First in vivo evaluation of the radiolabelled Hsp90 inhibitor [11C]Onalespib for cancer and brain PET imaging.

Background : Heat shock protein 90 (Hsp90) is a key molecular chaperone involved in maintaining proteostasis, and its function and aberrant expression are linked to tumour progression and to neurodegeneration. Its overexpression in cancers and the clinical development of Hsp90 inhibitors, including the recently approved pimitespib (TAS-116, trade name Jeselhy®), highlights the need for non‑invasive imaging tools to assess Hsp90 expression. Current PET/SPECT tracers show limited performance. Onalespib, a clinically evaluated inhibitor with favourable chemical flexibility and identified as a promising radiosensitizer in glioblastoma, suggesting blood-brain barrier penetration, offers an attractive alternative. In this work, we report the first preclinical evaluation of [¹¹C]Onalespib to assess its suitability as an Hsp90‑targeted PET radiotracer for tumour and brain imaging. Results : [¹¹C]Onalespib was produced in high purity, with an appropriate radiochemical profile and a molar activity suitable for biological studies. In vitro autoradiography demonstrated strong Hsp90‑specific binding in U‑87 MG, MDA‑MB‑231, PC-3 tumour tissues and mouse brain sections, with the signal markedly reduced after preincubation with onalespib or the structurally unrelated inhibitor HSP990. Cell binding assays further confirmed saturable uptake in all three cancer cell lines, with substantial inhibition in the presence of onalespib and other Hsp90 inhibitors. Ex vivo biodistribution studies revealed pronounced abdominal activity and moderate uptake in U‑87 MG xenografts, whereas MDA‑MB‑231 tumours showed only minimal uptake, both with negligible brain signal under the same conditions. PET/CT imaging in both tumour models corroborated these findings and demonstrated effective in vivo blocking by HSP990 in U‑87 MG xenografts, together with limited penetration of [¹¹C]Onalespib into the brain. Plasma metabolite analysis in healthy mice indicated rapid metabolic degradation, with only a small fraction of intact tracer remaining at all measured time points, despite the good in vitro stability in phosphate buffer, mouse serum and human serum. Conclusions : [¹¹C]Onalespib was obtained with high purity and showed Hsp90-specific binding in tumour tissues, cells and brain tissue in vitro. In vivo PET/CT and biodistribution confirmed high abdominal accumulation, moderate tumour uptake and negligible brain penetration. Despite rapid metabolism, the tracer demonstrates clear Hsp90 specificity and requires further optimisation to develop a Hsp90-targeted radiotracers.