Long-term frozen tetracosactide acetate maintains adrenal steroid induction in healthy Beagles

The adrenocorticotropic hormone (ACTH) stimulation test is a critical diagnostic tool used to assess adrenal function, particularly in cases of suspected adrenal insufficiency or hyperfunction. Current standard-of-care relies on fresh or short-term stored tetracosactide acetate (TCA), a synthetic ACTH analogue, to stimulate cortisol production. However, challenges related to supply chain stability, cost, and practical storage limitations often hinder widespread accessibility and consistent test performance. The stability of TCA under extended freezing conditions, specifically when stored in common clinical materials like plastic syringes, has remained an unexplored gap, posing logistical hurdles for veterinary and potentially human clinical settings.

This prospective case-crossover study investigated the biological activity of long-term frozen tetracosactide acetate (TCA) in eight adult experimental Beagles (2 males, 2 females per group). Each dog received 5 µg/kg IV of TCA on two occasions, separated by a 4-week washout period. Group 1 received frozen TCA followed by fresh TCA, while Group 2 received fresh TCA followed by frozen TCA. Pre- and post-administration blood samples were collected at both study periods. These samples were subsequently analyzed for cortisol and other steroid metabolites using liquid chromatography-tandem mass spectrometry to precisely quantify the adrenal response. A general linear mixed model was employed to compare responses, accounting for TCA condition (fresh vs. frozen), period, sequence, timing, and individual dog variation.

Frozen tetracosactide acetate (TCA), stored for a median time of 1.8 years (interquartile range 1.5-1.9 years) in plastic syringes, demonstrated comparable biological activity to fresh TCA in stimulating adrenal steroid production. The study found that frozen TCA induced similar overall steroid metabolite responses. Specifically, median T1 cortisol concentrations were 248 nmol/L (IQ range 231-272 nmol/L) with frozen TCA, which was not significantly different from the 254 nmol/L (IQ range 249-262 nmol/L) observed with fresh TCA (P = .037). Similarly, median T1 17-OHP concentrations were 0.73 µg/L (IQ range 0.67-0.85 µg/L) with frozen TCA, compared to 0.79 µg/L (IQ range 0.71-1.17 µg/L) with fresh TCA (P = .09), indicating no statistically significant difference in this key precursor. These results collectively confirm the long-term stability and efficacy of frozen TCA.

Frozen tetracosactide acetate stored for a median of 1.8 years induced steroid metabolite responses statistically similar to those of fresh TCA, demonstrating preserved biological activity.