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.     

Production and enhanced biological activity of a novel GHRH analog, hGHRH with an N-terminal Pro-Pro extension

Growth Hormone Releasing Hormone (GHRH) is one of the most important hormones in life. Because of its potential clinical importance, its short half-life, and its expensive chemical synthesis, an analog of hGHRH with a prolonged half-life and better activity has been studied for clinical application, especially for the treatment of muscle wasting, type II diabetes, or sleep disorders. The Pro-Pro-hGHRH(1-44) peptide has better activity. The fusion partner gene with 127 amino acid residues of the C-terminus from l-asparaginase was recombined with asp-pro-pro-hGHRH(1-44) gene synthesized by PCR method to form a fusion protein with the unique acid labile linker Asp-Pro. The recombinant protein was expressed to high levels in Escherichia coli BL21 (DE3). The Pro-Pro-hGHRH(1-44) peptide was purified to homogeneity by means of cell disruption, washing, ethanol precipitation, acid hydrolysis, and SP-Sephadex C-25, and Sephadex G-25 column chromatography. The fold of the purification was about 88 times and the yield was 1.1% of the total protein weight of the inclusion body. The peptide molecular mass of 5235.25 Da was determined by ESI mass spectroscopy. Its purity was determined by SDS-PAGE. In the study of the activity, we measured GH release of rat pituitary by using the antiserum kit against human GH. The peptide doses of 0.01, 0.1, 1.0, 7.72, and 20.9 microg/ml used, respectively, released the GH values of 0.1+/-0.1, 12.5+/-7.3, 16.6+/-5.8, 49.8+/-7.6, and 79.5+/-5.7 ng/ml whereas their blank controls, respectively, were 0.5+/-0.8, 4.1+/-2.6, 3.1+/-3.1, 4.7+/-1.8, and 1.2+/-0.3 ng/ml. The activity results of all dose groups except 0.01 microg/ml Pro-Pro-hGHRH(1-44) group and hGHRH(1-40) group showed that there were significant differences between GH released by the peptide and that by its blank control. With the increase of dosage, the differences were more significant. hGHRH(1-40) showed no measured GH release when the dose was up to 2 microg/ml. The activity results show that the Pro-Pro-hGHRH(1-44) peptide is a potential GH releasing analog.     

Nasal administration of a novel recombinant human parathyroid hormone (1-34) analog for the treatment of osteoporosis of ovariectomized rats

Synthetic human parathyroid (1-34) (hPTH (1-34)) is known to have the full biological activity of the holohormone for osteoporosis. This study is about designing a novel analog of hPTH (1-34) which is more suitable for intranasal administration. We likewise evaluate effectiveness of the nasal drops against osteoroporosis. Through fusion expression of combining gene, cell disruption, inclusion body washing, ethanol fraction precipitation, acid hydrolysis, and CM-52 ion exchange column chromatography Pro-Pro-[Arg11] hPTH (1-34)-Pro-Pro was designed and produced. Nasal drops of Pro-Pro-[Arg11] hPTH (1-34)-Pro-Pro were prepared and administrated to ovariectomized rats. After 12 weeks of raising, Bone Material Densities (BMD) of vertebrae were examined by Dual Energy X-Ray Absorptiometry (DEXA). The average BMD of these groups treated with nasal drops of the peptide were 28.0%-47.2% (P<0.01) higher than that of the group treated with normal saline (NS). The subchondral bone plates of the femoral heads were examined by scanning electron microscopy and a defined planar section was photographed. Percentage of the area of the cancellous bone was calculated. Percentages of the groups treated with nasal drops of the peptide increased; values were significantly different to that of the group treated with NS (P<0.001) and were even equivalent to that of normal groups. These results show that nasal drops of Pro-Pro-[Arg11] hPTH (1-34)-Pro-Pro are effective against osteoporosis.     

Anabolic effects of recombinant human parathyroid hormone (1 - 84) and synthetic human parathyroid hormone (1 - 34) on the mandibles of osteopenic ovariectomized rats with maxillary molar extraction.

In rodent osteoporosis models such as ovariectomized (OVX) rats, intermittently administered human parathyroid hormone (hPTH) has an anabolic effect in vertebrae and long bones. In the present experiments, subcutaneously injected hPTH(1 - 34) or hPTH(1 - 84) dose- and time-dependently increased bone mineral density (BMD) as measured by dual energy X-ray absorptiometry in mandibles, L2 to L4 vertebrae and femurs of such rats. The highest dose (15.9 nmol/kg, s. c.) of either peptide given four times weekly for 10 weeks completely reversed the effects of overiectomy on BMD. Significant elevation in lumbar BMD after 10 weeks was observed with hPTH(1 - 34) or hPTH(1 - 84) at 1.1 nmol/kg, whereas hPTH(1 - 34) at 1.1 and 4.2 nmol/kg significantly increased BMD of the whole bone and the metaphysis of the femur and the diaphysis of the bone, respectively. In contrast, significant effects of hPTH(1 - 84) administration on BMD increase in the femur were observed at 4.2 and 15.9 nmol/kg in the whole bone and the metaphysis, and in the diaphysis, respectively. Maxillary molar extraction left mandibular BMD in rats with intact ovaries unchanged, but significantly decreased mandibular BMD in OVX rats. Administration of hPTH(1 - 84) for 10 weeks in OVX rats without or with extraction significantly increased BMD in the mandibular molar region at doses of 15.9 and 4.2 nmol/kg, respectively, indicating that efficacy was increased by extraction. A significant BMD increase in the molar region in OVX rats with extraction occurred at only 1.1 nmol/kg of hPTH(1 - 34) and 4.2 nmol/kg of hPTH(1 - 84). Also, BMD of the ramus region was increased by administration of both peptides to a lesser extent than that of the molar region in these rats. Thus, intermittent administration of hPTH, especially hPTH(1 - 34), has an anabolic effect on bone, particularly alveolar bone. Such treatment may increase alveolar bone mass in postmenopausal women with osteoporosis.     

Expression-level dependent activation of recombinant human parathyroid hormone/parathyroid hormone-related peptide receptor: effect of human parathyroid hormone (1-34), (1-31), and (28-48)

A stable recombinant chinese hamster ovary (CHO) cell model system expressing the human type-1 receptor for parathyroid hormone and parathyroid hormone-related peptide (hPTH-R) was established for the analysis of human PTH (hPTH) variants. The cell lines showed receptor expression in the range from 10(5) to I.9 x 10(6) receptors per cell. The affinity of the receptors for hPTH-(1-34) was independent of the receptor number per cell (Kd approximately = 8 nmol/1). The induction of cAMP by hPTH-(1-34) is maximal in clones expressing >2x10(5) receptors per cell and Ca++ signals were maximal in cell lines expressing >1.4x10(6) receptors per cell. Second messenger specific inhibitors demonstrated that PTH-induced increases in intracellular cAMP and Ca++ are independent and Ca++ ions are derived from intracellular stores. The cAMP-specific receptor activator hPTH-(1-31) showed also an increase in intracellular Ca++. Even in cell lines expressing more than 10(6) receptors per cell the Ca++/PKC specific activator hPTH-(28-48) did not activate hPTH-Rs. Based on these results, synthesis of further derivatives of PTH is required to identify pathway-specific ligands for the type-1 hPTH-R.     

Large-scale preparation and biological activity of recombinant human parathyroid hormone

Human parathyroid hormone (hPTH) has been bacterially expressed in bioreactors as cro-β-galactosidase-hPTH fusion protein. We have developed a large-scale purification scheme that exploits the pH-dependent differential solubility of hPTH and a two-step Chromatographie procedure. We demonstrate that in a number of assay systems, the recombinant material obtained by this procedure is biologically active.     

Solution structures of human parathyroid hormone fragments hPTH(1-34) and hPTH(1-39) and bovine parathyroid hormone fragment bPTH(1-37)

Parathyroid hormone (PTH) is involved in regulation of the calcium level in blood and has an influence on bone metabolism, thus playing a role in osteoporosis therapy. In this study, the structures of the human PTH fragments (1-34) and (1-39) as well as bovine PTH(1-37) in aqueous buffer solution under near physiological conditions were determined using two-dimensional nuclear magnetic resonance spectroscopy. The overall structure of the first 34 amino acids of these three peptides is virtually identical, exhibiting a short NH(2)-terminal and a longer COOH-terminal helix as well as a defined loop region from His14 to Ser17, stabilized by hydrophobic interactions. bPTH(1-37), which has a higher biological activity, shows a better-defined NH(2)-terminal part. In contrast to NH(2)-terminal truncations, which cause destabilization of helical structure, neither COOH-terminal truncation nor elongation significantly influences the secondary structure. Furthermore, we investigated the structure of hPTH(1-34) in 20% trifluoroethanol solution. In addition to its helix-stabilizing effect, trifluorethanol causes the loss of tertiary hydrophobic interactions.     

Large scale preparation of recombinant human parathyroid hormone 1-84 from Escherichia coli

Human parathyroid hormone (hPTH) is a promising agent in the treatment of osteoporosis. The intact recombinant human parathyroid hormone [rhPTH(1-84)] was prepared in a large scale from Escherichia coli using a soluble fusion protein strategy. With degenerate codons, gene of hPTH(1-84) was synthesized, ligated with pET32a(+) vector, and then expressed in E. coli BL21(DE3) cells. The soluble fusion protein His(6)-thioredoxin-hPTH(1-84) was harvested after purification by immobilized metal affinity chromatography (IMAC). Following enterokinase cleavage, ion-exchange-chromatography (IEC) and size-exclusive-chromatography (SEC) were used, and finally, over 300mg/l intact hPTH(1-84) with high purity up to 99% was obtained. The purified rhPTH(1-84) was confirmed by mass spectrometry and N-terminal/C-terminal amino-acid sequence analysis. Additionally, this product stimulated adenylate cyclase in Rat Osteosarcoma Cell UMR-106 at the same extent as hPTH standards, indicating that the purified rhPTH(1-84) has full biological activity. The efficient procedure for expression and purification of rhPTH(1-84) may be useful for the mass production of this important protein.     

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 1h 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.     

Pilot study with technosphere/PTH(1-34)--a new approach for effective pulmonary delivery of parathyroid hormone (1-34)

Sequential subcutaneous PTH injection therapy (repeated 14 days of PTH administration and a subsequent treatment pause for a few weeks) is known to increase bone mineral density in patients with osteopenic disorders. Alternative methods of drug delivery may be beneficial in increasing compliance. A pilot study was performed in 10 healthy volunteers (4 female/6-male, age: 25.6 +/- 3.5 years, BMI: 22.3 +/- 2.4 kg/m 2, mean +/- SD) to assess the pharmacokinetic profiles of 1600 IU of PTH(1 - 34) using the pulmonary Technosphere drug delivery system in comparison to a subcutaneous injection of 400 IU. The treatments were administered in the morning after an overnight fast and blood samples for measurement of PTH(1 - 34), PTH(1 - 84), and calcium and calcitonin were taken over a period of 6 hours. Both injection and pulmonary application of PTH(1 - 34) were well tolerated. After pulmonary administration of Technosphere/PTH(1 - 34), PTH(1 - 34) appeared in the serum with a faster concentration increase (T max: pulmonary 10 +/- 5 min vs. subcutaneous 28 +/- 8 min, p < 0.001) and with higher maximal concentrations (C max : pulmonary 309 +/- 215 pmol/l vs. subcutaneous 102 +/- 45 pmol/l, p < 0.05) as compared to the subcutaneous injection. The relative bioavailability of pulmonary Technosphere/PTH(1 - 34) was calculated to be 48 %. No differences were seen between pulmonary and subcutaneous application with regard to the PTH(1 - 84), calcitonin and calcium concentrations. In conclusion, pulmonary application of Technosphere/PTH(1 - 34) appears to be an effective and thus attractive candidate for PTH substitution therapy in osteoporosis and other conditions leading to a decrease in bone mineral density.     

Two-dimensional 1H-NMR study of the 1-34 fragment of human parathyroid hormone

The complete assignment of resonances in the proton nmr spectrum of the 1-34 amino acid fragment of human parathyroid hormone [hPTH(1-34)], determined using a combination of one- and two-dimensional nmr techniques at 500 MHz, is described. In particular, homonuclear Hartmann-Hahn experiments, recorded in H2O and D2O, are used to resolve ambiguities in the connectivities between the highly overlapped resonances in the aliphatic region of the spectrum. One-dimensional multiple quantum filtering experiments are used to identify serine and phenylalanine spin systems. Analyses of the through-bond and through-space connectivities in the alpha H-NH fingerprint regions of the correlated spectroscopy (COSY) and nuclear Overhauser effect spectroscopy (NOESY) spectra lead to the assignment of resonances to specific amino acid residues in the polypeptide. Examination of the observed NOE cross peaks indicates that hPTH(1-34) has no detectable secondary structural elements in aqueous solution.     

Crystal structure of human parathyroid hormone 1-34 at 0.9-A resolution

The N-terminal fragment 1-34 of parathyroid hormone (PTH), administered intermittently, results in increased bone formation in patients with osteoporosis. PTH and a related molecule, parathyroid hormone-related peptide (PTHrP), act on cells via a common PTH/PTHrP receptor. To define more precisely the ligand-receptor interactions, we have crystallized human PTH (hPTH)-(1-34) and determined the structure to 0.9-A resolution. hPTH-(1-34) crystallizes as a slightly bent, long helical dimer. Analysis reveals that the extended helical conformation of hPTH-(1-34) is the likely bioactive conformation. We have developed molecular models for the interaction of hPTH-(1-34) and hPTHrP-(1-34) with the PTH/PTHrP receptor. A receptor binding pocket for the N terminus of hPTH-(1-34) and a hydrophobic interface with the receptor for the C terminus of hPTH-(1-34) are proposed.     

1,25-Dihydroxyvitamin D production after stimulation with synthetic human parathyroid hormone (1-34) in hypoparathyroid and renal tubular disorders

1,25-dihydroxyvitamin D production in response to two successive infusions of synthetic active 1-34 fragment of human PTH [hPTH-(1-34)] was evaluated in order to develop an understanding of the vitamin D metabolism and the rationale of vitamin D therapy in calcium disorders. Five normal controls, six hypoparathyroid patients, two patients with hypophosphatemic vitamin-D-resistant rickets, one patient with Lowe's synd. and one patient with primary Fanconi's synd. were investigated, and the following results were obtained. All normal controls showed a significant increase in serum 1,25(OH)2D[43 +/- 3.8 (m +/- SEM, n = 5, basal), 53 +/- 4.3 (three hours after the first PTH infusion), 65 +/- 7.7 (six hours) and 66 +/- 4.4 (nine hours) pg/ml]. All patients with PTH-deficient hypoparathyroidism showed a significant increase in serum 1,25(OH)2D, and serum 1,25(OH)2D values were within the normal range after hPTH-(1-34) stimulation. Serum 1,25(OH)2D remained low after hPTH-(1-34) infusions in a patient with pseudohypoparathyroidism type I who showed a significant increase in this value after infusion of dibutyryl cyclic AMP. On the other hand, a patient with normocalcemic pseudohypoparathyroidism type I had a high basal 1,25(OH)2D value, which increased further after hPTH-(1-34) infusions. An almost normal increase in serum 1,25(OH)2D was observed in two patients with hypophosphatemic vitamin-D-resistant rickets, one with Lowe's syndrome and the other with primary Franconi's syndrome. We conclude that these results ae important in obtaining an understanding of calcium and vitamin D metabolism in these disorders and that this PTH stimulation test is a useful method to use in evaluating renal responsiveness in 1,25(OH)2D production to PTH in various calcium disorders.     

Human parathyroid hormone (1-34) increases urinary excretion of lysosomal enzymes in rats

The kidney is the major target of parathyroid hormone (PTH), and PTH influences the urinary excretion of calcium, phosphate and hydrogen ions. It was previously reported that the urinary, excretion of N-acetyl-beta-D-glucosaminidase (NAG), a lysosomal enzyme, transiently increases after human PTH (hPTH) (1-34) infusion in normal subjects and idiopathic hypoparathyroidism patients, but not in pseudohypoparathyroidism type I patients. Here we report that intravenous infusion of hPTH(1-34) to rats transiently increased the urinary excretion of various lysosomal enzymes, such as beta-glucuronidase and acid phosphatase as well as NAG. However, it did not affect the urinary excretion of tubular brush border membrane enzymes, i.e. alkaline phosphatase, leucine aminopeptidase and gamma-glutamyl transpeptidase. Human PTH(1-34) dose-dependently increased the urinary excretion of NAG in rats with a peak at 30 min, which returned to a baseline within 60 min. The increase in the urinary NAG excretion caused by hPTH(1-34) positively correlated with the increase in the urinary cAMP excretion (r = 0.844, p < 0.01), and infusion of dibutyryl cAMP at a dose of 20 mg/kg similarly increased the urinary excretion of NAG. These results suggested that the increase in the urinary excretion of lysosomal enzymes caused by hPTH(1-34) may be a functional response to hPTH(1-34) occurring in the renal tubules via PTH signaling pathway.     

Activation of avian medullary bone osteoclasts by oxidized synthetic parathyroid hormone (1-34)

Synthetic bovine parathyroid hormone (1-34) [bPTH(1-34)] has been treated with hydrogen peroxide and assayed for the effect of such treatment on the ability of bPTH(1-34) to activate medullary bone osteoclasts during their quiescent period in the early phase of the ovulatory cycle in Japanese quail. In addition, the same batches of oxidized and unoxidized bPTH(1-34) were assayed for their hypercalcemic activity in Japanese quail and their capacity to stimulate renal adenylate cyclase activity in the same species. Three groups, each consisting of five 5-month-old egg-laying Japanese quail (Coturnix coturnix japonica), were used. Between 4 to 5 hr after oviposition the three groups were injected intraperitoneally with acid saline (control) solution, bPTH(1-34) at 40 micrograms/bird, or oxidized bPTH(1-34) at 40 micrograms/bird, respectively. Twenty minutes after injection, the femoral bones were removed, split, fixed, and appropriately processed for examination by electron microscopy. Both oxidized and unoxidized bPTH(1-34) stimulated the development of osteoclast ruffled borders within 20 min after injection of the hormone preparations. As anticipated from previously published work from this laboratory, oxidized bPTH(1-34) retained its hypercalcemic activity and lost its capacity to stimulate renal adenylate cyclase activity in the Japanese quail. These results support, but do not prove, the contention that bPTH(1-34) exhibits its responses in the Japanese quail through the mediation of more than one type of receptor.     

Preparation of a photoreactive analog of parathyroid hormone [Nle8, Lys(N-epsilon-4-azido-2-nitrophenyl)13,Nle18,Tyr34]bovine parathyroid hormone-(1-34)NH2, a selective, high-affinity ligand for characterization of parathyroid hormone receptors

Photoaffinity radiolabeling techniques have been widely used to characterize the properties of peptide hormone receptors. However, the identity of authentic receptors is often uncertain because many macromolecules are labeled. These ambiguities are due, in part, to the use of a heterogeneous mixture of photoreactive photoligands, many of which have no or low affinity for the relevant hormone receptor. In this report, we describe the synthesis, purification, and structural analysis of the photoreactive parathyroid hormone analog, [Nle8,Lys(N-epsilon-4-azido-2-nitrophenyl)13,Nle18,Tyr34]-bovine parathyroid hormone-(1-34)NH2. The sulfur-free, oxidation-resistant, synthetic analog of bovine parathyroid hormone (PTH), [Nle8,Nle18,Tyr34]bovine PTH-(1-34)NH2 (NlePTH), was reacted with 4-fluoro-3-nitrophenylazide under nonaqueous conditions to yield several derivatives which were separated by reverse-phase high-performance liquid chromatography and analyzed by amino acid compositional analysis, thin-layer chromatography, and ultraviolet and visible absorption spectroscopy. Among the NlePTH derivatives generated, one of the least hydrophobic was shown to retain the highest potency as assessed in the canine renal cortical membrane radioreceptor assay. Sequence analysis of this peptide, after it had been derivatized with 4-fluoro-3-nitro-[2,6-3H]phenylazide and purified to homogeneity, permitted us to determine that the structure of this analog is [Nle8,Lys(N-epsilon-4-azide-2-nitrophenyl)13,Nle18,Tyr34]bovine PTH-(1-34)NH2. We emphasize the importance of using photoreactive ligands which are purified and subjected to detailed chemical and biological analyses for characterizing the properties of parathyroid hormone receptors and receptors for other peptide hormones.