(W1530-02-08) Non-invasive Molecular Imaging of V3 Receptors to Localize ACTH-Dependent Cushing's Syndrome and Precision Surgery of Corticotropinoma

Purpose: Most often the ACTH-dependent Cushing's syndrome (CS) is due to pituitary adenoma (Cushing disease or CD). The corticotropinoma (pituitary adenomas) produce excess ACTH, which act on adrenals to produce cortisol. The localization and lateralization of corticotropinoma, specially microadenoma of size < 6mm with currently available diagnostic modalities is very challenging. The AVPR1b (V1b or V3) receptors are over-expressed on corticotrophs of the pituitary adenoma but are infrequent on normal pituitary. Therefore, molecular imaging of V3 receptors with PET-CT using Gallium-68 labelled V3 receptors ligand may play a key role in localization of microadenomas for precisely removal of adenoma and preservation of normal functioning pituitary.

Methods: Desmopressin, a ligand of V3 receptors, was designed for conjugation with a bifunctional chelator as DOTA-mDesmo, to enable radiolabeling with a positron emitter, Gallium-68 (⁶⁸Ga). The target specificity of designed molecule, DOTA-mDesmo, was compared by in-silico binding study of desmopresin, DOTA-mDesmo and ⁶⁸Ga-mDesmo with V3 receptors using AlphaFold2 (3D structures prediction), GROMACS (250ns MD simulation to verify the stability of structures), Autodock-GPU (Docking), PyMOL (Visualization), LigPlot⁺ (Interaction analysis). A novel DOTA-mDesmo has been custom synthesized. The radiolabeling of DOTA-mDesmo was standardized and quality control tests were performed for the suitability of ⁶⁸Ga-mDesmo for intravenous administration. In-vitro binding affinity was determined using human V3 receptor protein. Biodistribution study was carried out in healthy male wistar rats at 30, 60, and 120 min post-injection of ⁶⁸Ga-mDesmo (7-9 MBq). A pilot study was performed after obtaining clearance from institutional ethics committee and informed written consent from the patients. Sixteen patients (women-14, Men-2) underwent regional brain PET/CT imaging 35 minutes after injecting 105 ± 36 MBq of ⁶⁸Ga-mDesmo. Low-dose CT image (120 keV, 40 mA) was acquired for whole-body PET/CT aquisition. CE-MRI was performed as routine investigation. All patients underwent trans-sphenoidal surgery of pituitary adenoma. Post-surgical histopathological examination was used as the gold standard.

Results: In-silico studies of DOTA-mDesmo with V3 receptors demonstrated the same binding site of desmopresin and DOTA-mDesmo. The binding energies of both ligands with V3 receptors were -14.55 kCal/mol and -13.98 kCal/mol, respectively. A total of 9 hydrogen bonds (140 non-bonded interactions) and 14 hydrogens (148 non-bonded interactions) were recorded for V3-Desmo and V3-DOTA-mDesmo, respectively. Radiolabelling of DOTA-mDesmo with ⁶⁸GaCl₃, (⁶⁸Ga-mDesmo) eluted from commercially available ⁶⁸Ge/⁶⁸Ga generator, demonstrated high radiochemical yield  ( >95%) and radiochemical purity  ( >99%) at pH 4.2, temp 100^oC and reaction time10 min. The quality control of ⁶⁸Ga-mDesmo demonstrated formulation as sterile; shelf-life and in-vitro stability in human serum (37^oC) up to 4 h; endotoxins (7.3 ± 1.3 EU/V) i.e. within the permissible limit (175 EU/V). ⁶⁸Ga-mDesmo showed good binding, more than 40% with V3 receptors. The V3 receptors pretreated with excess DOTA-mDesmo resulted in more than 50% reduction in binding of ⁶⁸Ga-mDesmo with V3 receptors. The animal biodistribution study showed a high %ID/g in the kidneys (7.37 ± 0.58) and urinary bladder (4.32 ± 0.48) 30 min post intravenous injection, suggested renal route of excretion. Negligible counts (Pituitary: 73 ± 7; Injected counts: 1376689 ± 6356) were observed in the pituitary, suggested non-expression of the V3 receptor in the healthy pituitary. however, the %ID/g in the pituitary was high (0.5 ± 0.03) due to the denominator effect resulting from the small mass of the pituitary gland (~0.008 g). Blood pool %ID/g (1.61±0.22) can be attributed to the moderate plasma protein binding of ⁶⁸Ga-mDesmo, as determined by the TCA precipitation method. Mild physiological uptake (%ID/g) was noted in various organs including muscles (0.74 ±0.24), lungs (0.70 ± 0.28), adrenals (0.47 ± 0.11), and liver (0.46 ± 0.05). However, clearance of ⁶⁸Ga-mDesmo from these organs occurred within 120 minutes following administration. A low uptake was also noted in stomach (0.36 ± 0.08), pancreas (0.30 ±0.10), spleen (0.30 ± 0.09), heart (0.28 ± 0.04), and thyroid (0.28 ± 0.10). The recruited patients in were: macroadenoma-5 (positive control), microadenoma-9, and acromegaly-2 (negative control, ACTH independent). CE-MRI was not able to localize microadenoma in 2 patients. ⁶⁸Ga-mDesmo PET-CT imaging showed good uptake in all accept acromegaly patients. The SUV_max of adenoma and background was 1.0-3.4 and 0.12 ± 0.08. ⁶⁸Ga-mDesmo was not seen in sella of acromegaly (negative controls) patients. The combined anatomical and functional imaging helped surgeons to remove microadenoma precisely and preservation of normal pituitary. The histopathology, of surgical tissue, revealed ACTH immunostaining positive pituitary adenoma in CD patients, while patients with negative ⁶⁸Ga-mDESMO scan (acromegaly) showed GH immunopositivity on histopathological examination.

Conclusion: In-silico and in vitro studies showed high binding affinity of DOTA-⁶⁸Ga-mDesmo for V3 receptor. A good uptake of ⁶⁸Ga-mDesmo on PET-CT of ACTH dependent pituitary micro suggested ⁶⁸Ga-mDesmo PET-CT a non-invasive molecular imaging tool for functional characterization and anatomical localization.

Acknowledgements: The Study was financially supported by Department of Science & Technology (DST)-Biomedical Device and Technology Department Programme, Govt. of India and Endocrine society of India.

48-year-old female presented with blurring in both eyes. CE-MRI showed pituitary macroadenoma (size 38 x 25 x 29 mm). The patient underwent [68Ga]Ga-DOTA-mDesmo (124 MBq) brain PET/CT after 35 minutes of intravenous administration. CT (A) and fused PET/CT (B) images localized the tracer uptake on the pituitary with SUVmax 3.3 (Background SUVmax 0.35).