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Title: Measurement of human PTH (1-34) and human PTHrP (1-36) using liquid-chromatography tandem mass spectrometry : application to therapeutic studies and drug monitoring
Author: Al Riyami, Sulaiman
ISNI:       0000 0004 7973 2385
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2019
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Background: Teriparatide, a recombinant human parathyroid hormone (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. PTH (1-34) can also be used as replacement therapy in hypoparathyroidism and to accelerate fracture healing. PTH (1-34) has also been used to assess response to PTH in conditions such as pseudohypoparathyroidism (PHP) (Ellsworth-Howard test (EHT)). Abaloparatide, a synthetic peptide analog of human parathytoid hormone-related peptide (PTHrP) (1-34), is also an osteoanabolic agent recently approved for treatment of osteoporosis by the FDA. The amino acid sequence of abaloparatide is identical to that of PTHrP in the first 21 amino acids, while eight of the remaining amino acids are different to enhance its affinity for PTHR1. PTHrP (1-36) induces beta cell proliferation and improves glucose-stimulated insulin secretion. Ongoing studies are exploring the potential of PTHrP (1-36) to enhance the functional beta cell mass in the setting of diabetes. Aims: To develop a sensitive method for quantification of PTH (1-34) and PTHrP (1-36) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). To develop assays in accordance with published guidelines on bioanalytical method validation. To perform a comparison study on human PTH (1-34) with a commercial immunoassay. To review the use of PTH (1-34) measurements in drug development studies, and in the drug monitoring during diagnosis of patients with PHP. Method: Sample extraction was developed using a Waters (Milford, MA, USA) Oasis® HLB μElution solid phase extraction. Quantification m/z transitions of 589.2>656.1 and 609.5>682.7 were used on Waters/Micromass® Quattro Ultima™ Pt mass spectrometer to measure PTH (1-34) and PTHrP (1-36), respectively, in human plasma using rat PTH (1-34) as internal standard. Validation criteria were carried out against industry standards. hPTH (1-34) results obtained by LC-MS/MS (n=390) were compared against results obtained from an immunoassay (IDS; Boldon Tyne and Wear. UK). Pharmacokinetic (PK) profiles from human subjects given either single subcutaneous (sc) injection of 20 μg Teriparatide (n=10) or 0.69 mg (n=5), 2.07 mg (n=10) oral PTH (1-34) (EnteraBio) were analysed using the validated LC-MS/MS method for hPTH (1-34). 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-assay precision (CV%) and accuracy (%RE) of the method were < 9.8% and < 14.9%, respectively, for four QCs (20, 100, 200, and 800 pg/mL). While, the intra-assay precision and accuracy were < 7.8% and < 6.9%, respectively. The mean recovery of PTH (1-34) was 107.2%. For PTHrP (1-36), LC-MS/MS produced a linear calibration curve from 25 to 2000 pg/mL (r2 >0.96). The LLoQ and LLoD for PTHrP (1-36) were 25 pg/mL and 2.5 pg/mL respectively. The inter- and intra-assay variations of the precision were < 11.8% and 12.4%, respectively, while those for accuracy were < 9.1 and 10.7%, respectively, at four QC concentrations (50, 100, 200, and 800 pg/mL). The assay showed efficient recovery of hPTHrP (1-36) from plasma with an average recovery of 103.7%. Method comparison between the LC-MS/MS and immunoassay for PTH (1-34) 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 oxidized form of PTH (1-34) does not interfere with the LC-MS/MS method. PK profiles 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 20 ug teriparatide injection. Conclusion: The LC-MS/MS methods for measurement of PTH (1-34) and PTHrP (1-36) demonstrated linearity over the calibration range, good precision and accuracy, excellent analyte recovery, and negligible matrix effects. The methods have a capability of measuring oxidized and non-oxidized forms of PTH (1-34) that may offer new insights into the physiology and pathophysiology of PTH, aid studies in the therapeutic use/efficacy of osteoanabolic agents and may facilitate in the development of combination therapy with other anti-resorptive/anti-remodeling agents.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available