Measurements of Osteoanabolic agents PTH (1-34) and PTHrP (1-36) in therapeutic studies and clinical diagnostics

Background: Teriparatide PTH (1-34) is an osteoanabolic agent for treatment of osteoporosis. PTH (1-34) can also be used as replacement therapy in hypoparathyroidism and to accelerate fracture healing. Abaloparatide, PTHrP (1-36) analogue is a novel anabolic drug for treatment of osteoporosis. PTH (1-34) has also been used to assess response to PTH in conditions such as pseudohypoparathyroidism (PHP) (Ellsworth-Howard test (EHT))  Aims: To review the use of PTH (1-34) measurements in drug development studies, and in the diagnosis of patients with PHP. To highlight the potential use of measurement of PTHrP (1-36) using our LC-MS/MS method for measurement of intact PTHrP (1-36), intact PTH (1-34) and their respective oxidised forms simultaneously.  Methods: Pharmacokinetic (PK) profiles from human subjects given either single subcutaneous (sc) injection of 20 ug Teriparatide (n=6) or 0.69 mg (n=4), 2.07 mg (n=6) oral PTH (1-34) (EnteraBio) were analysed using a validated LC-MS/MS method for intact/oxidised PTH (1-34). In EHT, urinary phosphate (spectrophotometric assay; Roche, Germany) and urine/plasma cyclic adenosine 3’,5’–monophosphate (cAMP) (LC-MS/MS) were performed on samples before and after 20ug sc adminstration of Teriparatide.  Results/Discussion: PK profiles (Figure 1A) of oral PTH (1-34) showed a rapid absorption then rapid elimination. In contrast, teriparatide injection showed a slower rate of plasma clearance, possibly due to continuous absorption from the site of administration. C­max was proportional to oral dosage given and the 2.07 mg of oral PTH (1-34) produced comparable Cmax to that produced by 20ug teriparatide injection (Figure 1B). We found the oxidised form of PTH (1-34) represent 20-30% of intact PTH (1-34) in the studied subjects. The EHT profile from a patient suspected of PHP showed a lack of cAMP response despite significant increase in plasma PTH (1-34) concentration. On the contrary, a post dose plasma cAMP concentration of >100 pmol/L and an increase from baseline level of urine cAMP exclude the diagnosis of PHP. Our LC-MS/MS PTHrP (1-36) assay produced a linear calibration curve from 10-2000 pg/mL (r2 >0.990), inter-/ intra-assay CV of 98.6% (CV 3.6%).  Conclusion: Our method for measurements of intact/oxidised form PTH (1-34) and PTHrP (1-36) can offer new insights into the therapeutic use of osteoanabolic agents, and the development of combination therapy with other anti-resorptive/anti-remodelling agents

The calcium-sensing receptor regulates parathyroid hormone gene expression in transfected HEK293 cells

<p>Abstract</p> <p>Background</p> <p>The parathyroid calcium receptor determines parathyroid hormone secretion and the response of parathyroid hormone gene expression to serum Ca²⁺in the parathyroid gland. Serum Ca²⁺regulates parathyroid hormone gene expression <it>in vivo </it>post-transcriptionally affecting parathyroid hormone mRNA stability through the interaction of <it>trans</it>-acting proteins to a defined <it>cis </it>element in the parathyroid hormone mRNA 3'-untranslated region. These parathyroid hormone mRNA binding proteins include AUF1 which stabilizes and KSRP which destabilizes the parathyroid hormone mRNA. There is no parathyroid cell line; therefore, we developed a parathyroid engineered cell using expression vectors for the full-length human parathyroid hormone gene and the human calcium receptor.</p> <p>Results</p> <p>Co-transfection of the human calcium receptor and the human parathyroid hormone plasmid into HEK293 cells decreased parathyroid hormone mRNA levels and secreted parathyroid hormone compared with cells that do not express the calcium receptor. The decreased parathyroid hormone mRNA correlated with decreased parathyroid hormone mRNA stability <it>in vitro</it>, which was dependent upon the 3'-UTR <it>cis </it>element. Moreover, parathyroid hormone gene expression was regulated by Ca²⁺and the calcimimetic R568, in cells co-transfected with the calcium receptor but not in cells without the calcium receptor. RNA immunoprecipitation analysis in calcium receptor-transfected cells showed increased KSRP-parathyroid hormone mRNA binding and decreased binding to AUF1. The calcium receptor led to post-translational modifications in AUF1 as occurs in the parathyroid <it>in vivo </it>after activation of the calcium receptor.</p> <p>Conclusion</p> <p>The expression of the calcium receptor is sufficient to confer the regulation of parathyroid hormone gene expression to these heterologous cells. The calcium receptor decreases parathyroid hormone gene expression in these engineered cells through the parathyroid hormone mRNA 3'-UTR <it>cis </it>element and the balanced interactions of the <it>trans</it>-acting factors KSRP and AUF1 with parathyroid hormone mRNA, as <it>in vivo </it>in the parathyroid. This is the first demonstration that the calcium receptor can regulate parathyroid hormone gene expression in heterologous cells.</p

Enhanced bioavailability and reduced pharmacokinetic variability of Oral PTH (1-34) in man

An orally administered PTH may have prodigious advantages in the treatment of hypoparathyroidism and osteoporosis. Unfortunately, the oral delivery of biologic macromolecules is characterized by a negligible bioavailability and a high dose-to-dose variability in absorption, resulting in difficulty in accurately titrating the drug effect. We present clinical study data of a novel oral peptide delivery technology demonstrating an enhanced bioavailability with reduced Cmax variability. Methods: A Phase I, open label crossover pharmacokinetic (PK) study to assess the safety and PK of oral PTH (1-34) in ten healthy male adult volunteers was conducted. The PK profile of a fixed dose - 1.5mg PTH (1-34) of three different oral formulations was compared. PTH (1-34) levels in the plasma of subjects was analyzed at a number of time points post administration, utilizing a PTH (1-34) immunoassay (IDS; Bolden, UK). In parallel, to assess the pharmacodynamic (PD) effect, serum calcium of subjects receiving the different formulations of oral PTH (1-34) was analyzed. Results: PK profiles of all oral PTH (1-34) formulations were characterized by a rapid absorption and elimination. The systemic exposure (AUC) of the basic oral formulation and two modified formulation versions were 3481 ±1843 pg*min/mL, 7976 ±2556 pg*min/mL and 11369 ±3719 pg*min/mL (mean ± SE). The maximal plasma concentration (Cmax) of these formulations were 145 ±56pg/mL, 375 ±108pg/mL, and 481 ±101pg/mL, respectively. Cmax coefficients of variation (CV%) of the same formulations were 123%, 91% and 67%, respectively. Similarly to the drug absorption, PD response of the modified formulations, presented as the maximal relative increase in albumin adjusted calcium, was improved from 0.07 ±0.29mg/dL to 0.32 ±0.24mg/dL. Discussion: Inherent to oral drug delivery of biopharmaceuticals is the extremely low bioavailability and high absorption variability. The current results indicate that Entera’s delivery technology can overcome these two principal obstacles by achieving repeatable, clinically relevant systemic drug exposure. Entera’s proprietary delivery platform was optimized and achieved anenhancement in drug bioavailability in parallel with the significant decrease in its absorption variability. Similarly, its effect on blood calcium was enhanced by the novel oral formulation of PTH (1-34) pointing out the potential of the drug to be a first line treatment of hypoparathyroidism and osteoporosis

Development and Validation of a LC-MS/MS Assay for Quantification of Parathyroid Hormone (PTH 1-34) in human Plasma

Background: Teriparatide [recombinant human PTH (1-34)] is an osteoanabolic agent for treatment of osteoporosis. The effect on bone decreases the risk of vertebral and non-vertebral fractures and increases bone mineral density (BMD) in post-menopausal women with osteoporosis. Measurement of PTH (1-34) is valuable in assessing treatment response and concordance with therapy.Aim: To develop and validate a method for quantification of PTH (1-34) using Liquid chromatography tandem mass spectrometry (LC-MS/MS) and to perform comparison with a commercial immunoassay.  Method: Sample extraction was developed using a Waters (Milford, MA, USA) Oasis® HLB µElution solid phase extraction. Quantification m/z transition 589>656 was used on Waters/Micromass® Quattro Ultima™ Pt mass spectrometer to measure PTH (1-34) in human plasma using rat PTH (1-34) as internal standard. Validation criteria were carried out against industry standards. PTH (1-34) results obtained from human subjects given Teriparatide (Fortsteo, Eli Lilly, IN, USA) (n=390) were compared against results obtained from an immunoassay (IDS; Boldon Tyne and Wear. UK).  Results and Discussion: LC-MS/MS produced a linear calibration curve from 10 to 2000 pg/mL (r2 >0.990). The LLoQ and LLoD for PTH (1-34) were 10 pg/mL and 2.1 pg/mL respectively. The inter- /intra-assay precision (CV%) of the method were 98.3% for four QCs (20, 100, 200, and 800 pg/mL). The mean recovery of PTH (1-34) was 107.2%. Method comparison between the LC-MS/MS and immunoassay using human EDTA plasma samples showed a high correlation (r2 = 0.950). A concentration-dependent, negative bias of 35.5% was observed across the range of 0 – 800 pg/mL. The immunoassay showed a 7% cross reactivity to human PTH (1-84) and 44% to rat PTH (1-34), no interference was observed in the LC-MS/MS method. Matrix effect and cross reactivity to human PTH (1-84) in the immunoassay were the likely contributing factors to the bias between the methods. The oxidised form of PTH (1-34) does not interfere with our LC-MS/MS method.  Conclusion: Our LC-MS/MS method demonstrated linearity over the calibration range, good precision and accuracy, excellent analyte recovery, and negligible matrix effects. The method was successfully used for measurements of PTH (1-34) in rat and human plasma

First in Man Studies of Pharmacokinetic Profiles of a Novel Oral PTH(1-34)

Background: PTH(1-34) (Teriparatide) is an anabolic agent used in treatment of osteoporosis. It promotes bone formation and reduces the risk of vertebral and some non-vertebral fractures. The route of administration by daily subcutaneous (sc) injection can cause problems in certain patients. A new oral delivery system for human PTH(1-34) has been developed as a possible treatment option. Galitzer et al. presented pre-clinical data (ASBMR 2012, MO0402) and first-in-human results (ASBMR 2013, FR0378) on safety, tolerability and absorption dynamics of oral PTH(1-34) in various dosages. We now describe the pharmacokinetics (PK) of oral PTH(1-34) compared to sc and placebo in healthy subjects. Objective: A single-center, double blinded, triple crossover study was designed to compare the 1.8 mg optimal dose of oral PTH(1-34) against standard dosage of teriparatide injection and oral placebo. Method: The study was conducted following and in accordance with the Hadassah Medical Center ethical approval committee. 12 healthy volunteers (6m/6f), 18-50y, received three treatments: single sc injection of 20µg FORTEO®, 1.8 mg oral PTH(1-34), or placebo. Blood samples were collected at time 0, 10, 15, 20, 30, 45, 60, 75, 90, 120, 180, 240, 300 minute post dose. Plasma concentration of PTH(1-34) (IDS, Tyne and Wear, UK) and cyclic adenosine 3’,5’monophosphate (cAMP) were measured on all samples. Results: All 12 subjects on oral PTH(1-34) showed rapid, post dose increase then decrease of PTH(1-34), from baseline mean (±SD) of 5.9 (1.8) pg/mL to peak mean of 185.3 (±128.8) pg/mL. PK profiles of oral PTH(1-34) showed Cmax (pg/mL), Tmax (mins), AUC0-last of 238.3 (110.8), 17.5 (5.4) and 6161.7 (2726.7), respectively; whereas sc showed mean Cmax (pg/mL), Tmax (mins), AUC0-last of 172.3 (55.7), 20.8 (8.7) and 13965.9 (2984.8), respectively. Plasma cAMP increased in all subjects in response to oral PTH(1-34) and sc treatment. Serum adjusted calcium in all subjects remained within normal limits throughout the studies. Conclusion: PK profiles showed a single oral dose of 1.8 mg PTH(1-34) is rapidly absorbed, and no significant difference in Cmax and Tmax when compared with 20µg of sc teriparatide. A significant difference in the rate of plasma clearance and AUC0-last value was observed (fig.1). These differing profiles and modality of administration of PTH(1-34) could offer unique advantages in the treatment of calcium and metabolic bone disorders

The combined effect of permeation enhancement and proteolysis inhibition on the systemic exposure of orally administrated peptides: Salcaprozate sodium, soybean trypsin inhibitor, and teriparatide study in pigs

Oral delivery of peptides and proteins is hindered by their rapid proteolysis in the gastrointestinal tract and their inability to permeate biological membranes. Various drug delivery approaches are being investigated and implemented to overcome these obstacles. In the discussed study conducted in pigs, an investigation was undertaken to assess the effect of combination of a permeation enhancer – salcaprozate sodium, and a proteolysis inhibitor – soybean trypsin inhibitor, on the systemic exposure of the peptide teriparatide, following intraduodenal administration. Results demonstrate that this combination achieves significantly higher Cmax and AUC (~10- and ~ 20-fold respectively) compared to each of these methodologies on their own. It was thus concluded that an appropriate combination of different technological approaches may considerably contribute to an efficient oral delivery of biological macromolecules

A LC-MS/MS method for the diagnostic measurement of cAMP in plasma and urine

Background: Parathyroid hormone (PTH) plays a key role in calcium and phosphate homeostasis. Upon binding to its receptor, it signals via a second messenger, cyclic adenosine 3, 5’ monophosphate (cAMP). Lack of increase in plasma and urinary cAMP concentrations in response to PTH are used as diagnostic markers for pseudohypoparathyroidism (PHP), a condition primarily associated with resistance to PTH (Ellsworth-Howard Test).  Aims: 1) Develop and validate a LC-MS/MS method for the quantification of cAMP in plasma and urine. 2) Investigate assay performance in a rat pharmacokinetic study investigating the response to an oral dose of PTH (1-34) and the response to subcutaneous (sc) PTH administration in a patient with suspected PHP.  Method: cAMP and 13C5-cAMP internal standard were extracted from EDTA plasma using a weak anion exchange solid phase extraction. Chromatography was performed in positive electrospray ionisation mode, using a pentafluorophenyl column with a 10 mins 2% formic acid water:acetonitrile gradient. Transitions were m/z 330/136 for cAMP and 335/136 for 13C5-cAMP. Over concentrations ranging from 4.6 (lower limit of quantification) to 293.5 nmol/L, the calibration curve was linear (mean curve fits of >0.95, 5 repeats) and intra- and inter-assay precisions were <12% and <8%, respectively. Spiked recovery was 98±5%.  Application: A single oral dose of 5 mg/kg PTH (1–34) or placebo was administered to Sprague-Dawley rats after an overnight fast. cAMP was analysed in EDTA samples obtained at baseline, prior to dosing and every 15 min for 1h and then hourly for another 3h after dosing. In the suspected PHP patient, urinary cAMP was measured after a standard 20µg sc injection of teriparatide (Forsteo).  Results: In rats, plasma PTH (1-34) and cAMP increased significantly within 15min of dosing, reaching peak values between 15 and 30 mins. PTH (1–34) concentration increased significantly by up to 6770-fold, although response to PTH (1-34) varied between animals. Plasma cAMP typically tripled, from 36.5±3.7 nmol/L at baseline to 108.9±26.3 nmol/L. Placebo had no effect.Urine cAMP from the suspected PHP patient did not change significantly reflecting a lack of biological response to sc PTH (1-34) despite a significant increase in plasma PTH (1-34) (27.8 to 101.1 pmol/L).  Conclusion: The present method was robust and selective. It also showed utility in determining cAMP in biological systems and the ability to study the effect of drugs such as Forsteo

Safety and efficacy of oral human parathyroid hormone (1-34) in hypoparathyroidism: An open-label study

The standard treatment of primary hypoparathyroidism (hypoPT) with oral calcium supplementation and calcitriol (or an analog), intended to control hypocalcemia and hyperphosphatemia and avoid hypercalciuria, remains challenging for both patients and clinicians. In 2015, human parathyroid hormone (hPTH) (1-84) administered as a daily subcutaneous injection was approved as an adjunctive treatment in patients who cannot be well controlled on the standard treatments alone. This open-label study aimed to assess the safety and efficacy of an oral hPTH(1-34) formulation as an adjunct to standard treatment in adult subjects with hypoparathyroidism. Oral hPTH(1-34) tablets (0.75 mg human hPTH(1-34) acetate) were administered four times daily for 16 consecutive weeks, and changes in calcium supplementation and alfacalcidol use, albumin-adjusted serum calcium (ACa), serum phosphate, urinary calcium excretion, and quality of life throughout the study were monitored. Of the 19 enrolled subjects, 15 completed the trial per protocol. A median 42% reduction from baseline in exogenous calcium dose was recorded (p =.001), whereas median serum ACa levels remained above the lower target ACa levels for hypoPT patients (>7.5 mg/dL) throughout the study. Median serum phosphate levels rapidly decreased (23%, p =.0003) 2 hours after the first dose and were maintained within the normal range for the duration of the study. A notable, but not statistically significant, median decrease (21%, p =.07) in 24-hour urine calcium excretion was observed between the first and last treatment days. Only four possible drug-related, non-serious adverse events were reported over the 16-week study, all by the same patient. A small but statistically significant increase from baseline quality of life (5%, p =.03) was reported by the end of the treatment period. Oral hPTH(1-34) treatment was generally safe and well tolerated and allowed for a reduction in exogenous calcium supplementation, while maintaining normocalcemia in adult patients with hypoparathyroidism

LC-MS/MS measurement of parathyroid hormone PTH (1-34): Use in studying oral PTH (1-34) administration and possible diagnostic application in pseudohypoparathyroidism.

Background: Teriparatide PTH(1-34) is an osteoanabolic agent used in the treatment of osteoporosis. Measurement of PTH(1-34) can be useful in osteoporosis treatment and in the diagnosis of pseudohypoparathyroidism (PHP) by confirming administration of PTH(1-34) Aims: 1) To assess PTH(1-34) profiles obtained using standard Forsteo treatment compared to using a novel oral administration. 2) To confirm the PTH(1-34) in a PHP patient receiving PTH(1-34) as part of an Ellsworth Howard Test (EHT). 3) To perform a method comparison of oxidised/non oxidised forms of PTH(1-34) detected by LC-MS/MS with immunoassay. Methods: Using a LC-MS/MS method, PTH(1-34) was measured in Pharmacokinetic (PK) profiles from a human double blinded study. Participants were given teriparatide either by a single SC injection (Forsteo,20µg) (n=6); or in an oral dose of 0.69mg (n=4), or 2.07mg (n=6) (EnteraBio). In an EHT, PTH(1-34), urinary PO4, and urine/plasma cyclic adenosine 3’5’-monophosphate (cAMP) were measured on samples before/after 20µg Forsteo injection. Oxidised/non-oxidised forms of PTH(1-34) (n=390) measured by LC-MS/MS were compared against immunoassay (IDS; Boldon, UK). Results: PK profiles showed rapid absorption of PTH(1-34) in plasma. The 2.07 mg oral dose achieved Cmax of 271pg/mL comparable to that of 20µg Forsteo, but the injection form showed slower rate of plasma clearance (T½(injection)¬=37.7min, T½(oral)=12.5min). The EHT profile from a PHP patient showed a lack of cAMP response despite significant increase in plasma PTH(1-34) concentration. Method comparison showed LC-MS/MS results were correlated (r2=0.950), but biased (-35.5%) against the immunoassay. The bias was caused partly by a matrix effect (14.6±18.4%), cross reactivity of the immunoassay with PTH(1-84) (7.1±0.45%) and to oxidised forms of PTH(1-34) (23.9±6.1%). Conclusion: Our LC-MS/MS method for PTH(1-34) can help validate the therapeutic use of osteoanabolic agents; confirm the lack of response to exogenous stimulation in EHT; and may explain the differences in responses to treatment due to oxidation of PTH(1-34)

The parathyroid is a target organ for FGF23 in rats

Phosphate homeostasis is maintained by a counterbalance between efflux from the kidney and influx from intestine and bone. FGF23 is a bone-derived phosphaturic hormone that acts on the kidney to increase phosphate excretion and suppress biosynthesis of vitamin D. FGF23 signals with highest efficacy through several FGF receptors (FGFRs) bound by the transmembrane protein Klotho as a coreceptor. Since most tissues express FGFR, expression of Klotho determines FGF23 target organs. Here we identify the parathyroid as a target organ for FGF23 in rats. We show that the parathyroid gland expressed Klotho and 2 FGFRs. The administration of recombinant FGF23 led to an increase in parathyroid Klotho levels. In addition, FGF23 activated the MAPK pathway in the parathyroid through ERK1/2 phosphorylation and increased early growth response 1 mRNA levels. Using both rats and in vitro rat parathyroid cultures, we show that FGF23 suppressed both parathyroid hormone (PTH) secretion and PTH gene expression. The FGF23-induced decrease in PTH secretion was prevented by a MAPK inhibitor. These data indicate that FGF23 acts directly on the parathyroid through the MAPK pathway to decrease serum PTH. This bone-parathyroid endocrine axis adds a new dimension to the understanding of mineral homeostasis