PTHrP N15 Human

What is PTHrP N15 Human and how does it differ from regular PTHrP?

PTHrP N15 Human is a recombinant human Parathyroid Hormone-Related Protein that has been labeled with the stable isotope N15. It is a single, non-glycosylated polypeptide chain containing 86 amino acids with a molecular weight of approximately 10033 Da . The key difference from regular PTHrP is the incorporation of the stable isotope N15, which allows for specific tracking and quantification in experimental settings, particularly in mass spectrometry applications. This isotopic labeling does not alter the biological function of the protein but provides a distinct mass signature that can be exploited in various analytical techniques, particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS) .

What are the primary receptor interactions of PTHrP N15 Human?

PTHrP acts as a powerful and discriminating agonist of PTH2R (Parathyroid Hormone 2 Receptor). Through this interaction, it participates in adenyl cyclase activation and elevation of intracellular calcium levels . It encourages protein kinase C beta activation, recruitment of beta-arrestin, and PTH2R internalization . Additionally, PTHrP inhibits cell proliferation through its contribution to PTH2R activation. It has been noted to activate nociceptors and nociceptive circuits and can act as a neuropeptide in spermatogenesis . Unlike PTH, which exhibits both anabolic and catabolic effects, PTHrP appears to function as a pure skeletal anabolic agent when administered intermittently .

How does PTHrP N15 Human differ from PTH (1-34) in therapeutic applications?

PTH (1-34), or teriparatide, is currently the only approved skeletal anabolic agent for osteoporosis treatment, but it functions as a mixed anabolic and catabolic agent . In contrast, PTHrP displays features suggesting it may be a pure anabolic agent when intermittently administered, even at doses as high as 750 μg/day . Clinical trials have shown that PTHrP administered at 500 and 625 μg/day for 3 weeks is well-tolerated and appears to act solely as a skeletal anabolic agent . In a 3-month study, daily subcutaneous PTHrP at 400 μg/day increased lumbar spine BMD by approximately 5%, comparing favorably with PTH . Importantly, unlike PTH, markers of bone resorption were not activated with PTHrP treatment, and even minor hypercalcemia was not observed in early studies .

What analytical methods are most effective for quantifying PTHrP N15 in experimental samples?

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) represents the gold standard for quantifying PTHrP N15 in research samples. A validated LC-MS/MS method has been developed with quantification m/z transitions of 609.5>682.7 for PTHrP (1-36) . Sample extraction can be performed using Waters Oasis® HLB μElution solid phase extraction, followed by analysis on a mass spectrometer such as Waters/Micromass® Quattro UltimaTM Pt . This method has demonstrated high precision with inter- and intra-assay variations <11.8% and 12.4%, respectively, and accuracy variations <9.1% and 10.7% at concentrations ranging from 50-800 pg/mL . The recovery efficiency from plasma averages 103.7%, indicating excellent extraction performance .

How should PTHrP N15 Human be properly stored and reconstituted for experimental use?

For optimal stability, lyophilized PTHrP N15 Human should be stored desiccated below -18°C, although it remains stable at room temperature for up to 3 weeks . Prior to opening, it is recommended to briefly centrifuge the vial to bring contents to the bottom . For reconstitution, use 10mM HAc (acetic acid) to achieve a concentration of 0.1-1.0 mg/mL, with further dilutions made in appropriate buffered solutions . After reconstitution, PTHrP N15 should be stored at 4°C if used within 2-7 days, or below -18°C for longer-term storage . For extended storage periods, adding a carrier protein (0.1% HSA or BSA) is recommended to enhance stability . Importantly, freeze-thaw cycles should be avoided to maintain protein integrity .

What are the key considerations when designing dose-response studies with PTHrP N15 Human?

When designing dose-response studies with PTHrP N15 Human, researchers should consider the therapeutic window established in clinical trials. Studies have shown that PTHrP is well-tolerated at doses up to 625 μg/day, while mild hypercalcemia may develop at 750 μg/day in some subjects . A dose-escalating design is recommended, starting with lower doses (e.g., 500 μg/day) and progressively increasing by increments of 125 μg . Monitoring for adverse events is critical, with defined protocols for subject discontinuation if hypercalcemia develops . Researchers should assess both markers of bone formation and resorption to characterize the anabolic/catabolic profile at each dose level. The duration of administration should also be carefully considered, with clinical studies ranging from 1 day to 3 months showing different outcomes .

How can researchers differentiate between the effects of PTHrP and PTH in experimental models?

To differentiate between PTHrP and PTH effects, researchers should implement a comprehensive analytical approach:

• Simultaneous measurement of formation and resorption markers: While PTH increases both formation and resorption markers, PTHrP primarily increases formation markers without activating resorption .

• Calcium homeostasis monitoring: PTHrP has been shown to have less effect on serum calcium levels compared to equivalent doses of PTH .

• Receptor binding studies: Although both hormones act via the common PTH-PTHrP receptor (PTH1R), their binding kinetics and downstream signaling pathways may differ .

• Comparative time-course studies: The temporal profile of effects may differ between the two hormones.

• Method specificity verification: Ensure analytical methods (particularly immunoassays) do not cross-react between PTH and PTHrP. LC-MS/MS offers superior specificity in this regard, as demonstrated in comparison studies showing that while immunoassays may have 7% cross-reactivity to human PTH (1-84) and 44% to rat PTH (1-34), LC-MS/MS methods show no such interference .

What controls should be incorporated in PTHrP N15 Human experimental protocols?

Rigorous experimental design with PTHrP N15 Human should include:

• Vehicle controls: Placebo groups receiving saline or appropriate vehicle solution to account for injection effects .

• Acetic acid controls: When using acetic acid in reconstitution, include controls with acetic acid alone to identify potential dermatological reactions or other effects from the diluent .

• PTH positive controls: Include PTH treatment arms to directly compare anabolic and catabolic effects .

• Dose gradient: Include multiple dose levels to establish dose-response relationships .

• Time-course sampling: Collect samples at multiple timepoints (e.g., 6, 12, 18, and 24 hours post-injection) to characterize the pharmacokinetic and pharmacodynamic profiles .

• Internal standards for quantification: When using LC-MS/MS, incorporate appropriate internal standards such as rat PTH (1-34) for accurate quantification .

How can researchers optimize LC-MS/MS methods for PTHrP N15 detection and quantification?

Optimization of LC-MS/MS methods for PTHrP N15 requires:

• Efficient extraction protocol: Implement solid phase extraction using appropriate sorbents such as Waters Oasis® HLB μElution for optimal recovery from biological matrices .

• Specific quantification transitions: Use specific m/z transitions (e.g., 609.5>682.7 for PTHrP (1-36)) to ensure selective detection .

• Appropriate internal standards: Utilize isotopically labeled internal standards that closely match the analyte structure to correct for extraction and ionization variability .

• Validation against industry standards: Ensure the method meets validation criteria for precision (<12% variation) and accuracy (<11% variation) across the relevant concentration range (e.g., 50-800 pg/mL) .

• Cross-reactivity assessment: Verify that the method does not exhibit cross-reactivity with related peptides such as PTH (1-84) or oxidized forms of the peptide .

• Matrix effect evaluation: Assess and correct for potential matrix effects that may influence ionization efficiency .

What are the comparative outcomes between PTHrP and PTH in clinical bone density studies?

In clinical studies, PTHrP has demonstrated promising effects on bone mineral density (BMD). A 3-month study administering daily subcutaneous PTHrP at 400 μg/day increased lumbar spine BMD by approximately 5%, which compares favorably with results observed with PTH treatment . The table below summarizes baseline characteristics of participants in a clinical trial comparing different doses of PTHrP:

A key difference between PTHrP and PTH treatments is that PTHrP appears to function as a pure anabolic agent, increasing bone formation markers without activating bone resorption markers, even at doses as high as 750 μg/day . This contrasts with PTH, which increases both formation and resorption markers, reflecting its mixed anabolic/catabolic profile .

What are the established therapeutic windows and dose-limiting toxicities for PTHrP in human studies?

Clinical trials have helped define the therapeutic window and dose-limiting toxicities of PTHrP. Studies have shown that PTHrP can be safely administered to healthy adults and postmenopausal women at doses ranging from 50 to 2000 μg/day for periods between 1 day and 3 months . Specifically, PTHrP administered at 500 and 625 μg/day for 3 weeks has been shown to be well-tolerated .

The dose-limiting toxicity appears to be hypercalcemia, which was observed in some subjects receiving 750 μg/day . In a dose-escalation study, three of six subjects receiving 750 μg/day developed mild hypercalcemia, leading to termination of the study drug in these individuals . This established 625 μg/day as the maximum well-tolerated dose in the study population .

Interestingly, unlike with PTH treatment, even minor hypercalcemia was not observed in several earlier studies with PTHrP at various doses . This suggests that PTHrP may have a more favorable safety profile regarding calcium homeostasis compared to PTH.

How do immunoassays compare with LC-MS/MS for quantifying PTHrP N15 Human in research samples?

Comparison studies between LC-MS/MS and immunoassay methods for quantifying PTH-related peptides reveal important differences. Method comparison for PTH (1-34) using human EDTA plasma samples showed a high correlation (r² = 0.950) between LC-MS/MS and immunoassay, but a concentration-dependent negative bias of 35.5% was observed across the range of 0-800 pg/mL with the immunoassay .

Key differences between the methods include:

• Specificity: The immunoassay showed 7% cross-reactivity to human PTH (1-84) and 44% to rat PTH (1-34), while no such interference was observed with the LC-MS/MS method .

• Matrix effects: Matrix effects in the immunoassay were identified as likely contributing factors to the bias between methods .

• Interference from modified forms: The oxidized form of PTH (1-34) does not interfere with the LC-MS/MS method, whereas immunoassays may not distinguish between native and modified forms .

These findings suggest that LC-MS/MS offers superior specificity and accuracy for quantification of PTHrP N15 Human in research applications, particularly when precise quantification is required or when samples may contain related peptides or modified forms of the target analyte.

What are the purity requirements for PTHrP N15 Human in experimental applications?

For research applications, PTHrP N15 Human should meet stringent purity criteria to ensure experimental rigor and reproducibility. Commercial preparations typically specify purity greater than 95.0% as determined by complementary analytical techniques :

These purity assessments are critical for ensuring that experimental outcomes are attributable to the PTHrP N15 Human itself rather than contaminants or degradation products. Researchers should verify the purity information provided by manufacturers and consider performing their own quality control analyses before using the product in critical experiments.