A hybrid IA-LC-MS/MS method for adrenocorticotropic hormone(1–24) to support interpretation of low-dose cosyntropin-stimulation test

Abstract

Adrenocorticotropic hormone 1–24 (ACTH[1–24]) has a similar effect as endogenous ACTH(1–39) to generate cortisol by targeting the MC2R receptor on the adrenal gland. A new investigational ACTH receptor antagonist drug is being developed to treat diseases of ACTH excess (e.g., Cushing's disease) by binding to the MC2R receptor. Administration of ACTH(1–24) was used in a Phase I clinical study to assess the ability of this drug candidate to suppress the cortisol response to ACTH stimulation. A hybrid immunoaffinity-LCMS assay measuring ACTH(1–24) with a concentration range of 10 to 400 pg/ml was developed to support the study. Consistent and acceptable A&P results were achieved. The assay development and qualification will be discussed.

Article highlights

Background

• Mechanistic PK/PD modeling of hypothalamic–pituitary–adrenocortical axis with an adrenocorticotropic hormone (ACTH) competitive antagonist drug benefits from distinguishing endogenous ACTH(1–39) and exogenous ACTH(1–24) in the context of ACTH stimulation test.

• Immunoassays able to measure ACTH(1–24) without cross-reactivity to ACTH(1–39) were not found.

• A highly sensitive method was needed due to the low-dose ACTH(1–24) administration and rapid clearance from circulation.

Experimental

• A fit-for-purpose, bead-based, hybrid immunoaffinity-LC-MS/MS method was developed for intact measurement of ACTH(1–24) in human plasma.

• Pre-conjugation of capture antibody to magnetic beads provides tolerance to high levels of endogenous biotin, a problem reported for clinical ACTH(1–39) assays.

• A surrogate matrix calibration strategy is employed, which enables potential future multiplexing of endogenous ACTH counterparts.

Results & discussion

• Suitable sensitivity was achieved with an LLOQ of 10 pg/ml.

• Quantitation of ACTH(1–24) was accurate and precise in the presence of physiological levels of ACTH(1–39).

• The stability of ACTH(1–24) needs further investigation.

Conclusion

• The assay is considered suitable for measurement of ACTH(1–24) in human plasma samples from the clinical study.

Keywords: :

• ACTH(1–24)
• ACTH(1–39)
• bead conjugation
• cosyntropin-stimulation test (CST)
• fit-for-purpose
• hybrid IA-LC-MS