In vitro metabolism of cabozantinib in an adrenocortical carcinoma cell line

Mai Le, Oliver Scherf-Clavel

Department of Pharmacy, Ludwig-Maximilians-Universität (LMU) München, 81377 Munich, Germany

 

Background

Adrenocortical carcinoma (ACC) represents a rare tumor of the adrenal cortex with poor prognosis in unresectable/metastatic stages [1]. First-line treatment typically includes mitotane, often combined with etoposide, doxorubicin and cisplatin, though optimal scheduling and drug interactions remain uncertain [2]. A prospective phase II single-arm trial in patients with advanced ACC demonstrated that treatment with the tyrosine kinase inhibitor cabozantinib (CAB) resulted in promising efficacy and a manageable safety profile [3]. CAB is metabolized via CYP3A4. Consequently, prior mitotane therapy may influence CAB plasma concentrations due to CYP3A4 induction. [4] Furthermore, disease characteristics could lead to variable responses [3]. An intrinsic CYP3A4 activity of the tumor is assumed and has not been further investigated, yet.

Methods

This study examines the in vitro metabolism of CAB in the ACC cell line NCI-H295R. Cells were seeded in 96-well plates (40,000 cells/well) and incubated with CAB (500 ng/ml, 100 µl) for a total duration of 72 h. Supernatants were collected at 1, 24, 48 and 72 h, and subsequently stored at -75°C for future analysis. Cells exposed to equivalent DMSO concentrations served as vehicle control. HepG2 and HEK-293 cells were included as positive and negative controls, respectively, and treated identically.

CAB concentrations in cell supernatants were measured by LC-MS/MS (API3200). Sample preparation and measurements were carried out using a published method with minor modifications [5].

Results

CAB levels in supernatants decreased over time in all three cell lines, most notably in HepG2 (to 55% at 72 h), followed by NCI-H295R (61%) and HEK-293 (78%). No CAB metabolites were detected in the supernatant.

Conclusion

The reduction in CAB concentration in the supernatant warrant additional investigations regarding the fate of CAB. Analysis of intracellular CAB and potential metabolites should be the next step.

References

[1] Libé, R.; Huillard, O. Adrenocortical carcinoma: Diagnosis, prognostic classification and treatment of localized and advanced disease. Cancer treatment and research communications, 2023, 37, 100759.

[2] Rowell, N.P. Oncological Management of Adrenocortical Carcinoma: An Update and Critical Review. Oncology and therapy, 2025, 13, 307–323.

[3] Campbell, M.T.; Balderrama-Brondani, V.; Jimenez, C.; Tamsen, G.; Marcal, L.P.; Varghese, J.; Shah, A.Y.; Long, J.P.; Zhang, M.; Ochieng, J.; Haymaker, C.; Habra, M.A. Cabozantinib monotherapy for advanced adrenocortical carcinoma: a single-arm, phase 2 trial. The Lancet. Oncology, 2024, 25, 649–657.

[4] Corso, C.R.; Acco, A.; Bach, C.; Bonatto, S.J.R.; Figueiredo, B.C. de; Souza, L.M. de. Pharmacological profile and effects of mitotane in adrenocortical carcinoma. British journal of clinical pharmacology, 2021, 87, 2698–2710.

[5] Aghai, F.; Zimmermann, S.; Kurlbaum, M.; Jung, P.; Pelzer, T.; Klinker, H.; Isberner, N.; Scherf-Clavel, O. Development and validation of a sensitive liquid chromatography tandem mass spectrometry assay for the simultaneous determination of ten kinase inhibitors in human serum and plasma. Analytical and bioanalytical chemistry, 2021, 413, 599–612.

 

Keywords: Adrenocortical carcinoma, NCI-H295R, Cabozantinib, CYP3A4