Background/Objectives: NANOBODY® molecules (VHHs) are attractive vectors for radiopharmaceuticals due to their small size and high target affinity, but rapid clearance and pronounced kidney retention limit their therapeutic applicability. Binding to serum albumin is a widely used strategy to prolong circulation, yet the respective contributions of albumin-binding affinity and molecular format remain insufficiently defined. This study aimed to systematically evaluate how affinity and valency modulate VHH pharmacokinetics. Methods: Four monovalent albumin-binding VHHs spanning nanomolar to micromolar affinities and two bivalent constructs were engineered, generated by fusing an albumin-binding VHH to an irrelevant non-binding VHH. All constructs incorporated a site-specific cysteine for DFO* conjugation, enabling uniform zirconium-89 labeling with high radiochemical purity. Pharmacokinetics were assessed in healthy mice using serial blood sampling and positron emission tomography. Blood and kidney exposure were quantified by non-compartmental analysis. Results: All albumin-binding constructs showed increased systemic exposure and reduced kidney uptake relative to a non-binding control. Nanomolar-affinity binders reached maximal exposure, and further affinity increases (KD < ~100 nM) did not improve pharmacokinetics, suggesting a threshold. The micromolar binder showed intermediate exposure but still reduced renal retention compared with control. Valency effects were affinity-dependent. They were negligible at high affinity but pronounced at low affinity, where bivalency reduced systemic exposure and increased kidney uptake toward control levels. Conclusions: Albumin binding enables tuning of VHH pharmacokinetics in an affinity-dependent manner. Above an apparent affinity threshold, pharmacokinetics become format independent, whereas below this threshold, molecular format substantially influences systemic and renal disposition.

Fluorine-18-labeled somatostatin receptor (SSTR) tracers are increasingly adopted as an alternative to generator-produced gallium-68 compounds for imaging neuroendocrine neoplasms (NENs). While [¹⁸F]AlF-NOTA-octreotide ([¹⁸F]AlF-OC) has shown comparable or even superior diagnostic performance in controlled clinical studies, real-world evidence remains limited, particularly for underrepresented NEN subtypes. This study evaluates the clinical implementation, biodistribution, and uptake characteristics of [¹⁸F]AlF-OC PET/CT in routine practice across the full NEN spectrum. In this retrospective single-center analysis, all patients referred for SSTR PET/CT between March 2023 and January 2024 were included. Relevant clinical and demographic data, along with procedural details, were collected. Lesions were segmented semi-automatically using MIM version 7.3.4 and quantitatively assessed. Uptake patterns were analyzed according to tumor grade, primary tumor origin, organ site and clinical indication. A total of 322 scans were performed in 288 patients (median age 64 years). The cohort included 93 Grade 1 (G1), 84 G2, 17 G3 NETs and 3 neuroendocrine carcinomas of gastroenteropancreatic (GEP) or unknown origin, alongside 15 phaeochromocytoma/paraganglioma (PPGL) cases and 40 lung NETs. In total, 4,677 lesions were evaluated. Mean injected activity was 208.5 ± 73.6 MBq with an average uptake time of 121 ± 14 min. G3 NETs demonstrated sufficient uptake (mean SUVmax 19.0), comparable to low-grade NETs. Uptake varied by primary origin, with the highest mean SUVmax values observed in gastric (53.3), sigmoidal (22.0), rectal (20.5), small intestinal (19.1) and pancreatic NETs (17.1). Lung NETs (17.0) also have comparable uptake. Strong tracer avidity was observed in lesions within the liver (19.8) and bone (12.1). Physiological distribution was consistent with known SSTR-expressing tissues. This large real-world series confirms that [¹⁸F]AlF-OC PET/CT is a robust and broadly applicable SSTR imaging modality across diverse NEN subtypes, including tumor groups traditionally underrepresented in prospective trials of SSTR tracers. The tracer demonstrates reliable uptake across grades and organ sites, supporting its routine clinical use as a practical, high-throughput alternative to ⁶⁸Ga-labeled SSAs.

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. Clinical trial number:not applicable.

Purpose Fluorine-18-labeled somatostatin receptor (SSTR) tracers are increasingly adopted as an alternative to generator-produced gallium-68 compounds for imaging neuroendocrine neoplasms (NENs). While [& sup1;F-8]AlF-NOTA-octreotide ([& sup1;F-8]AlF-OC) has shown comparable or even superior diagnostic performance in controlled clinical studies, real-world evidence remains limited, particularly for underrepresented NEN subtypes. This study evaluates the clinical implementation, biodistribution, and uptake characteristics of [& sup1;F-8]AlF-OC PET/CT in routine practice across the full NEN spectrum. Materials and methods In this retrospective single-center analysis, all patients referred for SSTR PET/CT between March 2023 and January 2024 were included. Relevant clinical and demographic data, along with procedural details, were collected. Lesions were segmented semi-automatically using MIM version 7.3.4 and quantitatively assessed. Uptake patterns were analyzed according to tumor grade, primary tumor origin, organ site and clinical indication. Results A total of 322 scans were performed in 288 patients (median age 64 years). The cohort included 93 Grade 1 (G1), 84 G2, 17 G3 NETs and 3 neuroendocrine carcinomas of gastroenteropancreatic (GEP) or unknown origin, alongside 15 phaeochromocytoma/paraganglioma (PPGL) cases and 40 lung NETs. In total, 4,677 lesions were evaluated. Mean injected activity was 208.5 +/- 73.6 MBq with an average uptake time of 121 +/- 14 min. G3 NETs demonstrated sufficient uptake (mean SUVmax 19.0), comparable to low-grade NETs. Uptake varied by primary origin, with the highest mean SUVmax values observed in gastric (53.3), sigmoidal (22.0), rectal (20.5), small intestinal (19.1) and pancreatic NETs (17.1). Lung NETs (17.0) also have comparable uptake. Strong tracer avidity was observed in lesions within the liver (19.8) and bone (12.1). Physiological distribution was consistent with known SSTR-expressing tissues. Conclusion This large real-world series confirms that [& sup1;F-8]AlF-OC PET/CT is a robust and broadly applicable SSTR imaging modality across diverse NEN subtypes, including tumor groups traditionally underrepresented in prospective trials of SSTR tracers. The tracer demonstrates reliable uptake across grades and organ sites, supporting its routine clinical use as a practical, high-throughput alternative to Ga-6(8)-labeled SSAs.

Background/Objectives: The chemokine receptor CXCR4 plays a pivotal role in tumor progression, metastasis, and therapy resistance and is frequently overexpressed in hematologic malignancies, including multiple myeloma and lymphoma. This study investigates a CXCR4-targeted theranostic platform comprising a PET imaging agent and two therapeutic radioconjugates derived from the high-affinity CXCR4 antagonist LY2510924. Methods: The PET tracer [18F]AlF-NOTA-SC and therapeutic radioconjugates [177Lu]Lu-BL02 and [161Tb]Tb-BL02 were synthesized and evaluated. Radiolabeling efficiency, molar activity, and in vitro binding affinity were assessed. Specificity and uptake were evaluated in CXCR4-expressing U87.CD4.CXCR4 and MM.1S cells, with cytotoxic potential being analyzed via clonogenic survival assays. In vivo biodistribution and pharmacokinetics were evaluated in MM.1S xenograft mouse models, supported by longitudinal SPECT imaging. Results: All radioconjugates were obtained with high radiochemical purity (>98%). The constructs showed nanomolar affinity for human CXCR4; in vitro assays confirmed specific uptake in CXCR4-positive cells, and both therapeutic agents demonstrated dose-dependent cytotoxicity. In vivo, all compounds displayed comparable tumor uptake with low off-target accumulation. Co-injection studies confirmed consistent pharmacokinetics across agents, while SPECT/CT imaging demonstrated gradual tumor clearance of [177Lu]Lu-BL02 over seven days. Conclusions: The radiopharmaceutical trio [18F]AlF-NOTA-SC, [177Lu]Lu-BL02, and [161Tb]Tb-BL02 demonstrates the feasibility of a CXCR4-targeted theranostic approach for imaging and treating hematologic malignancies. The observed tumor washout highlights the need for further structural optimization to enhance tumor retention and therapeutic efficacy, providing a clear direction for future development toward clinical translation.

Chronic myeloid leukemia (CML) remains a therapeutic challenge, particularly in patients who develop resistance to standard tyrosine kinase inhibitors (TKIs) such as imatinib. Here, we present the first demonstration of the potent anti-leukemic activity of the histone deacetylase (HDAC) inhibitor martinostat in both TKI-sensitive and TKI-resistant CML. Structural and biochemical analyses confirmed the efficient and selective binding of martinostat to HDAC isoenzyme ligand-binding pockets, resulting in histone and tubulin hyperacetylation in both imatinib-sensitive and resistant CML cells, outperforming vorinostat, a clinically used HDAC inhibitor (HDACi). It selectively impaired CML cell proliferation and viability and induced apoptosis across various CML models, including resistant cell models and patient blasts, with minimal toxicity to healthy cells and low developmental toxicity in zebrafish. In addition to its single-agent efficacy, martinostat demonstrated enhanced anticancer effects when combined with imatinib, both in vitro and in vivo, significantly reducing tumor growth in resistant CML xenograft models. Mechanistically, mRNA-seq data showed that martinostat disrupted key survival signaling pathways and amplified apoptotic responses, contributing to its anticancer activity. These findings highlight the potential of martinostat as a selective, low-toxicity HDACi that, combined with TKIs, could provide an effective strategy to overcome drug resistance in CML and improve therapeutic outcomes.

[11C]CHDI-00485180-R ([11C]CHDI-180R) is a novel PET radioligand developed to image aggregated mutant huntingtin (mHTT). Data from mouse models of Huntington’s disease (HD) and biodistribution studies in healthy volunteers suggested that [11C]CHDI-180R is a promising candidate for in vivo determination of cerebral aggregated mHTT levels using PET. In the iMagemHTT study reported here, we investigated [11C]CHDI-180R kinetic properties and suitability to quantify aggregated mHTT in brains of people with HD (pwHD). A total of 12 pwHD (53.7 ± 6.9y, 5 M/ 7 F, Shoulson-Fahn stage 2) and 12 healthy controls (HC; six young [26.8 ± 3.2y], 2 M/ 4 F; six age-matched [53.7 ± 6.1y],2 M/ 4 F) were included. We conducted dynamic 90 min [11C]CHDI-180R PET imaging with arterial sampling and radiometabolite quantification, and delineated volumes of interest (VOIs) using individual 3D T1-MRI. We calculated total distribution volumes (VT) using 2-compartment modelling (2TCM) as well as Logan graphical analysis and determined distribution volume ratios relative to cerebellum (DVRCBL). We applied partial volume correction, and assessed test-retest variability in pwHD. VT showed considerable intersubject variability among HC (VT(cortex) = 0.68 ± 0.22) and pwHD (VT(cortex) = 0.75 ± 0.26), without any regional significant differences between the groups. VT test-retest variability was high if test and retest scans were performed on the same day, but low (< 10 https://clinicaltrials.gov/study/NCT03810898?term=NCT03810898 rank=1

C-X-C chemokine receptor type 4 (CXCR4) is highly expressed in a range of pathologies, including cancers like multiple myeloma and non-Hodgkin lymphoma, inflammatory diseases such as rheumatoid arthritis, and viral infections like HIV. Currently, the most advanced radiotracer for CXCR4 imaging in clinics is [68Ga]PentixaFor. However, its structure is prone to modifications, complicating the development of a specific CXCR4 fluorine-18-labeled tracer with good pharmacokinetic properties. This study aimed to screen multiple CXCR4-targeting variable domains of heavy-chain-only antibody (VHH or single-domain antibody (sdAb)) constructs to identify the most promising sdAb as a vector molecule for the future development of a CXCR4 fluorine-18 tracer. We have generated five CXCR4-specific sdAb constructs with a cysteine-containing C-terminal tag (C-Direct tag) (VUN400-C-Direct, VUN401-C-Direct, VUN410-C-Direct, VUN411-C-Direct, and VUN415-C-Direct) and one probe (VUN400-C) without. The reduced sdAbs were coupled to maleimide-DOTAGA for 111In-labeling. Their binding affinity against human CXCR4 (hCXCR4) was assessed by using a previously described BRET-based displacement assay. The in vivo profile was assessed using naive mice. Based on the plasma stability (60 min post injection (p.i.)), we selected VUN400-C-Direct and its derivative VUN400-C for further evaluation. These compounds ([111In]In-DOTAGA-VUN400-C-Direct and [111In]In-DOTAGA-VUN400-C) were tested in mice bearing xenografts derived from U87.CD4, U87.CXCR4, and U87.CD4.CXCR4 cells through ex vivo biodistribution studies and SPECT/CT imaging. The six sdAb constructs were labeled with a high radiochemical conversion (75-97%) and purity (>95%). In radioactive binding assays using U87.CD4.CXCR4 cells, [111In]In-DOTAGA-VUN400-C-Direct and [111In]In-DOTAGA-VUN401-C-Direct displayed the highest cellular uptake, achieving 10.4 ± 1.6% and 11.5 ± 1.1%, respectively. In naive mice, [111In]In-DOTAGA-VUN400-C-Direct showed the most favorable biodistribution profile, with low uptake across all organs except the kidneys (Standardized Uptake Value (SUV) > 50, n = 3, 60 min p.i.), but average plasma stability (40.6 ± 9.4%, n = 3, 60 min p.i.). In a xenografted tumor model, [111In]In-DOTAGA-VUN400-C-Direct showed only minor uptake (SUVU87.CXCR4 0.71 ± 0.002, n = 3, 60 min p.i.). [111In]In-DOTAGA-VUN400-C demonstrated nearly identical plasma stability (41.08 ± 5.45%, n = 4) but showed high and specific uptake in the CXCR4-expressing xenografted tumor (SUVU87.CD4.CXCR4 3.75 ± 1.08 vs SUVU87.CD4 = 0.64 ± 0.19, n = 5, 60 min p.i.), which could be blocked by coinjection of AMD3100 (5 mg/kg) (SUVU87.CD4.CXCR4 0.55 ± 0.32 vs SUVU87.CD4 = 0.39 ± 0.07, n = 2, 60 min p.i.). In conclusion, all six sdAbs exhibited high in vitro affinity against hCXCR4. Among these, [111In]In-DOTAGA-VUN400-C showed high CXCR4-specific tumor uptake and favorable pharmacokinetic properties, indicating VUN400-C's potential as a promising vector for future CXCR4 PET imaging applications with fluorine-18.

Heat shock protein 90 (Hsp90) is a critical chaperone in the protein quality control system, essential for maintaining cellular proteostasis. Aberrant Hsp90 function has been implicated in cancer and neurodegenerative disorders, making it an attractive therapeutic target and a potential biomarker for disease characterisation and progression using PET imaging. In this study, we aimed to develop the first fluorine-18 labelled brain permeable PET imaging agent, [18F]FEHSP990, suitable for imaging Hsp90 in both brain and tumour tissue. The radiosynthesis of [18F]FEHSP990 was achieved with a radiochemical yield of 48 ± 29%, high radiochemical purity of > 99% and a molar activity of 213 ± 101 GBq/μmol at the end of synthesis. Competition binding studies in healthy mouse brain homogenate samples indicated a Ki value of approximately 200 nM. In vitro tracer binding to rodent brain and glioblastoma tumour tissue slices was high and deemed Hsp90-specific, as demonstrated by autoradiography blocking studies, whereas binding to living glioblastoma U87 cells was notably low. Ex vivo biodistribution and in vivo PET imaging studies in healthy rodents demonstrated limited brain exposure of the tracer, potentially due to insufficient affinity for Hsp90 and/or restricted blood-brain barrier permeability. Further development of fluorine-18 labelled Hsp90 tracers is warranted.

Therapeutic antibodies for the treatment of neurological disease show great potential, but their applications are rather limited due to limited brain exposure. The most well-studied approach to enhance brain influx of protein therapeutics, is receptor-mediated transcytosis (RMT) by targeting nutrient receptors to shuttle protein therapeutics over the blood–brain barrier (BBB) along with their endogenous cargos. While higher brain exposure is achieved with RMT, the timeframe is short due to rather fast brain clearance. Therefore, we aim to increase the brain half-life of antibodies by binding to myelin oligodendrocyte glycoprotein (MOG), a CNS specific protein. Alpaca immunization with mouse/human MOG, and subsequent phage selections and screenings for MOG binding single variable domain antibodies (VHHs) were performed to find mouse/human cross-reactive VHHs. Their ability to increase the brain half-life of antibodies was evaluated in healthy wild-type mice by coupling two different MOG VHHs (low/high affinity) in a mono- and bivalent format to a β-secretase 1 (BACE1) inhibiting antibody or a control (anti-SARS-CoV-2) antibody, fused to an anti-transferrin receptor (TfR) VHH for active transport over the BBB. Brain pharmacokinetics and pharmacodynamics, CNS and peripheral biodistribution, and brain toxicity were evaluated after intravenous administration to balb/c mice. Additional binding to MOG increases the Cmax and brain half-life of antibodies that are actively shuttled over the BBB. Anti-SARS-CoV-2 antibodies coupled with an anti-TfR VHH and two low affinity anti-MOG VHHs could be detected in brain 49 days after a single intravenous injection, which is a major improvement compared to an anti-SARS-CoV-2 antibody fused to an anti-TfR VHH which cannot be detected in brain anymore one week post treatment. Additional MOG binding of antibodies does not affect peripheral biodistribution but alters brain distribution to white matter localization and less neuronal internalization. We have discovered mouse/human/cynomolgus cross-reactive anti-MOG VHHs which have the ability to drastically increase brain exposure of antibodies. Combining MOG and TfR binding leads to distinct PK, biodistribution, and brain exposure, differentiating it from the highly investigated TfR-shuttling. It is the first time such long brain antibody exposure has been demonstrated after one single dose. This new approach of adding a binding moiety for brain specific targets to RMT shuttling antibodies is a huge advancement for the field and paves the way for further research into brain half-life extension.

Heat shock protein 90 (Hsp90) is a molecular chaperone that interacts with other co-chaperones to stabilize, mature and (re)fold proteins1. Its overexpression in cancer cells makes it attractive as a chemotherapeutic target. However, due to insufficient efficacy or toxicity, only TAS116 (pimitespib) was approved for clinical use in advanced gastrointestinal tumors in Japan2. A synergistic anti-cancer effect of Onalespib with external beam radiotherapy (EBRT) was preclinically observed in malignant gliomas3. This study evaluates the synergistic effect of Hsp90 inhibitor treatment combined with EBRT and temozolomide (TMZ) in glioblastoma models. Methodology: The Hsp90 inhibitors Geldanamycin, Onalespib, BIIB021, NVP-HSP990 and NMS-E973 were evaluated in U-87 MG-WT/IDH1 mutant glioblastoma cell models. IC50 values were determined via sulforhodamine B assay. Western blot was performed for Hsp90 expression evaluation following TMZ treatment. Clonogenic assays were conducted 10 days following 96-hour treatment with monotherapy or dual therapy using Geldanamycin, TMZ, and/or EBRT. In U-87 MG-WT cells, IC50 values were determined for Geldanamycin (53 nM), Onalespib (135 nM), BIIB021 (97 nM), NVP-HSP990 (35 nM), and NMS-E973 (193 nM). For U-87 MG-IDH1 mutant cells, IC50 values were slightly elevated. Preliminary clonogenic assays showed reduced colony formation following EBRT, with enhanced effects in combination with Geldanamycin or TMZ. U-87 MG cell viability assays showed nanomolar-range IC50 values for all Hsp90 inhibitors. A trend towards reduced colony formation was observed following monotherapy and combination therapy. Ongoing in vitro experiments include assays for viability, clonogenic survival, and DNA damage following monotherapy, dual, and triple combination treatments using Hsp90 inhibitors, TMZ and EBRT in additional cell lines. The best performing compounds will be tested in subcutaneous, patient-derived xenografts and orthotopic mouse models, focusing on tumor growth and survival. [1]Mol Pharmacol 95, 468-474 (2019). [2]Journal of Clinical Oncology 39, 11524-11524 (2021). [3]Uffenorde et al. Front Oncol 15 (2025).

The molecular chaperone heat shock protein 90 (Hsp90), essential for protein homeostasis and cellular stress response, has emerged as a promising therapeutic target across various diseases, including cancer, neurodegenerative disorders, and inflammatory conditions. Although numerous Hsp90 inhibitors have been developed and extensively evaluated in clinical studies, progress has been impeded by limited clinical efficacy, narrow therapeutic windows, and challenges in assessing target engagement. These limitations highlight the importance of developing complementary noninvasive molecular imaging tools to better understand Hsp90 function in vivo and optimize therapeutic strategies, including assessing target engagement, refining dosing strategies, monitoring treatment response, and enabling patient stratification. This review provides a comprehensive overview of the current landscape of Hsp90-targeted molecular imaging. We discuss imaging modalities applicable to Hsp90, optical imaging, single-photon emission computed tomography, and positron emission tomography, and highlight key molecular probes developed to visualize Hsp90 expression and function in vivo using these modalities. Furthermore, we summarize significant findings that have deepened our fundamental understanding of Hsp90’s role in disease, supported the development of novel therapeutic approaches, demonstrated imaging effectiveness in preclinical models, and suggested potential for integration into clinical research. We also address current challenges and propose future directions for the field. Through this review, we aim to illustrate the translational potential of molecular imaging in advancing our understanding of Hsp90 in disease and optimizing Hsp90-targeted therapeutics, thereby contributing to precision medicine approaches.

Heat shock protein 90 (Hsp90) is essential for maintaining cellular proteostasis and may play an important role in the development of neurodegenerative proteinopathies. Therefore, we aimed to develop an Hsp90-specific PET brain tracer to quantify Hsp90 expression in the brain in vivo in order to explore its potential as a biomarker for neurodegenerative disease characterization and to support Hsp90-targeted drug development. Methods: We developed the radiosynthesis of (R)-2-amino-7-(4-fluoro-2-(6-(methoxy-11C)pyridin-2-yl)phenyl)-4-methyl-7,8-dihydropyrido[4,3-d]pyrimidin-5(6H)-one, [11C]HSP990, and validated the tracer using in vitro autoradiography, in vitro brain homogenate saturation binding, ex vivo biodistribution, and in vivo PET imaging in rodent models of Alzheimer disease (AD) and Parkinson disease versus healthy age-matched and young controls. Human brain samples from AD patients and healthy subjects were included in our in vitro binding studies. A nonhuman primate PET brain study with arterial blood sampling was conducted under baseline and blocking conditions. Results: In vitro and in vivo [11C]HSP990 studies in rodents and a nonhuman primate revealed saturable Hsp90 binding pools in natural killer lymphocytes, bone marrow, and notably the brain, where the highest binding was observed, particularly in gray matter. Blocking studies indicated that saturable Hsp90 in natural killer lymphocytes considerably influences the pharmacokinetics of Hsp90-targeting probes, which is critical for Hsp90 drug development. In vitro [3H]HSP990 brain homogenate saturation binding assays suggested that the tracer binds a distinct subfraction of the total Hsp90 pool, which is significantly diminished in both rodent and human AD brain tissue compared with age-matched controls. In vivo PET imaging confirmed reduced [11C]HSP990 brain binding on aging and an even stronger decrease in AD mice, suggesting that Hsp90 depletion may impair protein quality control and accelerate proteinopathies. Conclusion: [11C]HSP990 is a promising Hsp90-specific tracer and reveals strong Hsp90 binding in the brain. Uniformly reduced tracer binding was observed in AD brain tissue compared with age-matched controls. [11C]HSP990 holds potential as a biomarker for neurodegenerative disease characterization and progression, and it may aid in patient stratification and therapy monitoring. Human [11C]HSP990 PET neuroimaging studies are under way to investigate whether these findings translate to humans.

This study compares inter- and intraobserver agreement between [18F]AlF-NOTA-octreotide ([18F]AlF-OC) and [68Ga]Ga-DOTA-somatostatin analogues (SSAs) in PET/CT imaging for neuroendocrine neoplasm (NEN) patients. This is a secondary endpoint analysis from our multicenter trial (clin trial.gov identifier: NCT04552847) including 75 NEN patients who received both [68Ga]Ga-DOTATATE (n = 56) or [68Ga]Ga-DOTA-NOC (n = 19) and [18F]AlF-OC PET imaging. Five readers assessed lesion detection and characterization across multiple organs, scoring lesions by number and conspicuity using a 5-point Likert scale. Agreement was measured using Gwet’s agreement coefficient. Results demonstrated nearly perfect interobserver agreement for lesion characterization across all organs for both tracers (0.921 for [18F]AlF-OC; 0.934 for [68Ga]Ga-DOTA-SSA). Similar agreement was observed for the number of lesions across organs (0.736 for [18F]AlF-OC and 0.749 for [68Ga]Ga-DOTA-SSAs). Organ-specific analysis revealed strong agreement for bone and liver lesions, with slightly lower agreement for lymph nodes. Both tracers also showed excellent agreement in determining Krenning scores (0.925 for [18F]AlF-OC and 0.927 for [68Ga]Ga-DOTA-SSAs). While mean lesion conspicuity was similar between tracers, [18F]AlF-OC had a higher global image quality score (4.22 vs. 3.86, p < 0.0001). Intraobserver agreement was consistent between tracers for lesion characterization (> 0.95 for both readers) and lesion count (> 0.80 for both readers). [18F]AlF-OC and [68Ga]Ga-DOTA-SSAs demonstrate comparable and excellent inter- and intraobserver agreement, reinforcing the clinical interchangeability of [18F]AlF-OC PET/CT with [68Ga]Ga-DOTA-SSAs in routine practice.