were prepared by the ethanol injection method. Further, these vesicles were characterized for % entrapment efficiency (%EE), particle size, zeta potential, and morphology by Cryo-SEM. The teriparatide was eluting at 4.8 min in the run. Further, for the method validation, the linear relationship between concentration and response was established over the concentration range of 50-250 µg/mL with the R2 > 0.999. The method sensitivity was shown with LOD and LOQ with the value of 100 ng/mL and 500 ng/mL, respectively. The method was found to be accurate and precise with the recovery ranging in 100 ± 2% and RSD <2%, respectively. Minor deliberate changes proved the robustness of the developed method. These results indicated that the developed and validated method is accurate, precise, rapid, reliable, and fit for the quantification of teriparatide in different formulations. The RP-HPLC method was developed and validated for the quantification of teriparatide from novel pegylated lipid nanovesicles.

Gli1+ progenitors mediate bone anabolic function of teriparatide via Hh and Igf signaling.

SUMMARYTeriparatide is the most widely prescribed bone anabolic drug in the world, but its cellular targets remain incompletely defined. The Gli1+ metaphyseal mesenchymal progenitors (MMPs) are a main source for osteoblasts in postnatal growing mice, but their potential response to teriparatide is unknown. Here, by lineage tracing, we show that teriparatide stimulates both proliferation and osteoblast differentiation of MMPs. Single-cell RNA sequencing reveals heterogeneity among MMPs, including an unexpected chondrocyte-like osteoprogenitor (COP). COP expresses the highest level of Hedgehog (Hh) target genes and the insulin-like growth factor 1 receptor (Igf1r) among all cell clusters. COP also expresses Pth1r and further upregulates Igf1r upon teriparatide treatment. Inhibition of Hh signaling or deletion of Igf1r from MMPs diminishes the proliferative and osteogenic effects of teriparatide. The study therefore identifies COP as a teriparatide target wherein Hh and insulin-like growth factor (Igf) signaling are critical for the osteoanabolic response in growing mice.

Proof of Concept on Functionality Improvement of Mesenchymal Stem-Cells, in Postmenopausal Osteoporotic Women Treated with Teriparatide (PTH1-34), After Suffering Atypical Fractures.

Osteoporosis long-term treatment with nitrogen-containing bisphosphonates, has been associated with uncommon adverse effects, as atypical femoral fractures (AFF). Thus, treatment with teriparatide (TPTD; fragment of human parathyroid hormone; PTH1-34) has been proposed for such patients. Besides its anabolizing effect on bone, TPTD may affect stem-cell mobilization and expansion. Bone marrow mononuclear cells (BMMNC) were isolated from five women that had suffered AFF associated to bisphosphonate treatment, before and after 6months of TPTD therapy. The presence of mesenchymal stromal cells (CD73, CD90 and CD105 positive cells), gene expression of NANOG, SOX2 and OCT4, proliferation, senescence and capacity to differentiate into osteoblasts and adipocytes were analyzed. After TPTD treatment, BMMNC positive cells for CD73, CD90 and CD105 increased from 6.5 to 37.5% (p < 0.05); NANOG, SOX2 and OCT4 were upregulated, being statistically significant for NANOG (p < 0.05), and cells increased proliferative capacity more than 50% at day 7 (p < 0.05). Senescence was reduced 2.5-fold (p < 0.05), increasing differentiation capacity into osteoblasts and adipocytes, with more than twice mineralization capacity of extracellular matrix or fat-droplet formation (p < 0.05), respectively. Results show that TPTD treatment caused BMMNC "rejuvenation", increasing the number of cells in a more undifferentiated stage, with higher differentiation potency. This effect may favor TPTD anabolic action on bone in such patients with AFF, increasing osteoblast precursor cells. Such response could also arise in other osteoporotic patients treated with TPTD, without previous AFF. Furthermore, our data suggest that TPTD effect on stromal cells may have clinical implications for bone-regenerative medicine. Further studies may deepen on this potential.

The first biosimilar approved for the treatment of osteoporosis: results of a comparative pharmacokinetic/pharmacodynamic study

SummaryTo demonstrate the clinical comparability between RGB-10 (a biosimilar teriparatide) and the originator, a comparative pharmacokinetic trial was conducted. The study was successful in establishing bioequivalence. Marketing authorisation for RGB-10 (Terrosa®) was granted by the European Medicines Agency in 2017.IntroductionTeriparatide, the first bone anabolic agent, is the biologically active fragment of human parathyroid hormone. The imminent patent expiry of the originator will open the door for biosimilars to enter the osteology market, thereby improving access to a highly effective, yet prohibitively expensive therapy.MethodsSubsequent to establishing comparability on the quality and non-clinical levels between RGB-10, a biosimilar teriparatide, and its reference product (Forsteo®), a randomised, double-blind, 2-way cross-over comparative study (duration: four days) was conducted in 54 healthy women (ages: 18 to 55 years) to demonstrate the pharmacokinetic/pharmacodynamic (PK/PD) equivalence and comparable safety of these products. Extents of exposure (AUC0-tlast) and peak exposure (Cmax), as measured by means of ELISA, were evaluated as co-primary PK endpoints, and serum calcium levels, as measured using standard automated techniques, were assessed for PD effects. Safety was monitored throughout the study.ResultsThe 94.12% CIs for the ratio of the test to the reference treatments, used due to the two-stage design (85.20–98.60% and 85.51–99.52% for AUC0-tlast and Cmax, respectively), fell within the 80.00–125.00% acceptance range. The calcium PD parameters were essentially identical with geometric mean ratios (GMRs) of 99.93% and 99.87% for AUC and Cmax, respectively. Analysis of the safety data did not reveal any differences between RGB-10 and its reference.ConclusionBased on the high level of similarity in the preclinical data and the results of this clinical study, marketing authorisation for RGB-10 (Terrosa®) was granted by the European Medicines Agency (EMA) in 2017.

Teriparatide treatment in fracture non-union: a case report

&lt;p class="abstract"&gt;Impaired healing of fractures delays the rehabilitation process, which in turn impacts quality of life. The associated costs cause an economic burden to both the society and the patients. Till date, no systemic treatment is approved for fracture healing. Teriparatide is a synthetic polypeptide hormone consisting of the 1–34 fragment of human parathyroid hormone. Apart from its recognized indication of osteoporosis, there is a growing body of evidence suggesting its ability to accelerate fracture healing and heal non-unions. The purpose of this case report is to elucidate the Indian experience of 5 cases of use of Teriparatide for fracture healing - delayed unions and non-unions and in the setting of osteoporotic fracture. The primary observation of our case report depicts that teriparatide is a viable therapy not only to treat osteoporosis but also to accelerate fracture healing. Teriparatide therapy accelerates healing, which allows patients to return to normal life and work faster, optimizes medical resource utilization, reduces chances for future second surgery, requirement of bone graft and overall chronic morbidity associated with long-term treatment. The advantages of teriparatide therapy are that it can be prescribed in any types of fractures, including those that will be treated non-surgically; it can be commenced at any time, and can be applied through the entire healing period. Our observations are in line with other studies showing the effects of Teriparatide on delayed union and non-unions in human subjects.&lt;/p&gt;

Administration of teriparatide improves the symptoms of advanced bisphosphonate-related osteonecrosis of the jaw: preliminary findings

Teriparatide is a synthetic polypeptide hormone that contains the 1–34 amino acid fragment of the recombinant human parathyroid hormone that stimulates bone formation. Currently, it is approved only for the treatment of osteoporosis. The outcomes of daily teriparatide injections for the treatment of bisphosphonate-related osteonecrosis of the jaw in 10 patients are reported here. Two of the 10 cases dropped out due to adverse events. Of the remaining eight cases, seven exhibited clinical improvement of the jaw-related symptoms of osteonecrosis and progression of the sequestration, while one case did not show improvement of the symptoms. Administration of teriparatide in patients with osteonecrosis of the jaw promotes bone formation and subsequent sequestration over a short period of time. These results suggest that adjunctive teriparatide therapy is a viable and effective option for treating osteonecrosis of the jaw.

Induction and antagonism of pica induced by teriparatide in rats

Intermittent subcutaneous injection of teriparatide, an active fragment of human parathyroid hormone, is clinically used for the treatment of osteoporosis. Patients suffer from nausea, which is one of the side effects teriparatide induces; however, the etiology of teriparatide-induced nausea remains unknown. We have reported pica, kaolin ingestion behavior, can be used as an assessment of nausea-related response in rats. In this study, we investigated the characteristics of teriparatide-induced pica and the abilities of anti-emetic drugs to inhibit teriparatide-induced pica. Male and female adolescent (4-week-old), young (8-week-old), and adult (30-week-old) naive rats, and ovariectomized (OVX: 17-week-old) and sham-operated (17-week-old) rats subcutaneously received teriparatide (0.4mg/kg, n=4), and their kaolin and food intakes were monitored for 24h after the injection. Among the tested rats, we found that OVX rats, rather than male, female, and sham-operated rats, showed marked teriparatide-induced pica (0mg/kg: 0.17±0.07g, 0.4mg/kg: 6.18±0.91g). Teriparatide-induced pica in OVX rats was inhibited by intraperitoneal pretreatment with serotonin 5-HT3 (granisetron 0.5mg/kg), dopamine D2 (prochlorperazine 0.5mg/kg), neurokinin NK1 (fosaprepitant 1mg/kg), and histamine H1 (diphenhydramine 10mg/kg) receptor antagonists to 70%, 11%, 19%, and 59% of that in vehicle-treated control, respectively. These results suggest that teriparatide-induced pica in OVX rats has the potential to reflect teriparatide-induced nausea; 5-HT3, D2, NK1, and H1 receptor activation is involved in the development of this behavior; antagonists of these receptors have the potential to be medical candidates used as treatments for teriparatide-induced nausea in human patients.

PEGylation site-dependent structural heterogeneity study of monoPEGylated human parathyroid hormone fragment hPTH(1-34).

The structures of C- and N-terminally monoPEGylated human parathyroid hormone fragment hPTH(1-34) as well as their unmodified counterparts, poly(ethylene glycol) (PEG) and hPTH(1-34), have been studied by small-angle neutron scattering (SANS). The scattering results show that free hPTH(1-34) in 100 mM phosphate buffer (pH 7.4) aggregates into clusters. After conjugation with PEG, the PEG-peptide conjugates self-assemble into a supramolecular core-shell structure with a cylindrical shape. The PEG chains form a shell around the hPTH(1-34) core to shield hPTH(1-34) from the solvent. The detailed structural information on the self-assembled structures is extracted from SANS using a model of the cylindrical core with a shell of Gaussian chains attached to the core surface. On the basis of the data, because of the charge-dipole interactions between the conjugated PEG chain and the peptide, the conjugated PEG chain forms a more collapsed conformation compared to free PEG. Moreover, the size of the self-assembled structures formed by the C-terminally monoPEGylated hPTH(1-34) is about 3 times larger than that of the N-terminally monoPEGylated hPTH(1-34). The different aggregation numbers of the self-assembled structures, triggered by different PEGylation sites, are reported. These size discrepancies because of different PEGylation sites could potentially affect the pharmacokinetics of the hPTH(1-34) drug.

High sensitivity LC–MS/MS method for direct quantification of human parathyroid 1–34 (teriparatide) in human plasma

Teriparatide, the 1-34 fragment of human parathyroid hormone, is used to treat osteoporosis patients with a high risk of fracture by stimulating new bone formation. Routinely teriparatide is quantified using radioimmunoassay however the LC-MS/MS described here has the potential to achieve greater accuracy and precision, higher specificity, and is readily implemented in routine bioanalytical laboratories. Hence a complete method combining effective sample prep with appropriate LC separation and selected reaction monitoring (SRM) MS detection was developed to selectively separate teriparatide from closely related endogenous peptides and to reduce interferences. Samples were concentrated without evaporation, minimizing the risk of adsorptive losses. Chromatography was performed on a sub 2μm particle charged surface hybrid column, which provided significantly higher peak capacity than a traditional C18 column when formic acid was used as the mobile phase modifier. Total LC cycle time was 6min. An LOD of 15pg/mL (3.6fmol/mL) from 200μL of human plasma was readily achieved and standard curves were accurate and precise from 15pg/mL to 500pg/mL. Mean QC accuracies ranged from 90% to 106%. Mean QC precision was better than 7%. The CV of matrix factors across 6 sources of human plasma was 5%. The assay presented here is the first LC-MS method which reaches clinically relevant detection limits for teriparatide.

Atrophic femoral nonunion successfully treated with teriparatide

Teriparatide is a synthetic polypeptide hormone that contains the 1-34 aminoacid fragment of the recombinant human parathyroid hormone. It has been approved for the treatment of postmenopausal women with osteoporosis who are at high risk for sustaining a fragility fracture. It has been shown that teriparatide also accelerates fracture healing by improving the biomechanical properties of the fracture callus, increasing endochondral ossification and bone remodeling in animal models. This effect has been observed in several case reports. Fracture healing disorders negatively affect the patient's quality of life and result in high healthcare costs, as a second surgery is required to stabilize the fracture and stimulate bone biology. Future biotechnologies that accelerate fracture healing may be useful tools. We present a case report of delayed union of a femoral fracture treated with teriparatide. She was diagnosed with right distal metaphyseal femoral fracture on total knee arthroplasty. She underwent surgery at our center consisting of ORIF with lateral femoral locking plate in October 2011. Radiologic controls at 5 and 7 months did not show any signs of healing. After 2 months of treatment with teriparatide, the X-ray showed the presence of bone bridges and a decreased gap between fragments and a different aspect of neoformed bone. After 3 months of treatment, healing was complete. Our case report seems to confirm the possible effect of TPTD as bone induction through a more rapid healing of fractures. The TPTD could have a potentially important role in treating some forms of nonunion and delay in consolidation. Thus, one could hypothesize the possibility of a medical treatment with TPTD both as a preventive way and also as a support to the synthesis in high risk of nonunion fractures and complexed fractures in osteoporotic bone.

Knochenanabole Therapie mit Teriparatid

The recombinant amino-terminal fragment of human parathyroid hormone (Teriparatide) is a bone anabolic agent which reduces fracture risk by increasing bone mass and improving bone microarchitecture. Teriparatide reduces vertebral fracture risk by 65 % and non-vertebral by 50 %. Its efficacy is higher as bisphosphonates to prevent corcicosteroid-induced osteoporosis. Teriparatide may also have a direct effect on bone pain. Teriparatide may be initiating immediately after an anticatabolic agent. However, it is not recommended to associate both treatments. At the end of teripatide treatment, an anticatabolic agent may be given. According to cost-effectiveness studies, Teriparatide should be considered as first line treatment for postmenopausal women and for men with severe osteoporosis.

Potential use of parathyroid hormone (PTH) in the treatments for oral diseases

Bone formation is the key for successful dental treatments including periodontal tissue regeneration osseointegration of implant placement in which new alveolar bone formation is mandatory. Parathyroid hormone (PTH) is a peptide hormone containing 84 amino acids. Accumulated results show that intermittent PTH administration increases bone mass. Accordingly, recombinant N-terminal fragment of human PTH (1-34) , Teriparatide, is used for the treatments of osteoporosis worldwide. It is, therefore, expected that PTH has the potential to be a new therapeutic intervention for oral diseases that require increased bone remodeling and new bone formation.

Indications to teriparatide treatment in patients with osteoporosis

To prevent osteoporotic fracture occurrence, a variety of treatment regimens with different mechanisms of action is available. The antiresorptive bisphosphonate drugs are currently the most commonly prescribed agents in the management of patients with osteoporosis. The recombinant amino-terminal fragment of human parathyroid hormone (Teriparatide) is a bone anabolic agent which reduces fracture risk by increasing bone mass and improving bone microarchitecture. Teriparatide treatment reduces vertebral and non-vertebral fracture risk markedly in women and men with idiopathic osteoporosis, or with glucocorticoid-induced osteoporosis. Teriparatide should thus be considered as first line treatment for postmenopausal women and for men with severe osteoporosis.

Biosynthesis of a novel recombinant peptide derived from hPTH(1–34)

A recombinant human parathyroid hormone fragment, Pro-Pro-[Arg 11]hPTH(1–34)-Pro-Pro (MW = 4550 Da), was developed by substituting Arg for Leu at position 11 and adding Pro-Pro at the carboxyl terminus. Following a single injection (0.5–13.5 μg/bird) of the rhPTH fragment, the serum calcium level in chickens increased 12–42% ( P < 0.05) after 1 h as determined by the Parson’s Chicken Assay. The functional activity of Pro-Pro-[Arg 11]hPTH(1–34)-Pro-Pro may be due to removal of the N-terminus Pro-Pro- by X-prolyl dipeptidyl peptidase IV (DPPIV) in vivo, increasing its activity compared to Pro-Pro-hPTH(1–34). This artificial rhPTH fragment could be used to increase calcium mobilization and potentially improve bone health.

Bone healing induced by local delivery of an engineered parathyroid hormone prodrug

Regenerative medicine requires innovative therapeutic designs to accommodate high morphogen concentrations in local depots, provide their sustained presence, and enhance cellular invasion and directed differentiation. Here we present an example for inducing local bone regeneration with a matrix-bound engineered active fragment of human parathyroid hormone (PTH 1–34), linked to a transglutaminase substrate for binding to fibrin as a delivery and cell-invasion matrix with an intervening plasmin-sensitive link (TGplPTH 1–34). The precursor form displays very little activity and signaling to osteoblasts, whereas the plasmin cleavage product, as it would be induced under the enzymatic influence of cells remodeling the matrix, was highly active. In vivo animal bone-defect experiments showed dose-dependent bone formation using the PTH–fibrin matrix, with evidence of both osteoconductive and osteoinductive bone-healing mechanisms. Results showed that this PTH-derivatized matrix may have potential utility in humans as a replacement for bone grafts or to repair bone defects.

Preparation and Characterization of a Novel Recombinant Human Parathyroid Hormone (1 – 34) Analog (Gly1-Gln26-rhPTH(1-34)) with Enhanced Biological Activity

A recombinant human parathyroid hormone (rhPTH) fragment (Gly1-Gln26-rhPTH(1-34)) which contains two amino acids substitutions (Gly1 and Gln26) was acquired through Escherichia coli expression system using a soluble fusion protein strategy. The soluble fusion protein MBP-Gly1-Gln26-rhPTH(1-34) was harvested after purification by Phenyl-Sepharose F.F and Q-Sepharose F.F chromatographies. Following tobacco etch virus (TEV) protease cleavage and further purification by SP-Sepharose F.F chromatography, 30.8 mg/L Gly1-Gln26-rhPTH(1-34) without tag was obtained with high purity up to 99%. Cyclic AMP (cAMP) stimulation assay suggested that Gly1-Gln26-rhPTH(1-34) could increase the biological activity by up to 13.89% and 6.34%. After daily subcutaneous injection (for 13 weeks) of 5, 10 and 20 microg of Gly1-Gln26-rhPTH(1-34)/1000g body weight, the mean Bone Material Density (BMD) of ovariectomized (OVXed) rats increased to 7.95-30.54% and 1.98-23.32%, compared to control-vehicle group (OVX, P<0.001) and sham- operated group (SHAM, P<0.01), respectively.

Study on preparation and activity of a novel recombinant human parathyroid hormone(1–34) analog with N-terminal Pro–Pro extension

A recombinant human parathyroid hormone fragment, Pro–Pro–hPTH(1–34), with molecular weight of 4311.46 was acquired through gene engineering. It was then isolated and purified. The homogeneity of this fragment was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), high performance liquid chromatography(HPLC), isoelectronic focusing (IEF) electrophoresis and mass spectrometry(MS) methods. Its isoelectric point is 8.0 which was determined by IEF. It was found that the hormone fragment significantly induced calcium increment as compared to the control group ( P < 0.001) in Parsons's Chicken Assay, an established bioassay for the evaluation of the PTH effect. After the 3-month-old ovariectomized (OVXed) rats, the OVXed rat is one of the two models required by the U.S. Food and Drug Administration for the preclinical assessment of drugs for treating osteoporosis [DeLuca PP, Dani BA. Skeletal effects of parathyroid hormone (1–34) in ovariectomized rats with or without concurrent administration of salmon calcitonin. Am Assoc Pharm Sci 2001;3(4):E27 [1]]. Sprague–Dawley rats were fed for 14 weeks, daily subcutaneous injections of Pro–Pro–hPTH(1–34) for 16 weeks (0.4, 0.6 or 0.9 nmol/100 g body weight), reduced the ovariectomy (OVX)-triggered mass loss of vertebral trabecular bone. The mean Bone Material Density (BMD) increased to 29.2–34.5% in 3-month-old OVXed rats compared to control-vehicle group ( P < 0.001) and increased to 17.5–22.3% compared to sham-operated groups ( P < 0.01). In short, A recombinant Pro–Pro–hPTH(1–34) was harvested in purified form and its physico-chemical characterization was determined. It showed significantly enhanced activity upon two typical models for PTH fragments. It can increase the mineral density of vertebral trabecular bone just as synthetic hPTH(1–34), and the functional activity of Pro–Pro–hPTH(1–34) should be due to the removing of Pro–Pro- by Dipeptidyl peptidase IV (DPPIV). This study opened out a simplified method which was cheaper, faster than the conventional one for producing active hPTH fragment, and its applied prospect would be good; Furthermore, it may open up our own path in finding new methods for post-processing of gene-engineering product.

Effects of cyclic vs. daily treatment with human parathyroid hormone (1–34) on murine bone structure and cellular activity

Previously, we demonstrated that the human parathyroid hormone (1–34) fragment (hPTH(1–34)) increased bone strength in proportion to its effects on BMD and cortical bone structure in the murine femur by comparing cyclic vs. daily administration of hPTH(1–34). Both cyclic and daily regimens increased vertebral BMD similarly at 7 weeks. Here, we have examined the effects of daily and cyclic PTH regimens on bone structure and cellular activity by static and dynamic histomorphometry. Twenty-week-old, intact female C57BL/J6 mice were treated with the following regimens ( n = 7 for each group): daily injection with vehicle for 7 weeks [control]; daily injection with hPTH(1–34) (40 μg/kg/day) for 7 weeks [daily PTH]; and daily injection with hPTH(1–34) (40 μg/kg/day) and vehicle alternating weekly for 7 weeks [cyclic PTH]. At days 9 and 10, and 2 and 3 prior to euthanasia, calcein (10 mg/kg) was injected subcutaneously. At the end of study, the lumbar vertebrae 1–3 and the left femora were excised, cleaned, and processed for histomorphometry. In the lumbar vertebrae, daily and cyclic PTH regimens significantly increased cancellous bone volume (BV/TV), trabecular number, trabecular osteoclast and osteoblast perimeters, trabecular mineral apposition rate (MAR) and bone formation rate (BFR), and periosteal MAR and BFR compared to control, with no significant difference between the two PTH-treated groups. Increased trabecular tunneling was observed in both PTH-treated groups. Both regimens tended to increase vertebral cortical bone formation parameters with the effects at the periosteum site being more marked than those at the endosteum site, resulting in a significant increase in cortical width. In the femur, the effects of cyclic PTH on BV/TV, trabecular width and number, trabecular and endocortical osteoblast and osteoclast perimeters, cortical width, and trabecular and periosteal BFR were less marked than those of daily PTH. A cyclic PTH regimen was as effective as a daily regimen in improving cancellous and cortical bone microarchitecture and cellular activity in the murine vertebra.

Short-term continuous infusion of human parathyroid hormone 1–34 fragment is catabolic with decreased trabecular connectivity density accompanied by hypercalcemia in C57BL/J6 mice

Parathyroid hormone (PTH) stimulates bone resorption as well as bone formation in vivo and in organ culture. The catabolic actions of PTH have been recognized in patients with hyperparathyroidism, or with acute infusion of the N-terminal 1-34 fragment of human PTH (hPTH1-34). Whereas the anabolic actions of daily injection with PTH have been well studied in both humans and mice, the catabolic actions of PTH on murine bone remain to be defined. To do this we sought to create a model with short-term, sustained hyperparathyroidism using osmotic infusion pumps. We treated 10-week-old female C57BL/J6 mice with continuous infusion of hPTH1-34 (8.1 pmol/0.25 microl per h, equivalent to 40 microg/kg per day) or vehicle for 2 weeks, using Alzet osmotic pumps. Bone mineral density (BMD), serum total calcium, hPTH1-34, mouse intact PTH (mPTH1-84), osteocalcin and mouse tartrate-resistant acid phosphatase (mTRAP) activity, and microarchitectural variables of the distal femur were measured. Separately, we compared the effects of intermittent daily injection of hPTH1-34 (40 microg/kg per day) with continuous infusion of hPTH1-34 on BMD and bone markers. Exogenous hPTH1-34 was detected only in the PTH-infused mice. Both intermittent and continuous treatment with hPTH1-34 markedly suppressed endogenous mPTH1-84, but only the latter induced hypercalcemia. Daily PTH injection significantly increased both serum osteocalcin and mTRAP, while continuous PTH infusion showed a strong trend to stimulate mTRAP, with a slight but non-significant increase in osteocalcin. There were significant differences in BMD at all sites between animals treated with the same daily dose of intermittent and continuous hPTH1-34. Micro-computed tomography (muCT) analysis of the distal femurs revealed that hPTH1-34 infusion significantly decreased trabecular connectivity density (P<0.05). Thus, the murine bone response to continuous PTH infusion was quite different from that seen with daily PTH injection. Short-term infusion of hPTH1-34 appears to be a good model to study the mechanisms underlying the catabolic action of PTH in mice.